长链非编码RNALINC00675在抑制胃癌发展中的作用及分子机制研究

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分类号R735.2密级公开学号1002014083学校代码90031博士学位论文长链非编码RNALINC00675在抑制胃癌发展中的作用及分子机制研究曾硕指导教师杨仕明教授导师组成员董辉教授王星教授柏健鹰教授培养单位第三军医大学第二附属医院消化科申请学位类别博士学术学位专业名称内科学（消化系病）论文提交日期2017年10月论文答辩日期2017年11月答辩委员会主席王春晖教授评阅人专家一专家二专家三房殿春教授赵晓晏教授二〇一七年十一月万方数据 万方数据 目录缩略语表....................................................................................................................................1英文摘要....................................................................................................................................3中文摘要....................................................................................................................................8论文正文长链非编码RNALINC00675在抑制胃癌发展中的作用及分子机制研究...12第一章前言............................................................................................................12第二章LncRNA芯片筛选并鉴定胃癌组织中显著低表达的lncRNALINC00675并分析其表达的临床意义....................................................................................152.1实验材料.........................................................................................................152.2实验方法.........................................................................................................172.3结果.........................................................................................................202.4讨论.........................................................................................................332.5小结.........................................................................................................34第三章LINC00675对胃癌细胞生物学行为的影响..................................................353.1实验材料.........................................................................................................353.2实验方法.........................................................................................................383.3结果.........................................................................................................513.4小结.........................................................................................................61第四章LINC00675在胃癌细胞中的定位..................................................................624.1实验材料.........................................................................................................624.2实验方法.........................................................................................................644.3实验结果.........................................................................................................654.4讨论.........................................................................................................65第五章LINC00675调控胃癌细胞生物学行为的分子机制......................................665.1实验材料.........................................................................................................665.2实验方法.........................................................................................................705.3结果.........................................................................................................815.4讨论.........................................................................................................93第六章LINC00675调节的信号通路..........................................................................946.1实验材料.........................................................................................................94万方数据 6.2实验方法.........................................................................................................956.3结果.........................................................................................................986.4讨论.......................................................................................................126全文结论........................................................................................................................128参考文献........................................................................................................................129文献综述LongNon-CodingRNAs:ThePromisingBiomarkersandTherapeuticTargetsinGastricCancer......................................................................................................139参考文献........................................................................................................................154附录................................................................................................................................163攻读博士学位期间发表的论文............................................................................................172致谢................................................................................................................................173万方数据 第三军医大学博士学位论文缩略语表英文缩写英文全称中文全称AMPAmpicillin氨苄青霉素APAmmoniumpersulfate过硫酸铵BpBasepair碱基对BSABovineserumalbumin牛血清白蛋白cDNAComplementaryDNA互补DNADEPCDiethylpyrocarbonate焦碳酸二乙酯DMEMDulbecco’sModifiedEagleMedium改良Eagle培养基DMSODimethylsulfoxide二甲基亚砜DNADeoxyribonucleicacid脱氧核糖核酸dNTPDeoxyribonucleosidetriphosphate脱氧核糖核苷三磷酸EBEthidiumbromide溴化乙锭E.coliEscherichiacoli大肠杆菌EDTAEthylenedominatetraaceticacid乙二胺四乙酸FCMFlowcytometry流式细胞术FCSFetalcalfserum胎牛血清GCGastriccancer胃癌HEHaematoxylin-eosin苏木素-伊红HRPHorseradishperoxidase辣根过氧化物酶IODIntegratedoptiondensity累积光密度kDaKilodalton千道尔顿LncRNALongnon-codingRNA长链非编码RNAMmol摩尔浓度mgMilligram毫克minMinute分钟mlMilliliter毫升mRNAMessengerRNA信使RNAncRNAnon-codingRNA非编码RNAntnucleotide核苷酸1万方数据 第三军医大学博士学位论文ODOpticaldensity光密度PBSPhosphatesalinebuffer磷酸盐缓冲液PCRPolymerasechainreaction聚合酶链反应RIPRNAbindingproteinRNA结合蛋白免疫沉淀immunoprecipitationRNaseRibonucleaseRNA酶RNasinRibonucleaseinhibitorRNA酶抑制剂RNARibonucleicacid核糖核酸RNAiRNAinterferenceRNA干扰rpmRevolutionsperminute每分钟转数RT-PCRReversetranscription-polymerasechain逆转录聚合酶链反应reactionSDSSodiumdodecylsulfate十二烷基硫酸钠siRNASmallinterferingRNA小干扰RNAshRNAShorthairRNA短发卡RNATAETris-Acetate-EDTA乙酸电泳缓冲液TaqThermusaquaticusDNAPolymerase栖热水生菌DNA聚合酶TBSTris-bufferedsalineTris-HCL缓冲盐溶液TBSTTrisbufferedsalinewithtween20含20%tween20TBS缓冲液TEMEDTetramethylethylenediamine四甲基乙二胺TrisTris（hydroxymethyl）aminomethane三（羟甲基）氨基甲烷WBWesternblotting蛋白免疫印迹2万方数据 第三军医大学博士学位论文TheroleandmolecularmechanismoflongnoncodingRNALINC00675ingastriccancerprogressionAbstractBackground:Gastriccancer（GC）isthemostcommonmalignancyworldwide.IntheUnitedStates,26,370newcasesand10,730deathsoccurredin2016.AworsesituationappearsinChina,wheretheincidenceandmortalityforGCbothranksecondamongtotalcancers.AlthoughtheincidenceofGChasdecreasedinwesterncountries,theoverall5-yearrelativesurvivalrateremainsapproximately20%inmostareasoftheworld;forstageIVdisease,thisrateislessthan5%.SurgicalresectionforearlyGChasacurableeffect,butmostpatientswithGChaveadvanced-stagediseasewhendiagnosed.Therefore,itisurgenttoexplorenovelbiomarkersandtherapeutictargetsforthediagnosisandtreatmentofGC.LongnoncodingRNAs（lncRNAs）areaclassofnoncodingRNAs>200nucleotidesinlengththatdonotencodeproteins.TheENCyclopediaofDNAElements（ENCODE）projecthasindicatedthatmorethan80%ofthehumangenometranscribesRNAproductsandhasbiologicaleffects,butlessthan2%ofthetotalgenomeencodesproteins,suggestingthatabundantnoncodingRNAshavepotentialbio-functions.Inrecentyears,increasingevidencehasshowntheroleoflncRNAsincancerdevelopment,suchasHOTAIR,H19,andXist.InGC,thelncRNAHOXA11-ASfunctionsasascaffoldtorecruitEZH2andLSD1,resultinginacomplex-integratedphenotypeaffectingcellgrowth,migration,invasion,andapoptosis.SeverallncRNAsinserumortissuewereconsideredasbiomarkersforthediagnosisandprognosisofGC.Untilnow,manylncRNAshavenotbeenfunctionallycharacterized,andthemechanismunderlyingtheirbiologicaleffectsonthedevelopmentofGCremainslargelyunknown.ThepresentstudyaimedtoidentifyanovellncRNAwithaberrantexpressioningastriccancer（GC）progression,andinvestigateitsroleandmolecularmechanisminGCprogression.Methods:1.ThelncRNAswithaberrantexpressionsinGCtissueswerescreenedusinglncRNAmicroarray.3万方数据 第三军医大学博士学位论文2.CandidatelncRNAswereselectedandidentifiedusingqRT-PCRexperiment,andfinallyanovellncRNA,termedLINC00675（gastriccancer-associatedsuppressorofVimentin）,waslockedtoinvestigate.3.LINC00675expressionwasanalyzedinlarge-scalesamplesofGCandadjacentnormaltissuesusingISHandqRT-PCR,andtherelationshipbetweenLINC00675expressionandclinicalcharacterizationwasassessed.4.GCcellswithstableexpressionofLINC00675wereconstuctedusinglentiviruscontainingpcDNA3.1vectorandshRNAsequence.5.TheroleofLINC00675inregulatingGCcellswasevaluatedusingCCK-8,colonyformation,Trans-well,nudemousemodelswithsubcutaneousinjectionorcaudalveininjectionorperitonealcavityinjection.6.pcDNA3.1-LINC00675-MS2andGFP-MS2vectorswereconstructedandtransfectedintoGCcells,andthecomplexinteractingwithLINC00675wereextractedusingRIPexperiment.7.ThecomplexweresubjecttoisolationusingSDS-PAGE,andstainedusingsilver,andfinallythecomponentwasanalyzedusingmassspectrum.8.TheproteininteratingwithLINC00675wasidentifiedusingWesternblotexperiment.9.ThecomplexinteratingwiththeproteinwasextractedusingRIPexperimentbasedonanantibodyoftheprotein,andtheenrichmentofLINC00675inthecomplexwasdetectedusingqRT-PCRexperiment.10.TheexpressionandphosphorylationlevelofthebindingproteinifregulatedbyLINC00675weredetectedusingWesternblotexperiment.11.ThegeneexpressionprofileinLINC00675-overexpressingGCcellswasanalyzedusinggenome-widetranscriptmicroarray.Results:1.TheaberrantexpressionprofileoflncRNAsinGCtissueswasscreenedusinglncRNAmicroarray.ThecandidatelncRNALINC00675wasselected,andtherelationshipbetweenitsexpressionandclinicalcharacterizationwasevaluated.（1）LncRNAmicroarrayexperimentrevealed154lncRNAswithaberrantexpressions,amongwhich59lncRNAswereoverexpressed,and95lncRNAsweredownregulated.InthedownregulatedlncRNAs,theexpressionofLOC400573wasdecreasedby5foldinGC4万方数据 第三军医大学博士学位论文tissuescomparedwithadjacentnormaltissues（rankedthethird）.Currently,theexpressionandroleinGChadnotbeenexplored.LOC400573wascalledLINC00675.（2）LINC00675expressionwasfoundtobesignificantlydecreasedinGCtissuescomparedwithadjacentnormaltissuesusingqRT-PCRandISHexperiments.ROCcurveanalysisshowedthatLINC00675expressionhadanAUCof0.7256indistinguishingGCtissuesfromadjacentnormaltissues,withsensitivityof78.05%andspecificityof72.5%.GCpatientswithdecreasedexpressionofLINC00675hadmorepoorsurvivalratesthanthosewithincreasedexpressionofLINC00675.（3）ClinicaldataanalysisshowedthatLINC00675expressionwascorrelatedwithtumorsize,TNMstage,lyphnodemetastasis,anddistalmetastasis.2.StudyontheroleofLINC00675invitro.（1）GCcells（MKN-45andSGC-7901）withstablyinhibitoryorenforcedexpressionofLINC00675wererespectivelyconstructed.（2）CCK-8andcolonyformationexperimentsshowedthatLINC00675-overexpressingMKN-45andSGC-7901cellshaddecreasedabilitiesofcellproliferationandcolonyformation,whereasLINC00675-knockdownMKN-45andSGC-7901cellshadincreasedabilitiesofcellproliferationandcolonyformation.（3）FlowcytometryexperimentshowedthatthenumberofLINC00675-overexpressingGCcellsinG1phasewassignificantlyincreased,andinSphasewassignificantlydecreased,whereasthenumberofLINC00675-knockdownGCcellsinG1phasewassignificantlydecreased,andinSphasewassignificantlyincreased,suggestingthatLINC00675suppressedtheG1/Stransition.（4）Trans-wellexperimentsshowedthatLINC00675-overexpressingMKN-45andSGC-7901cellshaddecreasedabilitiesofcellmigrationandinvasion,whereasLINC00675-knockdownMKN-45andSGC-7901cellshadincreasedabilitiesofcellmigrationandinvasion.（5）SubcutaneousinjectionmousemodelexperimentsshowedthatLINC00675upregulationresultedinsignificantlydecreasedsizeandweightofformativetumors,andtheexpressionofKi67wasdecreasedintheformativetumors.However,LINC00675downregulationcausedsignificantlyincreasedsizeandweightofformativetumors,andtheexpressionofKi67wasincreasedintheformativetumors.5万方数据 第三军医大学博士学位论文（6）CaudalveininjectionmousemodelexperimentsshowedthatLINC00675-upregulationGCcellshadasignificantlysubduedabilityoflungmetastasis,whereasLINC00675-knockdownGCcellshadasignificantlyenhancedabilityoflungmetastasis.IntraperitonealinjectionmousemodelexperimentsshowedthatLINC00675-upregulationGCcellshadasignificantlysubduedabilityoflivermetastasis,whereasLINC00675-knockdownGCcellshadasignificantlyenhancedabilityoflivermetastasis.3.ThemechanismbywhichLINC00675suppressesGCcellsproliferation,migrationandinvasion.（1）ThevectorsofpcDNA3.1-LINC00675-MS2andGFP-MS2weresuccessfullyconstructed,andco-transfectedintoSGC-7901cells.QRT-PCRexperimentshowedthattheexpressionofLINC00675wassignificantlyincreasedinthetransfectedSGC-7901cells,andWesternblotexperimentsrevealedthattheexpressionofGFPwassignificantlyincreasedthetransfectedSGC-7901cells.（2）LINC00675-interactingcomplexwereisolatedusingRIPexperiments.SDS-PAGEandsilverstainingrevealedthatsignificantlyaberrantexpressionsofproteinsexistedinGFPgroupcomparedwithIgGgroup.Thespecificbandwascutandunderwentmassspectrumexperiment,andtheresultsshowedthatVimentinprotein,whosemolecularweightwas57kDa,wasincludedinthecomplex.（3）WesternblotexperimentconfirmedthatVimentinproteinexistedinLINC00675-interatingcomplex.ByusingVimentinantibody,RIPandqRT-PCRexperimentsshowedthatLINC00675waslargelyenrichedintheisolatedcomplex.ThesedatasuggestedthatLINC00675interactedwithVimentin.（4）WesternblotexperimentshowedthatVimentinhadahighphosphorylationlevelonSer83inLINC00675-overexpressingSGC-7901cellscomparedwithparallelcontrolSGC-7901cells,whereasalowphosphorylationlevelonSer83wasfoundinLINC00675-knockdownSGC-7901cellscomparedwithparallelcontrolSGC-7901cells.However,theexpressionandphosphorylationlevelonSer39ofVimentinhadnotsignificantlychanged.（5）Genome-widetranscriptmicroarrayshowedthatmultiplesignalingpathwayswereactivated,includingp53,andsomewereinactiviated,includingTGF-beta,inLINC00675-overexpressingSGC-7901cells.Furthermore,theexpressionsofmultiple6万方数据 第三军医大学博士学位论文genesweredecreasedin“BENPPRATH_ES_WITH_H3K27ME3”and“ZWANG_TRANSIMENTLY_UP_BY_1ST_EGF_PULSE_ONLY”,andtheexpressionsofmultiplegeneswereincreasedin“MANALO_HYPOXIA_DN”and“GO_RIBONUCLEOPROTEIN_COMPLEX”.Conclusion:1.WefirstfoundthatLINC00675expressionwassignificantlydecreasedinGCtissues,andwasassociatedwithtumorsize,TNMstage,lymphnodemetastasis,distalmetastasis,andpatients’survivalrates.2.LINC00675suppresseSGCcellsproliferation,migrationandinvasion.3.LINC00675interactswithVimentinandenhancesitsphosphorylationonSer83,resultinginthecollapseofVimentinIF.4.LINC00675overexpressioncausesdecreasedexpressionsofmultipleoncogenesandincreasedexpressionsofmultipletumorsuppressors,andregulatesseveralcancer-relatedsignalingpathway.Inconclusion,LINC00675interactswithVimentinandenhancesitsphosphorylationonSer83tosuppressGCprogression.Keywords:longnoncodingRNA;Vimentin;phosphorylation;gastriccancer7万方数据 第三军医大学博士学位论文长链非编码RNALINC00675在抑制胃癌发展中的作用及分子机制研究摘要研究背景：胃癌是全球最常见的肿瘤之一。流行病学调查显示2016年美国新发胃癌病例26370，死亡病例10730，而我国的胃癌发病及死亡人数更多，在所有肿瘤中居第三位。尽管在西方国家胃癌的发病率有所降低，但患者5年生存率只有20%左右，而对于晚期（stageIV）患者，生存率低至5%。目前，手术切除术对早期胃癌具有治愈效果，但绝大部分患者被检时已为进展期。因此，探索新的胃癌诊断标志物及药物靶点具有重要意义。LncRNAs是一类长度大于200nt的单链RNA分子，不编码蛋白。ENCODE（ENCyclopediaofDNAEliments）计划研究表明人类80%以上的基因组是有功能的，并编码大量的RNA分子，而能编码蛋白的RNA分子不足2%，这就意味着人类基因组转录大量具有功能的非编码RNA分子。近年来，大量的证据已表明非编码RNA参与癌症发育的调控，如HOTAIR,H19，Xist等。在胃癌中，lncRNAHOXA11-AS作为支架，其两端分别结合EZH2和LSD1蛋白，影响胃癌细胞增殖、迁移、侵袭及凋亡。一些血浆或组织中出现的lncRNAs被认为是胃癌诊断的生物标志物。截至目前，胃癌中仍然存在大量差异表达的lncRNAs，其功能及调节机制未被鉴定。本研究的目的在于鉴定胃癌进展中差异表达的lncRNA，并研究其在胃癌进展中的作用及分子机制。研究方法：1.采用lncRNA芯片技术筛选胃癌组织中表达差异显著的lncRNAs；2.筛选候选lncRNAs，采用qRT-PCR进行鉴定，最终获得一条表达差异最显著且功能未被鉴定的lncRNA，即LINC00675；3.采用原位杂交、qRT-PCR技术大样本量分析LINC00675在胃癌及癌旁中的表达，并分析其与患者临床资料的相关性；4.采用慢病毒载体包装，pcDNA3.1载体稳定过表达或shRNA序列稳定干扰LINC00675的表达；5.采用CCK-8、平板克隆形成、Trans-well、裸鼠皮下成瘤模型、裸鼠尾静脉及腹腔注射荷瘤模型等研究LINC00675对胃癌细胞的调控；6.构建pcDNA3.1-LINC00675-MS2、GFP-MS2载体，8万方数据 第三军医大学博士学位论文共转染胃癌细胞，采用RIP技术分离与LINC00675结合的复合物；7.采用SDS-PAGE胶分离、银染，蛋白质谱分析复合物成分；8.采用Westernblot检测复合物中与LINC00675结合的蛋白；9.采用RIP技术分离结合蛋白及其复合物，qRT-PCR检测LINC00675的富集情况；10.采用Westernblot分析LINC00675对结合蛋白表达及磷酸化水平的影响；11.采用转录本芯片分析过表达LINC00675对胃癌细胞基因表达的影响。研究结果：一、LncRNA芯片筛选并鉴定胃癌组织中显著低表达的lncRNALINC00675并分析其在胃癌组织中的表达及临床意义。1.LncRNA芯片筛选发现有154个表达差异显著的lncRNAs，其中59个lncRNAs的表达显著上调，95个lncRNAs的表达显著下调；分析发现在下调的lncRNAs中，LOC400573的表达差异5倍（居第三位），其表达和功能在胃癌中未被证实，LOC400573后被命名为LINC00675。2.QRT-PCR及原位杂交实验发现LINC00675在胃癌组织中的表达显著低于癌旁组织；工作特征曲线（ROC）分析发现LINC00675鉴别胃癌及癌旁的曲线下面积为0.7256，敏感性为78.05%，特异性为72.5%；LINC00675低表达的胃癌患者生存率显著低于高表达患者。3.临床资料分析显示，LINC00675的低表达与胃癌肿瘤大小、TNM分期、淋巴结转移、远处转移密切相关。二、LINC00675对胃癌细胞生物学行为的影响。1.成功构建稳定干扰或过表达LINC00675的胃癌细胞株MKN-45和SGC-7901。2.CCK-8及平板克隆形成实验发现过表达LINC00675的胃癌细胞MKN-45和SGC-7901的增殖能力及克隆形成能力显著减弱，而干扰LINC00675表达的胃癌细胞MKN-45和SGC-7901的增殖能力及克隆形成能力显著增强。3.流式细胞仪器分析发现过表达LINC00675的胃癌细胞G1期显著增加，S期显著减少，而干扰LINC00675表达的胃癌细胞G1期显著减少，S期显著增加，提示LINC00675抑制G1期向S期转变。4.Trans-well实验发现过表达LINC00675的胃癌细胞MKN-45和SGC-7901的迁移和侵袭能力显著减弱，而干扰LINC00675表达的胃癌细胞MKN-45和SGC-7901的迁移和侵袭能力显著增强。5.裸鼠皮下成瘤实验发现过表达LINC00675的胃癌细胞SGC-7901的肿瘤形成体积及重量显著降低，以及形成瘤组织中Ki67的表达显著降低，而干扰LINC00675表9万方数据 第三军医大学博士学位论文达的胃癌细胞SGC-7901的肿瘤形成体积及重量显著增加，以及形成瘤组织中Ki67的表达显著增加。6.裸鼠尾静脉注射过表达LINC00675的胃癌细胞SGC-7901，发现细胞的肺转移能力显著减弱，而尾静脉注射干扰LINC00675表达的胃癌细胞SGC-7901，发现细胞的肺转移能力显著增强；裸鼠腹腔注射过表达LINC00675的胃癌细胞SGC-7901，发现细胞的肝转移能力显著减弱，而腹腔注射干扰LINC00675表达的胃癌细胞SGC-7901，发现细胞的肝转移能力显著增强。三、LINC00675抑制胃癌细胞增殖、迁移及侵袭的分子机制。1.成功构建pcDNA3.1-LINC00675-MS2、GFP-MS2载体，共转染至胃癌细胞SGC-7901，qRT-PCR检测发现LINC00675的表达显著增加，Westernblot检测发现GFP的表达显著增加。2.RIP实验分离与LINC00675结合的复合物；SDS-PAGE银染发现，与IgG组相比，GFP组在约60kDa处存在显著差异蛋白；将该蛋白切下行质谱分析发现该蛋白包含Vimentin蛋白，而Vimentin蛋白的分子量为57kDa。3.Westernblot检测与LINC00675结合的蛋白发现Vimentin蛋白呈阳性，而采用Vimentin的抗体行RIP、qRT-PCR实验发现LINC00675有大量富集，提示LINC00675与Vimentin发生相互作用。4.Westernblot检测发现过表达LINC00675的胃癌细胞SGC-7901中Vimentin的Ser83磷酸化水平显著增强，而干扰LINC00675表达的胃癌细胞SGC-7901中Vimentin的Ser83磷酸化水平显著降低，而Vimentin的表达没有变化及其Ser39磷酸化水平没有明显变化。5.转录组芯片分析发现过表达LINC00675的胃癌细胞SGC-7901中多个信号通路被激活，包括p53信号通路，多个信号通路被失活，包括TGF-beta信号通路；同时，与“BENPPRATH_ES_WITH_H3K27ME3”，“ZWANG_TRANSIMENTLY_UP_BY_1ST_EGF_PULSE_ONLY”相关基因的富集度显著降低，与“MANALO_HYPOXIA_DN”，“GO_RIBONUCLEOPROTEIN_COMPLEX”相关基因的富集度显著增多。研究结论：1.首次发现LINC00675在胃癌组织中低表达，且与胃癌的大小、TNM分期、淋巴结转移、远处转移呈负相关，与胃癌患者的生存率呈正相关。2.LINC00675抑制胃癌细胞的增殖、迁移及侵袭能力。3.LINC00675与Vimentin相互作用，促进Vimentin在Ser83位点磷酸化，导致Vimentin中间丝蛋白解聚。10万方数据 第三军医大学博士学位论文4.LINC00675的过表达导致多个癌基因表达降低，多个抑癌基因表达增加，并调节多个癌症相关信号通路。以上研究表明LINC00675通过与Vimentin相互作用增强Vimentin在Ser83位点磷酸化，抑制胃癌的进程。关键词：长链非编码RNA；波形蛋白；磷酸化；胃癌11万方数据 第三军医大学博士学位论文长链非编码RNALINC00675在抑制胃癌发展中的作用及分子机制研究第一章前言[1]2015年中国癌症统计，我国每年新发癌症病例约4292,000，每天约12,000人被诊断为癌症。男性最常见的5种癌症依次是肺癌、胃癌、食道癌、肝癌和结直肠癌，占所有癌症病例数的三分之二；女性最常见的5种癌症依次是乳腺癌、肺癌、胃癌、结直肠癌和食管癌占所有癌症病例数的60%。每年因癌症死亡的病例约2814,000，每天约7500人因癌症死亡。导致男性和女性死亡的5种癌症依次是肺癌、胃癌、肝癌、食管癌和结直肠癌，占所有癌症的四分之三。由此可见胃癌的发病和死亡率仅次于肺癌。胃癌的发病年龄主要集中在60-74岁之间。近10年来，尽管胃癌的发病和死亡率有下降趋势，但由于癌症人群，人口增长及老龄化人口增加所以导致2015年胃癌的新发病例数量是增加的。胃癌的发病机制十分复杂。目前认为慢性萎缩性胃癌和肠上皮化生是胃癌发病的[2]重要环节。胃粘膜的萎缩导致腺体丢失，并被化生细胞或成纤维细胞代替，因此导致胃酸分泌减少；肠上皮化生是胃粘膜转化成肠上皮样表型，布满杯状细胞和肠黏蛋白，被认为是癌前病变。肠上皮化生与同源转录因子CDX2的高表达密切相关，又被[3]分为三个亚型：肠型、胃型和肠胃混合型，而后者被认为具有胃癌发展的高风险。近十年来，几个研究团队的研究将胃癌的启动因素进行了综合的分类，包括基因突变、体细胞拷贝数的改变（sCNAs）、结构改变、表观遗传的改变、转录的改变，涉及mRNAs[4]和非编码RNA（noncodingRNA,ncRNA）。非编码RNA在胃癌的发病机制中呈现[5]出重要地位，包括不同类型的非编码RNA，主要是微小RNA（miRNA）和长链非[6,7]编码RNA（lncRNA）。人类基因组70%-90%的DNA转录RNA，但仅2%的RNA产物能编码蛋白，提示[8-10]RNA转录本中绝大部分属于非编码RNA。非编码RNA最初被认为是DNA转录[11-14]的“噪音”，但近年来大量的研究表明非编码RNA在人类疾病中发挥重要的作用。非编码RNA主要分为两大类：管家非编码RNA和调节性非编码RNA。管家非编码RNA包括核糖体RNA、转运RNA、小核RNA、小核仁RNA；调节性非编码RNA又可能分为短调节性非编码RNA和长调节性非编码RNA，短调节性非编码RNA包括12万方数据 第三军医大学博士学位论文[15]miRNA、小干扰RNA、Piwi相关RNA；长链非编码RNA（lncRNA）是最大的一类非编码转录本且具有重要意义。然而，目前对这方面的研究甚少，但每年仍有大量新的lncRNAs被注释。LncRNAs是一类长度大于200nt的单链RNA分子，不编码蛋白。其长度的阈值看似简单却实用于生物学功能的界定，区别于小的非编码RNA，如[16,17]miRNAs，siRNA以及其他的小RNA。一些lncRNAs被称为macroRNAs，其长[18,19]度可超过90Kb，如Airn和KCNQ1OT1。“Non-coding”是在转录本中指缺乏开放[20-22]阅读框和/或保守密码子，但仍保有争议。最近的研究提示lncRNAs能被核糖体[23,24]实用，以及一些转录本可以产生蛋白。当然，lncRNAs编码蛋白的能力是微不足道的，但也有研究表明一些转录本的双重功能，即同时具有蛋白编码和lncRNAs的功[25]能。与编码蛋白基因相比，lncRNAs在物种之间具有低保守性且大量存在于内含子[26,27]和基因间区域。总体来讲，lncRNAs的保守性需要从不同角度去考虑，如序列，结构，功能等，进而分析其在不同物种之间的进化关系。LncRNAs在细胞核内产生与编码蛋白的转录本的合成相似。LncRNA启动子通常[14]被组蛋白修饰所标记以及被相应的转录因子调节。许多lncRNAs同样通过PolII转录，而其他lncRNAs启动子具有PolIII转录的结构。这些调节允许lncRNAs在特定的时间和空间表达。通常细胞内lncRNAs的含量要比mRNAs低很多，并且它们的表[28,29]达更容易受到不同细胞的限制。大多数的lncRNAs同样具有5’端帽子结构及3’[30,31[32]端多聚腺苷酸化]。RNA剪切同样能改变非编码RNA的转录本。几乎所有的lncRNAs都有经典的剪切位点，导致至少两个转录本剪切体，其主要由两个外显子组[29]成。与mRNA不同，lncRNAs可出现在不同的细胞器。LncRNAs关键的特性是能够形成热力学稳定的结构。基于Wstson-Crick序列配对原则，lncRNAs可形成有功能[33]的结构，有利于与DNA或其他RNA分子相互作用，如mRNAs和miRNAs。二级结构包括螺旋线，发夹环，凸起，伪结等结构，其主要通过序列的配对或与核糖骨架[34]相互作用形成。大多数情况下，这些结构决定了lncRNA的功能，而不是其原始序列。例如，HOTAIR作为一个肿瘤抑制子，它的功能依赖于保守的二级结构元件，其[35]包围了蛋白结合基序。而RNA的三级结构主要出现在核酶中，如核糖体元件或剪[33]切体，而lncRNAs较少出现三级结构。尽管lncRNAs比它们编码蛋白的部分具有更复杂的结构，但它们结构的多能行可能是其与其它分子间相互作用的关键，这就提示lncRNA结构显著影响着它们的生物学功能。然而，关于lncRNA折叠的过程包括辅助因子的发现以及结构到功能的关系的研究才刚刚开始。1988年，Pachnis等首次报道了lncRNAH19，由5个外显子构成不同寻常的结构，13万方数据 第三军医大学博士学位论文[36][19][37][38]在肌细胞分化早期被激活。随后相继发现了Air、NRON、HOTAIR等lncRNAs。目前，超过32000个lncRNAs被记录，其中超过1000个lncRNAs被证实与疾病相关[39]（LncRNADiseasedatabase）。LncRNA的作用机制十分复杂，Guttman等总结了lncRNA的分子调节原则：（1）lncRNA有顺势作用元件功能，影响其临近的基因；（2）lncRNA作为反式调节因子调节其临近的基因；（3）lncRNA结合调节性蛋白并改变其活性，导致调节性蛋白修饰状态改变并影响其靶基因的表达；（4）lncRNA作为诱饵结合分子复合物，阻碍分子复合物与其他调节性靶标结合。总体上讲，lncRNA能与蛋白、RNA、DNA发生相互作用，进而影响染色体修饰，基因的表达，蛋白或mRNA的稳定性。越来越多的研究表明lncRNA在胃癌的发生和发展中发挥着重要的[40][41][6][42]调控作用，涉及胃癌细胞增殖、凋亡、迁移、侵袭等，但对于大量功能未被鉴定的lncRNAs来说，仍然是冰山一角。因此，本项目从临床样本出发，采用芯片筛选并鉴定出一条新的具有重要调控功能的lncRNA并探讨其在胃癌发展中的作用及分子机制。14万方数据 第三军医大学博士学位论文第二章LncRNA芯片筛选并鉴定胃癌组织中显著低表达的lncRNALINC00675并分析其表达的临床意义[43]课题组前期总结了在胃癌发生和发展中涉及的lncRNAs。目前，基于lncRNA芯片筛选病变部位表达差异的lncRNAs仍是主要手段之一。LncRNA芯片采用的探针几乎包含目前已记载的lncRNAs。课题组采用lncRNA芯片对5对人胃癌及癌旁组织样本进行分析，发现有154个表达差异显著的lncRNAs，其中59个lncRNAs的表达显著上调，95个lncRNAs的表达显著下调；分析发现在下调的lncRNAs中，LOC400573的表达差异5倍（居第三位），其表达和功能在胃癌中未被证实，该lncRNA后被命名为LINC00675。基于此，课题组针对LINC00675设计了扩增引物，采用qRT-PCR及原位杂交技术在两组胃癌人群中检测了LINC00675在胃癌及癌旁组织中的表达。2.1实验材料2.1.1临床样本用于芯片筛选的样本于2011年在第三军医大学第一附属医院普外科收集，共5例胃癌患者，已行病理诊断，样本为配对的胃癌和癌旁组织，所有患者均首次治疗，且在手术之前没有行放化疗。癌旁组织的取材位于癌组织边缘5cm之外，收集时间小于20分钟，取材样本迅速冻存于液氮中。用于检测LINC00675表达的胃癌组织样本收集于两组人群，第一组样本于2011-2014年收集于第三军医大学第一附属医院/第二附属医院普外科，共包含50对样本，其中45例患者资料齐全，随访3年。第二组样本购买上海芯超公司的胃癌组织芯片（HStm-Ade180Sur-04），共90对样本，其中80例患者资料齐全，随访6.6-7.3年（手术时间2007.5－2008.2，随访时间2014.9）。2.1.2主要试剂DEPC水上海碧云天生物技术有限公司TRIzol美国Thermo公司氯仿成都科龙化工试剂长异丙醇成都科龙化工试剂长无水乙醇重庆川东化工有限公司逆转录试剂盒日本TaKaRa公司15万方数据 第三军医大学博士学位论文Real-timePCR试剂盒日本TaKaRa公司原位杂交试剂盒博士德生物制品有限公司PBS博士德生物制品有限公司柠檬酸钠重庆川东化工有限公司双氧水重庆川东化工有限公司TritonX-100上海碧云天生物技术有限公司DAB显色试剂盒中杉金桥4%多聚甲醛上海溶剂厂伊红染液北京中杉金桥苏木素染液北京中杉金桥RNA无酶离心管（1.5ml）美国AXYGEN公司RNA无酶PCR离心管美国AXYGEN公司RNA无酶吸头美国AXYGEN公司2.1.3主要仪器电子分析天平300mc型，瑞典低温冰箱（4℃、-20℃）德国Siemens公司超低温冰箱（-80℃）德国Siemens公司ABSystem7500荧光定量PCR仪美国ABI公司高压灭菌锅上海压力容器设备厂分子杂交炉美国Thermo公司避光保湿盒常州市飞勒斯仪器有限公司移液器德国eppendorf公司光学显微镜及电子成像系统日本Olympus公司超净工作台（SW-CJ-1FD）苏州安泰空气技术有限公司ND-1000美国Thermo公司电热恒温水箱（DK-8B型）上海精宏实验室设备有限公司旋涡器（QL-901）江苏海门市麒麟医用仪器厂电热保湿干燥箱（S202B型）重庆实验设备厂台式低温离心机美国Thermo公司制冰机日本SANYO公司16万方数据 第三军医大学博士学位论文恒温磁力搅拌器（HWCB-2型）温州市医疗电器厂荧光倒置显微镜日本OLYMPUS公司液氮罐成都金凤液氮容器有限公司2.1.4主要试剂配制（1）DEPC水：500ml一级水中加入500μLDEPC原液，配制成DEPC水，摇床过夜后，高温高压灭活DEPC30min。（2）20×SSC溶液配制：800mLDEPC水中溶解175.3gNaCL和88.2g柠檬酸钠，加入10mol/LNaOH溶液将pH值调至7.0，加DEPC水定容至1L，分装后高压灭菌。（3）3%柠檬酸溶液配制：在100mL蒸馏水中溶解柠檬酸3g，pH2.0左右。1mL3%柠檬酸加2滴浓缩型胃蛋白酶，混匀。（4）3%H2O2：90mL蒸馏水中加入10mLH2O2，混匀。2.2实验方法2.2.1LncRNA芯片（1）将胃癌及癌旁组织切成2mm左右，采用液氮冰冻研磨，加入1mLTRIzol溶解组织粉末，收集至1.5mLRNA无酶离心管中。（2）将TRIzol溶解物送至上海康成生物工程有限公司行lncRNA芯片。（3）分析芯片结果，遴选表达差异显著的lncRNALINC00675。2.2.2提取总RNA（1）将冻存于液氮罐中的组织样品取出并置于含1mLTrizol的组织匀浆离心管中，将离心管置于冰上冷却2min以上。（2）设置组织匀浆仪参数为6000rpm，15s，间歇30s，共3次。（3）取出离心管置于冰上冷却2min以上，重复步骤（2）。（4）按照0.2mL氯仿/1mLTrizol的比例加入适量氯仿于上述离心管，漩涡10s。室温静止10min。（5）将离心管置于低温离心机，4℃，12000g离心15min，溶液分层三相，取上层水相溶液于新的离心管中。（6）加入吸取液等量的异丙醇溶液，充分混匀，室温放置8min，沉淀总RNAs。（7）将离心管置于低温离心机，4℃，12000g离心10min，弃上清。17万方数据 第三军医大学博士学位论文（8）加入1mL75%乙醇溶液，颠倒混匀数次以洗涤RNA沉淀，4℃，7500rpm离心10min，弃掉乙醇，自然干燥10min。（9）每管中加入40µLDEPC水分装，-20℃保存。2.2.3QRT-PCR（1）进入NCBI网站，获得LINC00675的RNA序列，采用Primer6设计特异扩增引物，选择一条合适的引物序列，委托上海生工合成。（2）随机引物逆转录1）准备PrimeScriptTMRTreagentKit，总RNAs，DEPC水。2）将总RNAs稀释为每个反应总量1µg。3）按下列体系加入反应试剂：试剂名称体积（µL）5×PrimeScriptbuffer2PrimeScriptEnzymeMix0.5OligodTPrimer（50µM）0.5Random6mers（100µM）2RNA（1µg）54）反应条件：37℃孵育15min，85℃酶灭活5s。（3）染料法扩增1）准备2×Real-timePCRMasterMIX，LINC00675上、下游引物，ddH2O。引物名称序列LINC00675sense:5'-GCCTACTGCTCTGGATCATCTGGTA-3'LINC00675anti-sense:5'-ACCTGCGTCTCTTCTCCTCTTCC-3'β-actinsense:5′-TTCCTTCCTGGGCATGGAGTCC-3′β-actinanti-sense:5′-TGGCGTACAGGTCTTTGCGG-3′18万方数据 第三军医大学博士学位论文2）以逆转率产物为模版，SYBRgreen法扩增LINC00675基因，反应体系如下：试剂名称体积（µL）2×Real-timePCRMasterMIX5上游引物（5µM）0.5下游引物（5µM）0.5ddH2O2cDNAs2（3）反应条件：95℃反应1min，47个循环于95℃，5s；60℃，5s；72℃，20s。在72℃时采集荧光，溶解曲线，从60℃升至95℃过程中，每个循环增加0.5℃，共71个循环。（4）采用ComparativeDelta-deltaCt法计算定量结果。2.2.4原位杂交2.2.4.1石蜡切片脱蜡至水。（1）将组织切片至于二甲苯溶液中浸泡10分钟，更换二甲苯后再浸泡10分钟。（2）将组织切片置于100%、75%、50%酒精溶液中，各放置5min，再放入蒸馏水中3min。2.2.4.2分子杂交。（1）30%H2O21份溶于10份蒸馏水中混匀，滴在组织切片上反应8min，以灭活内源性酶，蒸馏水洗涤3次，每次5min。（2）暴露RNA核酸片段：在切片上滴加3%柠檬酸溶液（1Ml3%柠檬酸加入2滴浓缩型胃蛋白酶，混匀），37℃消化20min。原位杂交用PBS洗涤3次，每次5min，蒸馏水洗涤1次。（3）后固定：胃蛋白酶消化后，用1%多聚甲醛/0.1MPBS，含有1/1000DEPC室温固定10分钟，蒸馏水洗涤3次，每次4min。（4）预杂交：湿盒底部加20%甘油20mL，以保持湿度。按每张切片20μL量加入与杂交液，恒温箱40℃孵育2-4小时，去掉多余的液体，不洗涤。（5）杂交：加入100μL杂交液于载玻片上，覆盖所用样品，恒温箱40℃杂交过夜。（6）杂交后洗涤：37℃水温的2×SSC洗涤2次，每次15分钟。19万方数据 第三军医大学博士学位论文（7）杂交后洗涤：37℃孵育30min，甩去多余液体，不洗涤。（8）滴加生物素化鼠抗地高辛：37℃孵育60分钟或室温120分钟，原位杂交用PBS溶液洗涤4次，每次5min。（9）滴加SABC：37℃孵育20分钟或室温30分钟，原位杂交用PBS溶液洗涤3次，每次5min。（10）滴加生物素化过氧化物酶：37℃孵育20min或室温30min，原位杂交用PBS溶液洗涤4次，每次5min。（11）DAB显色：使用DAB显色试剂盒，1mL蒸馏水加显色剂ABC各一滴，混匀后加至组织标本上，镜下控制显色深度，一般在30分钟内。（12）通过ImagePro软件对样本进行检测，检测值代表样本中表达含量。2.2.5统计分析所有数据采用均值±标准差表示。配对T检验用于比较配对组织中LINC00675的表达。曼-惠特尼U检验用于分析LINC00675的表达与临床参数的相关性。Kaplan-Meier法用于生存率分析。工作特征曲线用于评价LINC00675的表达在胃癌诊断中的意义。P值小于0.05具有统计学意义。所有统计采用SPSS19.0软件和GraphPadPrism6.0软件分析。Image-ProPlus软件用于分析原位杂交染色强度。2.3结果2.3.1LncRNA芯片筛选胃癌组织中显著低表达的lncRNALINC006752.3.1.1采用lncRNA芯片分析5对胃癌及癌旁组织中lncRNAs的表达。分层聚类分析发现一条lncRNALOC400573（被命名为LINC00675）在胃癌组织中显著低表达，见图1-1。20万方数据 第三军医大学博士学位论文图1-15对胃癌及癌旁组织中lncRNAs的表达分层聚类图2.3.1.2LncRNAs在胃癌组织中低表达倍数（部分结果），LncRRNNALINC00675的表达倍数为0.22倍，且其功能和分子机制在胃癌中不清楚，而PSCA和C11orf92在癌症中有少许研究。因此，课题组以LINC00675为研究对象，见表1-1：21万方数据 第三军医大学博士学位论文表1-1lncRNAs在胃癌组织中的表达倍数lncRNAtypep-valueFDRFold-change（G/N）regulationPSCAnon-coding0.01546140.210.17downC11orf92non-coding0.01784990.210.21downLOC400573non-coding0.03685540.2280.22down---non-coding0.00484790.210.27downMT1Lnon-coding0.04525990.2420.3down---non-coding0.0122670.210.32down---non-coding0.00181840.210.33down---non-coding0.016930.210.33downNCRNA00261non-coding0.03405640.2230.33downLOC728606non-coding0.00446930.210.38down---non-coding0.0073280.210.39downCYP2B7P1non-coding0.01836620.210.39downTESCnon-coding0.02117910.210.4downIGHV3-74non-coding0.03260740.2220.4down---non-coding0.01813740.210.42down---non-coding0.01027520.210.44down---non-coding0.02838410.2180.46downSULT1C2P1non-coding0.00520050.210.47downLOC100240735non-coding0.03840270.230.47down---non-coding0.00331670.210.49downMST1P9non-coding0.00701240.210.49down---non-coding0.00829760.210.5down---non-coding0.01660260.210.5down---non-coding0.03446010.2240.5downFCGR1Cnon-coding0.02480830.2140.53down---non-coding0.01253330.210.54down---non-coding0.00133650.210.56downPPAP2Bnon-coding0.01811520.210.56down---non-coding0.01943810.210.56downLOC643008non-coding0.02464140.2140.56down22万方数据 第三军医大学博士学位论文---non-coding0.00140590.210.57downLOC284191non-coding0.02179020.210.57downLOC643201non-coding0.00328820.210.58downMIR574non-coding0.00353650.210.58down---non-coding0.02104660.210.58downC6orf132non-coding0.04345630.240.58downC15orf21non-coding0.04618330.2450.58down---non-coding0.0342850.2230.59downMIR1295non-coding0.03554640.2270.59down---non-coding0.00987240.210.6downLOC283140non-coding0.01702610.210.6down---non-coding0.01893740.210.6downLOC100329109non-coding0.02333350.2120.6down---non-coding0.00428610.210.61down---non-coding0.01311640.210.61downMIRLET7A1non-coding0.03720610.2280.61down---non-coding0.00663660.210.63downZNF300P1non-coding0.00816310.210.63down---non-coding0.02768650.2180.63down---non-coding0.0029740.210.64downMIR29Cnon-coding0.00565860.210.64downMGC16703non-coding0.00896770.210.64downMIR4290non-coding0.02902640.2190.64down---non-coding0.03459490.2240.64down---non-coding0.03625960.2280.64downMIR518Bnon-coding0.03715850.2280.64down---non-coding0.04242130.2390.64down---non-coding0.04685960.2460.64down---non-coding0.04889770.250.64downFBXO22OSnon-coding0.00049370.210.65down---non-coding0.00295550.210.65downMIR647non-coding0.03100740.2210.65down---non-coding0.03703130.2280.65down23万方数据 第三军医大学博士学位论文---non-coding0.04246550.2390.65downLOC100289473non-coding0.01262650.210.66down---non-coding0.01473850.210.66downKIAA0125non-coding0.03095460.2210.66down---non-coding0.04255030.2390.66down---non-coding0.01040540.210.67down---non-coding0.03673650.2280.67downLOC285141non-coding0.03941610.2330.68downC1orf126non-coding0.04330140.240.68downNCRNA00173non-coding0.04388540.240.68down---non-coding0.00852540.210.69down---non-coding0.0250910.2140.69down---non-coding0.03290270.2220.69down---non-coding0.03344880.2220.69down---non-coding0.03740130.2280.69down---non-coding0.01028280.210.7down---non-coding0.03958890.2330.7down---non-coding0.04992710.2520.7downMIR770non-coding0.0192210.210.71downLOC283693non-coding0.02342330.2120.71down---non-coding0.02677150.2160.71downLOC401504non-coding0.0289720.2190.71downKRT42Pnon-coding0.0442360.240.71down---non-coding0.04649930.2450.71down---non-coding0.03196190.2210.72downLOC257396non-coding0.04623380.2450.72downAKR7A2P1non-coding0.04011450.2340.73down---non-coding0.02923180.2190.74down---non-coding0.01496020.210.76downECRPnon-coding0.0388770.2310.76down---non-coding0.04487910.2420.76down---non-coding0.04720720.2460.76down24万方数据 第三军医大学博士学位论文2.3.1.3在NCBI上搜索LINC00675的序列，LINC00675属于基因间的非编码RNA，全长1551个核苷酸，含有polyA尾，如下：>NR_036581.1Homosapienslongintergenicnon-proteincodingRNA675（LINC00675）,longnon-codingRNAGTGGCTCCAAGAAGCGCCAGCTCGCTCATTTGCTCACACCCAGCAGGCAGAGAAGGCAGCAGCAGGCAGGACCGCCACCCTCCCATGCAAATCACCCCCGGGAGTGCAGCTGGGCTCCTCCCGCTCCTCCTGGGCAATGCTCCTGGGGAGTCTGTGGGGAAGATGCCATCCAGGGCGCTGTGCGCTCTTCCTCATCCTCGCCCTCCTGCTGGACGCGGTCGGCCTGGTCCTTTTGCTGCTGGGGATCTTGGCCCCCCTGAGTTCCTGGGACTTCTTCATCTACACAGGTGCCCTGATCCTGGCTCTCAGCCTACTGCTCTGGATCATCTGGTATTCCCTCAACATTGAGGTGTCTCCTGAAAAACTGGACCTGTAATTTGGCCATGGGAAGAGGAGAAGAGACGCAGGTGCTGTATGCAGACATGTCTGTGAACCTGGGGCTCTTGGGCAGCAACACGTTGCAGCTTCCACCTAGCAAGCCACGCCGGGACCAGGTCCCATCTGATGGAGGAGAATCAATTCAAGGAGATGCCCTTCCTTTACAGAACACCCTTTAACAGCATCCAGGAGGAACGAGAGGCTGCAATACTGAGGCTTTCAAAGTACTCACGAGGATGTCCGAGAATGGCTGTGATGCCAGGCTTCTGGCAGGTTCCAGACTCCATCACAAGCCCAGCATCCCTGCACCAGATCTGACATCGCTGCTGTTGTGCCAGCTGTTTATGAAGGGCCTGAGTAGCTAGCAGGTTTTTATCAGGAGCCCTGCTGGGGGCTTAGACACCAAAAGAGAAGACTCATCCTCTGTAGTTCTTCTTGTGAATGTCCTTTTAGAAAAACAATTAGAACCAACCACAAGCACCAAAGTCCTAATGGGATCTCCTGCGAGCACATATCAAGCAGGATTGTTGCTATTTCTCCTCACTGGCTCTTTGGACAGACTGTGTGAGCTCCTGGAGGGTTCCACTGTATCTACCCTTTGACACTGACCAGTTGGCACATGGTGACATATTCCAATGTGTTGATTGCAAATGTGAACGTACAGCCAGTGCTGTGTGCGGGAGGACTCTCTCCTCCTCAGTGGGGCCACACCGTGCACTATTAATGGAGCCCCACTCCTTTGCACAGCCTGGCCATGCAGTGGCTCATATTGAGGTTTTAGCCAACTGAAATCTCCCGTGCATTTTTCTGACAAGCCAGCTAGGCCTCTGCTATGCTGTCCTTGTGTCTTTCATTTGATGACCTTAAGGGTGGGACTGTTTTATCTTAAGTTACAGGTGGTCAAGTCCAGCCCAAGGACAGCAACTCTGAGGGTCAAGCCTCATAGGCTAACTGGATAGATGTTCTCTGCTTTGCCACCCACTGGAGCCCGACCTGCCCCACTAATTTATATTTCCCCTGGTCTCATTTTGTACTTTTTATTTATAATTCACCCTTAAAGTGTATGTGTCTCTTATAAGCTGCCTCCGATCTTTCATGGTATGAGGTGGTTACCTAAATAAAGAAGGAGATTTGGCCTTTGTTTTTATGTAAAAAAAAAAA25万方数据 第三军医大学博士学位论文AAAAAAAAAAAA2.3.1.4LINC00675在基因组的定位，发现LINC00675位于17号染色体短臂1区3带1亚带至短臂1区2带之间，含2个外显子，见图1-2：图1-2LINC00675在染色体中的定位2.3.1.5ORF软件预测LINC00675编码蛋白的能力，发现仅有一个区域可能编码超过55个AA的蛋白（81AA），见图1-3：26万方数据 第三军医大学博士学位论文图1-3ORF软件预测LINC00675编码蛋白的能力2.3.2根据LINC00675的序列设计引物，验证引物扩增的特异性和有效性2.3.2.1采用Primer6.0设计LINC00675的引物，发现一条最适引物，见图2-1：图2-1Primer6.0设计LINC00675的引物2.3.2.2验证引物的有效性，见图2-2：图2-2LINC00675的扩增曲线27万方数据 第三军医大学博士学位论文2.3.2.3引物扩增的特异性，见图2-3：图2-3LINC00675扩增的溶解曲线28万方数据 第三军医大学博士学位论文2.3.3在组织样本中验证LINC00675的表达2.3.3.1采用qRT-PCR检测LINC00675在45对胃癌组织中的表达，发现LINC00675在胃癌组织中的表达显著低于癌旁组织（P=0.0004），见图2-4图2-4LINC00675在胃癌组织中显著低表达。NT（normaltissue）为癌旁正常组织GC（gastriccancer）为胃癌组织，样本例数为45对，采用β-actin作为内参。2.3.3.2采用原位杂交检测LINC00675在90对胃癌组织中的表达，发现LINC00675在胃癌组织中的表达显著低于癌旁组织，见2-5癌旁胃癌图2-5原位杂交代表性图片，LINC00675在胃癌组织中显著低表达。29万方数据 第三军医大学博士学位论文2.3.3.3采用Image-ProPlus软件分析发现LINC00675在65对胃胃癌组织中低表达，15对胃癌组织中高表达，在10对胃癌组织中变化不明显，见图2-66图2-6Image-ProPlus软件分析LINC00675在胃癌组织中的原位杂交结果GC（gastriccancer）胃癌，NT（normaltissue）癌旁正常组织。2.3.4LINC00675的表达在胃癌诊断中的意义2.3.4.1对于qRT-PCR检测组，采用ROC分析发现LINC00675的表达值在胃癌诊断中的曲线下面积为0.7256（0.6107tto0.8405），敏感性为78.05%，特异性为72.5%，见图2-7图2-5ROC曲线分析LINC00675的表达值在胃癌诊断中的意义。2.3.4.2对于qRT-PCR检测组，根据LINC00675表达的中位数，将胃癌患者分为两组，生存分析发现低表达LINC00675的胃癌患者生存率显著降低（P=0.0181），见图2-830万方数据 第三军医大学博士学位论文图2-6生存率分析发现低表达LINC00675的胃癌患者生存率显著降低。2.3.4.3对于qRT-PCR检测组，通过分析LINC00675的表达与胃癌患者临床资料的相关性，发现LINC00675的表达与肿瘤体积、远处转移数量及CEA值呈负相关，见表2-1表2-1LINC00675的表达与胃癌患者临床资料的相关性，“*”表示P<0.05。LINC00675expressionCharacterisiticsPvalueLowexpressionHighexpressionGennderMale13120.566Female1010Age（years）≤5511110.560＞551211Tumorsize（cm）≤129150.049*＞12147LymphnodemetastasisYes18140.22631万方数据 第三军医大学博士学位论文No58DistantmetastasisYes400.048*No1722TNMstageI-II350.324III-IV2017CEA（ng/μl）≤2.237160.005*＞2.231662.3.4.4对于原位杂交检测组，根据LINC00675表达的中位数，将胃癌患者分为两组，生存分析发现低表达LINC00675的胃癌患者生存率显著降低（P=0.0198），见图2-9图2-9生存率分析发现低表达LINC00675的胃癌患者生存率显著降低。2.3.4.5对于原位杂交检测组，通过分析LINC00675的表达与胃癌患者临床资料的相关性，发现LINC00675的表达与淋巴结转移、TNM分期呈负相关，与年龄、性别、肿瘤大小、肿瘤远处转移无明显关系，见表2-232万方数据 第三军医大学博士学位论文表2-2LINC00675的表达与胃癌患者临床资料的相关性，“*”表示P<0.05。LINC00675expressionCharacterisiticsPvalueLowexpressionHighexpressionGenderMale35350.600Female1010Age（years）≤66.522230.500＞66.52322Tumorsize（cm）≤3221240.337＞322421LymphnodemetastasisYes38290.026*No716DistantmetastasisYes220.692No4343TNMstageI-II14230.043*III-IV31222.4讨论本文基于lncRNA芯片筛选发现了大量存在差异表达的lncRNAs，而课题组选择LINC00675作为研究对象的原因在于：（1）LINC00675的低表达倍数位于第三位，前两位已被其他研究团队研究；（2）课题组拟从胃癌组织中筛选并鉴定一条新的lncRNA。通过qRT-PCR、原位杂交技术在两组不同人群共135对标本中对一条新的lncRNALINC00675在胃癌组织中的表达进行验证，并统计分析了LINC00675的表达值作为标志物在胃癌诊断、预后判断中的意义，提示LINC00675的表达值可能作为独立的胃癌诊断和预后判断的生物标志物。33万方数据 第三军医大学博士学位论文[44]分子标志物有不同的类型：（1）诊断标志物，用于部分疾病状态的诊断及亚型分型；（2）预后标志物，与患者的预后具有相关性，如总生存率、疾病未复发生存率；（3）预测标志物，能预测针对性治疗的效果以及指导更特异的治疗手段；（4）综合类标志物，可能用于诊断、预后判断、预测，但主要用于鉴定药物对一类患者的疗效。近年来的研究表明lncRNAs可能作为有效的诊断及预后判断标志用于诊断胃癌，[45]如ncRuPAR诊断胃癌的敏感性为88.41%，特异性为73.91%，准确度为81.16%；[46]AC096655-002诊断胃癌的敏感性为51.3%，特异性为87.2%，曲线下面积为0.731；[47]FER1L4预后判断胃癌的敏感性为67.2%，特异性为80.3%，曲线下面积为0.778。本文中，课题组发现LINC00675诊断胃癌的敏感性为78.05%，特异性为72.05%，曲线下面积为0.7256，提示LINC00675可能作为一个新的独立的胃癌诊断标志物；同时，低表达LINC00675的胃癌患者的总生存率显著低于高表达LINC00675的患者，LINC00675的低表达与肿瘤大小、TNM分期、淋巴结转移级远处转移呈显著负相关，提示LINC00675在胃癌预后判断中具有一定意义。本文采用不同的两组人群及两种不同的方法检测LINC00675在胃癌组织中的表达，结果均显示LINC00675在胃癌组织中显著低表达，且其低表达与胃癌患者的不良生存率显著相关。原位杂交技术广泛用于核酸分子表达及定位的检测，本文采用原位杂交技术观察90对胃癌组织芯片中LINC00675的表达，发现LINC00675的表达主要集中在胃腺体细胞，而癌细胞中LINC00675的表达并不广泛，并通过Image-ProPlus软件分析发现LINC00675在65对胃癌组织中低表达，15对胃癌组织中高表达，在10对胃癌组织中变化不明显，提示LINC00675在胃癌组织中低表达的普遍性。2.5小结2.5.1通过lncRNA芯片筛选发现一条新的lncRNALINC00675在胃癌组织中低表达。2.5.2通过qRT-PCR、原位杂交技术证实LINC00675的表达在两组胃癌人群胃癌组织中显著降低。2.5.3通过统计学分析发现LINC00675的表达值可能作为胃癌诊断、预后判断的生物标志物。34万方数据 第三军医大学博士学位论文第三章LINC00675对胃癌细胞生物学行为的影响在前期的研究中，课题组通过lncRNA芯片从胃癌组织中筛选到大量差异表达的lncRNAs，并采用qRT-PCR、原位杂交技术验证了LINC00675在两组胃癌人群胃癌组织中呈显著低表达，进一步统计分析发现LINC00675的表达与肿瘤大小、TNM分期、淋巴结转移及远处转移呈显著负相关，以及LINC00675低表达的胃癌患者具有不良的生存率。基于LINC00675的表达与胃癌大小及转移状态的相关性，课题研究了LINC00675对胃癌细胞增殖、侵袭和转移的影响。首先，课题组构建了慢病毒载体并包装慢病毒，感染胃癌细胞株以构建稳定过表达或干扰LINC00675的胃癌细胞株；进一步用这些细胞进行体外实验，即采用CCK-8实验检测胃癌细胞增殖，采用平板克隆形成实验检测胃癌细胞的克隆形成能力，采用Trans-well实验检测胃癌细胞的迁移和侵袭能力；进一步用这些细胞进行体内实验，即采用裸鼠皮下成瘤实验检测胃癌细胞皮下成瘤能力，采用裸鼠尾静脉注射实验观察胃癌细胞的肺转移能力，采用裸鼠腹腔注射实验观察胃癌细胞的肝转移能力。3.1实验材料3.1.1实验试剂慢病毒过表达质粒上海吉玛公司慢病毒干扰质粒上海吉玛公司慢病毒包装上海吉玛公司CCK-8试剂DMEM培养基美国Gibco公司PRMI1640培养基美国Gibco公司胰酶美国Hyclone公司PBS博士德生物制品有限公司胎牛血清美国Gibco公司酵母提取物上海生物工程公司胰蛋白胨上海生物工程公司琼脂糖西班牙进口分装Lipofectamin2000美国Invitrogen公司35万方数据 第三军医大学博士学位论文胰酶美国Hyclone公司DEPC水上海碧云天生物技术有限公司TRIzol美国Thermo公司氯仿成都科龙化工试剂长异丙醇成都科龙化工试剂长无水乙醇重庆川东化工有限公司逆转录试剂盒日本TaKaRa公司Real-timePCR试剂盒日本TaKaRa公司RNA无酶离心管（1.5ml）美国AXYGEN公司RNA无酶PCR离心管美国AXYGEN公司RNA无酶吸头美国AXYGEN公司Trans-well小室美国Corning公司6孔、12孔、96孔板美国Corning公司人胃腺癌细胞株AGS本实验室保存人胃腺癌细胞株MKN28本实验室保存人胃腺癌细胞株MGC-803本实验室保存人胃腺癌细胞株BGC-823本实验室保存人胃腺癌细胞株SGC-7901本实验室保存人胃腺癌细胞株MKN-45本实验室保存3.1.2主要仪器电子分析天平300mc型，瑞典低温冰箱（4℃、-20℃）德国Siemens公司超低温冰箱（-80℃）德国Siemens公司ABSystem7500荧光定量PCR仪美国ABI公司高压灭菌锅上海压力容器设备厂移液器德国eppendorf公司光学显微镜及电子成像系统日本Olympus公司超净工作台（SW-CJ-1FD）苏州安泰空气技术有限公司ND-1000美国Thermo公司电热恒温水箱（DK-8B型）上海精宏实验室设备有限公司36万方数据 第三军医大学博士学位论文旋涡器（QL-901）江苏海门市麒麟医用仪器厂电热保湿干燥箱（S202B型）重庆实验设备厂台式低温离心机美国Thermo公司制冰机日本SANYO公司恒温磁力搅拌器（HWCB-2型）温州市医疗电器厂荧光倒置显微镜日本OLYMPUS公司液氮罐成都金凤液氮容器有限公司DU-640紫外分光光度计美国Beckman公司制冰机日本SANYO公司细胞培养箱美国Sher-LabTM公司细菌培养箱美国Sher-LabTM公司3.1.3主要的试剂配制（1）DMEM双抗完全培养基：将50mL胎牛血清加入450mLDMEM培养基中，按100:1加入青霉素-链霉素溶液，混匀。（2）PBS溶液：袋装的PBS粉剂溶解于1000mL一级水中，高压灭菌后4℃保存。（3）胰酶消化液：将0.25g胰酶、0.02gEDTA溶于100mL经高压灭菌的PBS溶液中，采用0.22μm的过滤膜去除细菌及杂质，混匀后4℃保存。（4）细胞冻存液：按胎牛血清:DMSO=9:1的比例混匀，4℃保存。（5）75%乙醇：在750mL无水乙醇中加入250mL一级水，混匀室温保存。（6）CaCl2溶液：将0.83g氯化钙溶解于100mL一级水中，高压灭菌，4℃保存。（7）LB液体培养基：将10g胰蛋白胨、10gNaCl，5g酵母提取物溶解至1000mL一级水中，加入NaOH将pH调至7.0，高压灭菌，4℃保存。（8）LB固体培养基：将10g胰蛋白胨、10gNaCl，5g酵母提取物及15g琼脂糖溶解至1000mL一级水中，加入NaOH将pH调至7.0，高压灭菌，待冷却至约45℃时制作平板培养基。（9）在北京维通利华实验动物技术有限购买5周龄左右的雌性BALB/c裸鼠40只，饲养于新桥医院动物实验中心。37万方数据 第三军医大学博士学位论文3.2实验方法3.2.1细胞培养3.2.1.1细胞复苏：将冻存的细胞从液氮中取出，置于37℃恒温水浴中快速解冻，1分钟内完成，采用75%乙醇对细胞冻存管进行消毒。吸取4mL完全培养基于无菌离心管，加入冻存细胞液，混匀。将离心管置于台式离心机，800rpm离心5min。弃上清液，加入1mL完全培养基重悬，转移至细胞培养瓶中，补加3mL完全培养基，将瓶口拧松后置于细胞培养箱中培养。3.2.1.2细胞换液：当细胞培养数十小时后，观察培养基及细胞状态，如果培养基变黄或漂浮细胞较多，就需要给细胞换液。步骤是，取出细胞培养瓶，倒掉培养基，加入新鲜完全培养基，将瓶口拧松后置于细胞培养箱中培养。3.2.1.3细胞传代：当细胞汇合率达到80%左右，就需要对细胞进行换液。步骤是，取出细胞培养瓶，倒掉培养基，加入2mL无菌PBS润洗2次，倒掉PBS，加入1mL胰酶消化数分钟，显微镜下观察细胞的消化状态，如果细胞变圆变小、细胞间失去连接，或部分细胞漂浮，立即加入1mL完全培养基终止胰酶的作用，将液体转移至无菌离心管中，至于台式离心机，800rmp离心5min，倒掉上清液，加入2mL完全培养基重悬，分装至2个细胞培养瓶中，每瓶中补3mL完全培养基，将瓶口拧松后置于细胞培养箱中培养。3.2.2载体的构建3.2.2.1构建稳定过表达LINC00675的胃癌细胞株（1）经网站http://www.ncbi.nlm.nih.gov/nuccore/NR_036581.1获得LINC00675的cDNA序列，上海吉玛公司根据序列合成带有HandⅢ和EcoRⅠ酶切位点的DNA序列。（2）采用HandⅢ和EcoRⅠ酶切T载体，并回收鉴定，T载体图谱如下：38万方数据 第三军医大学博士学位论文T载体图谱（3）采用T4DNA连接酶将合成的DNA序列与酶切后的T载体进行连接，步骤是：取50ng载体和5倍摩尔量的DNA合成片段，加入双蒸水中，体积为10μL；加入10μLMbuffer，混匀；加入1μLT4DNA连接酶，充分而轻柔混匀；轻微离心后室温放置5分钟；冰上保存，带下一步转化。（4）制备感受态1）取出冻存于-70℃的DH5a，无菌接种环蘸取DH5a保种液，三线法划线接种于LB平板，37℃培养16h。2）从LB平板中挑取色泽较好的单个菌落接种于10mL的LB液体培养基中，37℃摇床培养16h。3）将过夜培养的DH5a按照1%比例接种至LB液体培养基中，37℃摇床培养至OD600为0.5时，置于冰上2min之后，4000g离心5min，弃上清。4）加入1mL预冷的0.1mol/LCaCl2重悬沉淀，置于冰水浴2min之后，4℃条件下4000g离心5min，弃上清。5）加入100µL预冷的0.1mol/LCaCl2重悬沉淀，置于冰水浴2min之后，4℃条件下4000g离心5min，弃上清，加入100µL预冷的0.1mol/LCaCl2悬浮沉淀，即为感受态。可冻存于-70℃。（5）转化1）取3管100µL感受态细胞，其中的1管加入连接产物，1管加入controlinsertDNA连接产物作为阳性对照，1管不加入载体作为阴性对照，置于冰水浴2min，擦干离心管后于42℃热休克90s，迅速置于冰水浴2min。39万方数据 第三军医大学博士学位论文2）每管加入900µLLB液体培养基，37℃摇床培养16h。3）5000g离心5min，吸弃500µL上清后混匀沉淀，取50µL细胞悬液均匀涂布于Amp+平板上，置于37℃浮箱培养过夜。（6）质粒提取1）吸取6mL菌液于离心管中，10000g离心3min，弃上清。2）加入250µL含RNaseA的SolutionⅠ，置于漩涡器上漩涡数s以充分裂解细菌。3）加入250µLSolutionⅡ，温和颠倒混匀数次至沉淀澄清，室温静止2min。4）加入350µLSolutionⅢ，立即温和颠倒混匀数次至出现白色沉淀，在室温条件下13000g离心10min。5）转移上清液于HiBind®MiniprepoColumn（I）中，在室温条件下10000g离心1min，弃掉流穿液。6）加入500µLHB溶液洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。7）加入700µLDNAWashBuffer洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。8）重复步骤7）一次。9）在室温条件下13000g离心2min，干燥吸附柱，弃掉废液收集管。10）取新的1.5mL离心管，将吸附柱套于管中，加入35µLElutionBuffer室温反应2min，13000g离心2min，洗脱质粒。（7）酶切1）准备HandⅢ和EcoRⅠ，10×Mbuffer，ddH2O，质粒。2）按照下列体系进行酶切反应：试剂名称体积（µL）HindⅢ0.5SpeⅠ0.510×Mbuffer2ddH2O12质粒2µg（5µL）3）反应条件：37℃条件下反应2h。（8）连接40万方数据 第三军医大学博士学位论文1）将慢病毒载体与目的片段进行连接，慢病毒表达载体（pGLV3/H1/GFP+Purovector，图谱如下）：慢病毒载体图谱2）准备目的片段，慢病毒载体，Ligationsolution试剂3）连接体系如下：试剂名称体积（µL）目的片段0.5µg（5µL）慢病毒载体0.2µg（4µL）Ligationsolution14）反应条件：16℃连接过夜。（9）转化1）取3管100µL感受态细胞，其中的1管加入连接产物，1管加入controlinsertDNA连接产物作为阳性对照，1管不加入载体作为阴性对照，置于冰水浴2min，擦干离心管后于42℃热休克90s，迅速置于冰水浴2min。2）每管加入900µLLB液体培养基，37℃摇床培养16h。3）5000g离心5min，吸弃500µL上清后混匀沉淀，取50µL细胞悬液均匀涂布于Amp+平板上，置于37℃浮箱培养过夜。（10）质粒提取41万方数据 第三军医大学博士学位论文1）吸取6mL菌液于离心管中，10000g离心3min，弃上清。2）加入250µL含RNaseA的SolutionⅠ，置于漩涡器上漩涡数s以充分裂解细菌。3）加入250µLSolutionⅡ，温和颠倒混匀数次至沉淀澄清，室温静止2min。4）加入350µLSolutionⅢ，立即温和颠倒混匀数次至出现白色沉淀，在室温条件下13000g离心10min。5）转移上清液于HiBind®MiniprepoColumn（I）中，在室温条件下10000g离心1min，弃掉流穿液。6）加入500µLHB溶液洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。7）加入700µLDNAWashBuffer洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。8）重复步骤7）一次。9）在室温条件下13000g离心2min，干燥吸附柱，弃掉废液收集管。10）取新的1.5mL离心管，将吸附柱套于管中，加入35µLElutionBuffer室温反应2min，13000g离心2min，洗脱质粒。（11）慢病毒质粒转染293T细胞1）采用6孔板培养293T细胞，至细胞贴壁生长汇合率达到70%时开始转染。TM2）准备Lipofectamine2000，Opti，慢病毒载体，离心管，无血清培养基。3）根据LipofectamineTM2000说明书推荐体系TM4）加入250µLOpti和6µLLipofectamine2000于离心管，标记为1号。5）加入250µLOpti和2µg慢病毒载体于离心管，标记为2号。6）室温静止5min。7）将1号和2号离心管溶液混合，充分混匀，室温静止15-20min。8）吸弃细胞培养基，PBS润洗2次，加入1mL无血清培养基。9）加入500µL7）步骤混合液于细胞培养基中，温和摇匀，将6孔板置于细胞培养箱中培养6h。10）取出6孔板，吸弃培养基，加入DMEM完全培养，置于细胞培养箱中培养48h。11）取出6孔板，收集细胞上清液，采用慢病毒提取试剂盒收集慢病毒，-80℃保存。（12）慢病毒感染胃癌细胞1）采用24孔板培养胃癌细胞SGC-7901和MKN-45，使其汇合率达到70%。2）吸弃培养基，将20µL慢病毒液与480µL无血清培养基混合，加至每孔中。3）置于细胞培养箱中培养24小时，在荧光显微镜下观察荧光的表达。42万方数据 第三军医大学博士学位论文4）将24孔板中的细胞传代至6孔板中，完全培养基培养至80%汇合率，在荧光显微镜下观察荧光的表达。5）如果表达荧光的细胞超过30%，每孔加入5µL嘌呤霉素用以筛选只表达荧光的细胞，完全培养基培养6-12h，细胞换液，荧光显微镜下观察细胞状态。如果表达荧光的细胞超过90%可用于后续研究，如果未达到90%，继续用嘌呤霉素药物筛选。3.2.2构建稳定干扰LINC00675表达的胃癌细胞株（1）经网站http://www.ncbi.nlm.nih.gov/nuccore/NR_036581.1获得LINC00675的cDNA序列，根据其序列设计三条shRNA干扰序列，并设计阴性对照，经BLAST比对分析其与人类基因序列无同源性，shRNA序列如下：LINC00675shRNA1:CTCGTGAGTACTTTGAAAGCCTCAGTLINC00675shRNA2:ATTCACAAGAAGAACTACAGAGGATGLINC00675shRNA3:TTAATAGTGCACGGTGTGGCCCCACTG（2）最佳干扰序列的挑选，将上述三条干扰序列及阴性对照序列分别采用脂质体2000转染至胃癌细胞MKN45中，采用qRT-PCR检测LINC00675的表达变化，挑选最佳干扰序列，克隆至慢病毒表达载体，进行病毒包装，如前所述。（3）将重组慢病毒感染MKN45和SGC-7901细胞，即获得2株4种胃癌细胞（MKN45lenti-control,MKN45lenti-shRNA;SGC-7901lenti-control,SGC-7901lenti-shRNA）。2.2.3鉴定稳定干扰或过表达LINC00675的胃癌细胞株（1）采用6孔板培养稳定干扰或过表达LINC00675的SGC-7901和MKN-45细胞。（2）吸弃培养基，加入无菌PBS润洗2次，每孔加入1mLTRIzol试剂。（3）按照0.2mL氯仿/1mLTrizol的比例加入适量氯仿于上述离心管，漩涡10s。室温静止10min。（4）将离心管置于低温离心机，4℃，12000g离心15min，溶液分层三相，取上层水相溶液于新的离心管中。（5）加入吸取液等量的异丙醇溶液，充分混匀，室温放置8min，沉淀总RNAs。（6）将离心管置于低温离心机，4℃，12000g离心10min，弃上清。（7）加入1mL75%乙醇溶液，颠倒混匀数次以洗涤RNA沉淀，4℃，7500rpm离心10min，弃掉乙醇，自然干燥10min。（8）每管中加入40µLDEPC水分装，-20℃保存。（9）随机引物逆转录43万方数据 第三军医大学博士学位论文TM1）准备PrimeScriptRTreagentKit，总RNAs，DEPC水。2）将总RNAs稀释为每个反应总量1µg。3）按下列体系加入反应试剂：试剂名称体积（µL）5×PrimeScriptbuffer2PrimeScriptEnzymeMix0.5OligodTPrimer（50µM）0.5Random6mers（100µM）2RNA（1µg）54）反应条件：37℃孵育15min，85℃酶灭活5s。（10）染料法扩增1）准备2×Real-timePCRMasterMIX，LINC00675上、下游引物，ddH2O。引物名称序列LINC00675sense:5'-GCCTACTGCTCTGGATCATCTGGTA-3'LINC00675anti-sense:5'-ACCTGCGTCTCTTCTCCTCTTCC-3'β-actinsense:5′-TTCCTTCCTGGGCATGGAGTCC-3′β-actinanti-sense:5′-TGGCGTACAGGTCTTTGCGG-3′2）以逆转率产物为模版，SYBRgreen法扩增LINC00675基因，反应体系如下：试剂名称体积（µL）2×Real-timePCRMasterMIX5上游引物（5µM）0.5下游引物（5µM）0.5ddH2O2cDNAs23）反应条件：95℃反应1min，47个循环于95℃，5s；60℃，5s；72℃，20s。在44万方数据 第三军医大学博士学位论文72℃时采集荧光，溶解曲线，从60℃升至95℃过程中，每个循环增加0.5℃，共71个循环。4）采用ComparativeDelta-deltaCt法计算定量结果。3.2.3CCK-8检测细胞增殖3.2.3.1原理：采用CCK-8试剂盒检测细胞增殖，CCK-8试剂盒是含有WST-8的化合物，用于检测细胞增殖能力，其作用原理类似于MTT，在电子螯合剂作用下，细胞线粒体的脱氢酶将其还原为水溶性的橙黄色甲臜。细胞增殖越快，反应生成的甲臜越多，颜色越深。采用酶标仪在450nm波长处测定吸光度，可定量评估细胞的数量。3.2.3.2操作步骤：（1）收集状态较好的胃癌细胞，并对细胞计数。（2）将胃癌细胞接种至96孔板中，每孔接种细胞2000个，加入200µL培养基置于细胞培养箱培养一定时间（24,48,72,96h）。（3）在既定时间点，吸弃培养板中培养基，取10µLCCK-8溶液与90µL无血清培养基混匀后加入培养板中，置于细胞培养箱反应2h。为每孔反应均匀，可以将所有孔需要的试剂一起配置，最后分别加入每孔中。（4）采用酶标仪测定在450nm处的吸光度。（5）如果暂时不能测定，向每孔中加入10µL0.1MHCl溶液并将培养板避光保存于室温，在24小时内吸光度不会发生变化。如果待测物有氧化性或还原性，需在加入CCK-8前更换新鲜培养基，去掉药物等影响。3.2.4流式检测细胞周期3.2.41原理：细胞周期是指从一次细胞分裂结束至下一次细胞分裂结束的一个周期，包含G1期、S期、G2期、M期四个阶段。细胞分裂过程中遗传物质复制并均等低分配至2个子细胞。细胞的周期状态反应了细胞的增殖速度。流式细胞计数对细胞周期的分析基于检测细胞中DNA的量，根据DNA的量可分为G0/G1期、S期、G2/M期。3.2.4.2操作步骤：（1）取出6孔板培养的细胞，每孔加入1mLPBS润洗1次，加入0.5mL胰酶消化，加入完全培养基终止，细胞悬液收集至离心管。（2）将离心管置于台式离心机，800rpm离心5min，弃上清。（3）加入5mL75%的乙醇，轻柔重悬细胞，4℃避光保存过夜。45万方数据 第三军医大学博士学位论文（4）将离心管置于台式离心机，800rpm离心5分钟，弃上清。（5）加入2mLPBS重悬细胞，润洗2次以去除乙醇。6（6）细胞染色：每管染色的细胞大约10数量，对于PI/RNase，加入PI/RNase染色液；对于7-AAD染色，加入0.1mL染色液同时加入20μL7-AAD染色液，室温避光孵育15分钟。（7）流式细胞仪检测，在1小时内完成。3.2.5平板克隆形成实验3.2.5.1克隆形成率：细胞贴壁的数量的比率表示细胞的存活率，但贴壁的细胞并一定能形成克隆。因此，形成克隆的细胞必然是贴壁且有增殖活力的细胞。克隆形成实验反映细胞增殖能力和群体细胞间相互作用两个方面。克隆形成率与细胞接种密度相关，在接种细胞时注意细胞应分散成单个细胞，持续一周随时监测直到细胞形成克隆。3.2.5.2平板克隆形成实验步骤：（1）取对数生长期的胃癌细胞，用胰酶消化成单细胞悬液，加入完全培养基，转入离心管中。（2）将离心管置于台式离心机，800rpm离心5min，弃上清，加入完全培养基备用。（3）将细胞悬液行梯度稀释，接种至6孔板中，每个孔细胞数量为200个，置于细胞培养箱培养。（4）每天观察细胞克隆形成状态，如果明显观察到克隆形成则可终止培养，胃癌细胞一般1周时间。（5）取出6孔板，吸弃培养基，每孔1mLPBS润洗2次，加入4%多聚甲醛固定10分钟。（6）吸取多聚甲醛，每孔1mLPBS润洗2次，加入结晶紫染色细胞，反应10分钟。（7）吸弃结晶紫，用水流润洗直到背景颜色全部退去，空气干燥。（8）在显微镜下观察细胞克隆形成状态，大于10个细胞的克隆视为1个克隆。（9）计算克隆形成率，克隆形成率=克隆数/接种细胞数×100%。3.2.6细胞迁移和侵袭实验3.2.6.1实验原理：采用Trans-well小室研究细胞的迁移和侵袭。小室内称上室，培养板内称下室。Trans-well小室采用聚碳酸酯膜，膜中具有8μm的孔，细胞正常状态无法穿过膜孔，而具有迁移或侵袭能力的细胞可以通过细胞变形穿过膜孔。由于下室中加入的是完全46万方数据 第三军医大学博士学位论文培养基，而上室中加入的是不完全培养基，因此，细胞会朝着营养浓度高的下室移动。穿过膜孔的细胞会贴在小室外侧的膜上，通过检测小室外侧膜上的细胞，可以计算细胞的迁移或侵袭能力。在细胞侵袭的研究中，还涉及到基质，细胞可以分泌酶将基质分解，从而实现其侵袭的目的。3.2.6.2细胞迁移实验步骤：（1）制备细胞悬液，先让胃癌细胞饥饿12h，去除血清的影响。（2）收集细胞，胰酶消化细胞，加入完全培养基终止消化作用，800rpm离心5min。（3）弃上清，加入PBS润洗2次，800rpm离心5min。（4）弃上清，加入1ml无血清培养基重悬，细胞计数板计数，将细胞密度调整5为10/ml。（5）将Trans-well小室置于24孔板中，24孔板下室加入600μL完全培养基，小室与培养基之间避免产生气泡。（6）接种细胞，取200μL细胞悬液加入Trans-well小室中。（7）培养细胞，将24孔板置于细胞培养箱培养24h。（8）取出Trans-well小室，吸弃上室中的培养基，PBS润洗2次，加入4%多聚甲醛固定10min。（9）结晶紫染色20min，清水洗去多余的染液，用棉签轻轻擦掉小室内层的未迁移的细胞，用PBS润洗3次。（10）显微镜下观察并计数细胞的迁移数量。3.2.6.3细胞迁移实验步骤：（1）将Matrigel置于4℃过夜融化。（2）用预冷的无血清培养基稀释Matrigel至终浓度1mg/mL，注意在冰上操作。（3）在Trans-well小室内加入100μL稀释后的Matrigel，37℃孵育4小时。（4）制备细胞悬液，先让胃癌细胞饥饿12h，去除血清的影响。（5）收集细胞，胰酶消化细胞，加入完全培养基终止消化作用，800rpm离心5min。（6）弃上清，加入PBS润洗2次，800rpm离心5min。（7）弃上清，加入1ml无血清培养基重悬，细胞计数板计数，将细胞密度调整5为10/ml。（8）将上述准备好的Trans-well小室置于24孔板中，24孔板下室加入600μL完全培养基，小室与培养基之间避免产生气泡。（9）接种细胞，取200μL细胞悬液加入Trans-well小室中。47万方数据 第三军医大学博士学位论文（10）培养细胞，将24孔板置于细胞培养箱培养24h。（11）取出Trans-well小室，吸弃上室中的培养基，PBS润洗2次，加入4%多聚甲醛固定10min。（12）结晶紫染色20min，清水洗去多余的染液，用棉签轻轻擦掉小室内层的未迁移的细胞，用PBS润洗3次。（13）显微镜下观察并计数细胞的迁移数量。3.2.7裸鼠皮下成瘤实验3.2.7.1裸鼠皮下成瘤实验主要研究肿瘤细胞在动物体内生长的过程，反应细胞的肿瘤形成能力。按照不同细胞的肿瘤形成能力不同，接种不同数量的细胞。对于胃癌7细胞SGC-7901和MKN-45，将10/mL的细胞浓度注射200μL于接种部位可以成瘤。接种部位，一般选择腋窝中部或外侧皮下，腋下成瘤率较高。接种时要确保与接种点的距离小于针头长度，不能刺破皮肤多次，不能刺破肌肉层，主要避免污染。裸鼠皮下成瘤，一般左右两侧各接种一群细胞，为对照组和处理组。3.2.7.2实验准备：（1）裸鼠的状态，采用5周龄大小的雌性裸鼠，在SPF级动物实验室饲养；（2）培养稳定干扰或过表达LINC00675的SGC-7901细胞，胰酶消化，完全培养基终止，800rpm离心后，加入1mLPBS重悬，细胞计数板计数，将细胞浓度调整7至10/mL备用。细胞状态决定细胞的活力，因此应选择对数生长期的细胞，且不要有污染；（3）准备胰岛素针、酒精棉球、75%酒精。3.2.7.3实验步骤：（1）采用胰岛素针吸取均匀的细胞悬液；（2）将裸鼠固定后注射细胞悬液至裸鼠腋下，一次性注射200μL细胞悬液；（3）用酒精棉球擦拭注射部位，防治感染；（4）将裸鼠放回培养槽中，做好标记；5）每周观察并测量一次成瘤情况。3.2.8裸鼠尾静脉注射肺转移实验3.2.8.1裸鼠尾静脉注射肺转移实验主要观察外周血肿瘤细胞转移至其他器官的情况，反映细胞的转移能力，包括侵袭、定植等。不同的细胞转移能力不同，对于胃7癌细胞SGC-7901，将5×10/mL的细胞浓度注射200μL至尾静脉可形成肺转移灶。尾静脉注射有一定难度，不容易一次成功，有时需要多次进针，需反复熟练操作。一般注射SGC-7901细胞4-6周处死裸鼠，取出肺组织行切片染色，可观察到肿瘤细胞的48万方数据 第三军医大学博士学位论文侵袭定植。操作时避免污染。3.2.8.2实验准备：（1）裸鼠的状态，采用5周龄大小的雌性裸鼠，在SPF级动物实验室饲养；（2）培养稳定干扰或过表达LINC00675的SGC-7901细胞，胰酶消化，完全培养基终止，800rpm离心后，加入1mLPBS重悬，细胞计数板计数，将细胞浓度调整至75×10/mL备用。细胞状态决定细胞的活力，因此应选择对数生长期的细胞，且不要有污染；（3）准备胰岛素针、酒精棉球、75%酒精。3.2.8.3实验步骤：（1）采用胰岛素针吸取均匀的细胞悬液；（2）将裸鼠固定后注射细胞悬液至裸鼠尾静脉，一次性注射200μL细胞悬液；（3）用酒精棉球擦拭注射部位，防治感染；（4）将裸鼠放回培养槽中，做好标记；（5）每周观察一次裸鼠状态。3.2.9裸鼠腹腔注射肝转移实验3.2.9.1裸鼠腹腔注射肝转移实验主要观察腹腔肿瘤细胞转移至临近器官的情况，反映细胞的转移能力，包括侵袭、定植等。不同的细胞转移能力不同，对于胃癌细胞7SGC-7901，将5×10/mL的细胞浓度注射200μL至腹腔可形成肝转移灶。一般注射SGC-7901细胞4-6周处死裸鼠，取出肝组织行切片染色，可观察到肿瘤细胞的侵袭定植。操作时避免污染。3.2.9.2实验准备：（1）裸鼠的状态，采用5周龄大小的雌性裸鼠，在SPF级动物实验室饲养；（2）培养稳定干扰或过表达LINC00675的SGC-7901细胞，胰酶消化，完全培养基终止，800rpm离心后，加入1mLPBS重悬，细胞计数板计数，将细胞浓度调整至75×10/mL备用。细胞状态决定细胞的活力，因此应选择对数生长期的细胞，且不要有污染；（3）准备胰岛素针、酒精棉球、75%酒精。3.2.9.3实验步骤：（1）采用胰岛素针吸取均匀的细胞悬液；（2）将裸鼠固定后注射细胞悬液至裸鼠腹腔，一次性注射200μL细胞悬液；（3）用酒精棉球擦拭注射部位，防治感染；49万方数据 第三军医大学博士学位论文（4）将裸鼠放回培养槽中，做好标记；（5）每周观察一次裸鼠状态。3.2.10H&E染色（1）将石蜡包埋的组织切片脱蜡至水：1）将切片置于二甲苯中浸泡10min，更换二甲苯浸泡10min；2）将切片置于100%乙醇溶液中浸泡10min，更换乙醇浸泡10min；3）将切片置于95%乙醇溶液中浸泡10min，更换95%乙醇溶液浸泡10min；4）将切片置于75%乙醇溶液中浸泡10分钟，更换75%乙醇溶液浸泡10分钟；5）将切片置于蒸馏水泡洗。（2）苏木素染色5分钟，自来水流洗3分钟，过蒸馏水再重复一次。（3）1%盐酸酒精洗2次，自来水冲洗，直到细胞核变蓝而胞浆无蓝色。（4）伊红染色2min，直到胞浆呈粉红色，自来水冲洗去多余的红色，过蒸馏水。（6）脱水：1）75%乙醇浸泡1min；2）95%乙醇浸泡2min，更换95%乙醇浸泡2min；3）100%乙醇浸泡2min，更换100%乙醇浸泡2min；4）二甲苯浸泡5min，更换二甲苯浸泡5min。（6）透蜜树脂封片。3.2.11免疫组化3.2.11.1基本原理：免疫组化基于抗原抗体结合的特性，在抗体上标记酶，借助酶对底物的催化作用，反应生成有色的不溶性产物，在光学显微镜下观察抗原的定位及表达。3.2.11.2.操作步骤：（1）将石蜡包埋的组织切片置于60℃烤箱中烘烤1h。（2）将切片脱蜡至水：①将切片置于二甲苯中浸泡10min，更换二甲苯浸泡10min；②将切片置于100%乙醇溶液中浸泡10min，更换乙醇浸泡10min；③将切片置于95%乙醇溶液中浸泡10min，更换95%乙醇溶液浸泡10min；④将切片置于75%乙醇溶液中浸泡10分钟，更换75%乙醇溶液浸泡10分钟；⑤将切片置于蒸馏水泡洗。（3）将30%H2O2用蒸馏水稀释10倍，即3%的H2O2，滴加在切片上室温反应10min，蒸馏水洗3次，每次3min。（4）微波修复：将切片浸泡于0.01M枸橼酸缓冲液中，在微波炉中98-100℃至溶液沸腾，冷却10min，反复2次。（5）将组织切片自然冷却至室温，用PBS洗涤3次，每次5min。（6）封闭，采用5%BSA溶液封闭，室温反应20min，甩去多余液体。50万方数据 第三军医大学博士学位论文（7）滴加一抗，在37℃反应1h或4℃过夜。（8）PBS润洗3次，每次3min。（9）滴加二抗，在37℃反应20min。（10）PBS润洗3次，每次3min。（11）滴加SABC，在37℃反应30min。（12）PBS润洗3次，每次5min。（13）配制DAB显色液，滴加至切片上，显微镜下观察直到底物变色时可终止反应。（14）自来水流洗至背景干净，过蒸馏水。（15）苏木素复染2min，自来水流洗。（16）脱水：30%乙醇3min，50%乙醇3min，70%乙醇3min，80%乙醇3min，90%乙醇3min，95%乙醇3min，100%乙醇3min，二甲苯20min。（17）树胶封片。3.3结果3.3.1成功构建稳定干扰或过表达LINC00675的胃癌细胞株为明确LINC00675在胃癌细胞株中的表达，进而选择合适的胃癌细胞进行下一步研究，课题组检测了LINC00675在6株细胞中的表达，分别是1株胃正常上皮细胞GES-1和5株胃癌细胞（MKN-28、MGC-803、BGC-823、SGC-7901和MKN-45）。与胃正常上皮细胞相比，LINC00675在4株胃癌细胞中呈显著低表达，其中LINC00675在SGC-7901和MKN-45细胞中的表达相对较低，见图4-1A。采用慢病毒载体在SGC-7901和MKN-45细胞中过表达LINC00675，qRT-PCR检测发现LINC00675在这两株细胞中显著高表达，见图4-1B和C，提示过表达LINC00675成功。图4-1成功构建稳定干扰或过表达LINC00675的胃癌细胞株。51万方数据 第三军医大学博士学位论文A：LINC00675在胃上皮及胃癌细胞中的表达，”*”,P<0.05,”**”,P<0.01；B：采用慢病毒载体在SGC-7901和MKN-45细胞中过表达LINC00675，qRT-PCR检测LINC00675的表达，”**”,P<0.01；C：采用慢病毒载体在SGC-7901和MKN-45细胞中干扰LINC00675的表达，qRT-PCR检测LINC00675的表达，”*”,P<0.05,”**”,P<0.01。3.3.2干扰或过表达LINC00675影响胃癌细胞的增殖能力采用已构建的干扰或过表达LINC00675的SGC-7901和MKN-45细胞分析LINC00675的表达对胃癌细胞增殖能力的影响。采用CCK-8试剂分析细胞1-5天的增殖状态，发现干扰LINC00675的表达显著增强了SGC-7901和MKN-45细胞的增殖能力，同时过表达LINC00675显著抑制了SGC-7901和MKN-45细胞的增殖能力，见图4-2。图4-2CCK-8检测胃癌细胞增殖。LINC00675过表达抑制SGC-7901细胞增殖，而干扰LINC00675的表达促进SGC-7901细胞的增殖；同时LINC00675过表达抑制MKN-45细胞增殖，而干扰LINC00675的表达促进MKN-45细胞的增殖，”**”,P<0.01。3.3.3干扰或过表达LINC00675影响胃癌细胞的克隆形成能力采用已构建的干扰或过表达LINC00675的SGC-7901和MKN-45细胞分析LINC00675的表达对胃癌细胞克隆形成能力的影响。采用平板克隆形成实验分析胃癌细胞1周的克隆形成情况，发现干扰LINC00675的表达显著促进了SGC-7901和MKN-45细胞的克隆形成能力，而过表达LINC00675显著抑制了SGC-7901和MKN-45细胞的克隆形成能力，见图4-3。52万方数据 第三军医大学博士学位论文图4-3平板克隆形成实验分析LINC00675对胃癌细胞克隆形成的影响。LINC00675过表达抑制SGC-7901细胞克隆形成，而干扰LINC00675的表达促进SGC-7901细胞的克隆形成；同时LINC00675过表达抑制MKN-45细胞克隆形成，而干扰LINC00675的表达促进MKN-45细胞的克隆形成。3.3.4干扰或过表达LINC00675影响胃癌细胞的周期采用已构建的干扰或过表达LINC00675的SGC-7901和MKN-45细胞分析LINC00675的表达对胃癌细胞周期的影响。采用流式细胞仪分析胃癌细胞的周期，发现干扰LINC00675的表达显著促进了SGC-7901和MKN-45细胞G1期向S期转变，而G2期没有明显变化；同时过表达LINC00675显著抑制了SGC-7901和MKN-45细胞G1期向S期转变，而G2期没有明显变化，提示LINC00675抑制细胞周期G1/S期转变，见图4-4。53万方数据 第三军医大学博士学位论文图4-4流式细胞仪分析胃癌细胞周期分布。LINC00675过表达显著抑制SGC-7901和MKN-45细胞G1/S期转变，而干扰LINC00675的表达显著促进SGC-7901和MKN-45细胞G1/S期转变，”**”,P<0.01。3.3.5干扰或过表达LINC00675影响胃癌细胞的侵袭转移能力采用已构建的干扰或过表达LINC00675的SGC-7901和MKN-45细胞分析LINC00675的表达对胃癌细胞周期的影响。采用Trans-well实验检测胃癌细胞的迁移和侵袭能力发现，过表达LINC00675显著抑制了SGC-7901和MKN-45细胞的迁移和侵袭能力，而干扰LINC00675的表达显著促进了SGC-7901和MKN-45的迁移和侵袭能力，见图4-5和4-6。54万方数据 第三军医大学博士学位论文图4-5干扰或过表达LINC00675显著抑制SGC-7901细胞的迁移和侵袭能力。A和C：干扰或过表达LINC00675显著抑制SGC-7901的迁移能力；B和D：干扰或过表达LINC00675显著抑制SGC-7901细胞的侵袭能力。图4-6干扰或过表达LINC00675显著抑制MKN-45细胞的迁移和侵袭能力。A和C：干扰或过表达LINC00675显著抑制MKN-45的迁移能力；B和D：干扰或过表达LINC00675显著抑制MKN-45细胞的侵袭能力。55万方数据 第三军医大学博士学位论文3.3.6干扰或过表达LINC00675影响胃癌细胞的体内成瘤能力3.3.6.1为进一步明确LINC00675对胃癌细胞皮下成瘤的影响，课题组采用裸鼠皮下成瘤模型，腋下注射干扰或过表达LINC00675的胃癌细胞SGC-7901，发现过表达LINC00675的SGC-7901和MKN45细胞形成的肿瘤的重量显著降低，肿瘤的体积显著降低，而干扰LINC00675的SGC-7901细胞形成的肿瘤的重量显著增加，肿瘤的体积显著增大，见图4-7和4-8。图4-7LINC00675抑制裸鼠体内肿瘤生长。A：干扰或过表达LINC00675的SGC-7901细胞接种至裸鼠腋下，4周后处死裸鼠，取腋下形成的肿瘤并拍照。B：称量形成的肿瘤，发现LINC00675的过表达显著抑制形成肿瘤的重量，而干扰LINC00675的表达显著促进形成肿瘤的重量。C：每周测量一次形成肿瘤的体积，发现LINC00675的过表达显著抑制了形成肿瘤的体积，而干扰LINC00675的表达显著促进了形成肿瘤的体积，”*”,P<0.05。图4-8LINC00675抑制裸鼠体内肿瘤生长。A：干扰或过表达LINC00675的MKN-45细胞接种至裸鼠腋下，4周后处死裸鼠，取腋下形成的肿瘤并拍照。B：称量形成的肿瘤，发现LINC00675的过表达显著抑制形成肿瘤的重量，而干扰LINC00675的表达显著促进形成肿瘤的重量。C：每周测量一次形成肿瘤的体积，发现LINC00675的过表达显著抑制了形成肿瘤的体积，而干扰LINC00675的表达显著促进了形成肿瘤的体积，”*”,P<0.05。56万方数据 第三军医大学博士学位论文3.3.6.2对形成的肿瘤组织行组织切片、H&E染色及Ki67免疫组化，发现过表达LINC00675的SGC-7901细胞形成的肿瘤中Ki67的表达显著降低，而干扰LINC00675的SGC-7901细胞形成的肿瘤中Ki67的表达显著增加，见图4-9和4-10。图4-9LINC00675抑制裸鼠体内形成肿瘤的增殖能力。A和B：Ki67免疫组化发现过表达LINC00675的SGC-7901细胞形成的肿瘤中Ki67的表达显著降低，而干扰LINC00675的SGC-7901细胞形成的肿瘤中Ki67的表达显著增加。57万方数据 第三军医大学博士学位论文图4-10LINC00675抑制裸鼠体内形成肿瘤的增殖能力。A和B：Ki67免疫组化发现过表达LINC00675的MKN-45细胞形成的肿瘤中Ki67的表达显著降低，而干扰LINC00675的MKN-45细胞形成的肿瘤中Ki67的表达显著增加。3.3.6.3采用qRT-PCR检测裸鼠体内形成肿瘤中LINC00675的表达，发现过表达LINC00675组LINC00675的表达显著增加，而干扰LINC00675的表达组LINC00675的表达显著降低，表明LINC00675在形成的肿瘤中的表达保持过表达或低表达水平，见图4-11。图4-11LINC00675在SGC-7901和MKN-45两株细胞形成肿瘤中的表达。QRT-PCR检测发现过表达LINC00675组LINC00675的表达显著增加，而干扰LINC00675的表达组LINC00675的表达显著降低。58万方数据 第三军医大学博士学位论文3.3.7干扰或过表达LINC00675影响胃癌细胞的肺转移能力为明确干扰或过表达LINC00675对胃癌细胞SGC-7901转移能力的影响，课题组采用裸鼠尾静脉注射模型，将稳定干扰或过表达的SGC-7901和MKN-45细胞通过尾静脉注射，6周后处死裸鼠，取出肺组织行H&E染色，显微镜下观察并计数转移灶的数量，发现过表达LINC00675显著抑制了转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了转移灶肿瘤的大小和数量，见图4-12和4-13。图4-12LINC00675抑制SGC-7901细胞肺转移能力。过表达LINC00675显著抑制了肺转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了肺转移灶肿瘤的大小和数量。图4-13LINC00675抑制MKN-45细胞肺转移能力。过表达LINC00675显著抑制了肺转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了肺转移灶肿瘤的大小和数量。59万方数据 第三军医大学博士学位论文3.3.8干扰或过表达LINC00675影响胃癌细胞的肝转移能力课题组进一步采用裸鼠腹腔注射模型明确干扰或过表达LINC00675对胃癌细胞SGC-7901转移能力的影响，将稳定干扰或过表达的SGC-7901细胞通过腹腔注射，6周后处死裸鼠，取出肝组织行H&E染色，显微镜下观察并计数转移灶的数量，发现过表达LINC00675显著抑制了转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了转移灶肿瘤的大小和数量，见图4-14和4-15。图4-14LINC00675抑制胃癌细胞肝转移能力。过表达LINC00675显著抑制了肝转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了肝转移灶肿瘤的大小和数量。图4-15LINC00675抑制胃癌细胞肝转移能力。过表达LINC00675显著抑制了肝转移灶肿瘤的大小和数量，而干扰LINC00675的表达显著促进了肝转移灶肿瘤的大小和数量。60万方数据 第三军医大学博士学位论文3.4小结近年来大量文献表明lncRNA在胃癌的发生和发展中发挥促癌或抑癌作用。[48]Brannan等首次报道了lncRNAH19以来，大量的lncRNAs被陆续鉴定。LncRNA由外显子和内含子组成，没有ORF区，具有不保守性。最近的研究预计人类约14880[29]个lncRNAs。过去十年间，越来越多的证据提示lncRNA参与癌细胞增殖、凋亡、[15]迁移及侵袭等。课题组聚焦在胃癌组织中表达下调的lncRNAs。转录组芯片及聚类分析指出大量的lncRNAs在胃癌组织中表达下调，其中lncRNALINC00675在胃癌组织中的表达和功能未被鉴定，课题组用定量PCR及原位杂交实验在两组人群共计135对样本中鉴定了LINC00675在胃癌组织中低表达，其低表达与胃癌组织大小、TNM分期、淋巴结转移及远处转移密切相关。另外，LINC00675的低表达还与胃癌患者的低生存率明显相关。这些数据提示LINC00675可能作为一个新的生物标志物来诊断或预后判断胃癌的进程。大量文献也表明有其他lncRNAs作为胃癌诊断的生物标志物，[6][49][50][52][53]如GAPLINC,H19,HOTAIR，LINC00152[51],AC096655.1-002,FER1L4,[47][45]HMlincRNA717,ncRuPAR等。由于lncRNA的不保守性，其作为生物标志物在胃癌诊断中具有重要意义。近年来，文献表明lncRNA在肿瘤的发生和发展中发挥重要作用，包括调节肿瘤[54][55][6][56][57]细胞增殖、凋亡、迁移、侵袭、表皮间质转化等。我们首次鉴定LINC00675在胃癌发展中的作用，结果表明LINC00675的低表达显著促进了胃癌细胞的增殖、克隆形成、迁移和侵袭，而恢复LINC00675的表达显著抑制了胃癌细胞的这些生物学行为，提示LINC00675在胃癌的发展中可能作为一个新的抑癌基因。在裸鼠皮下成瘤实验中，课题组构建了慢病毒构建的稳定过表达LINC00675的胃癌细胞株，通过裸鼠皮下注射胃癌细胞，每周观察肿瘤形成情况并测量肿瘤大小，发现稳定过表达LINC00675的胃癌细胞形成肿瘤的能力显著降低，而稳定干扰LINC00675表达的胃癌细胞形成肿瘤的能力显著增强。同时，在裸鼠尾静脉实验中，采用同样的胃癌细胞注射裸鼠尾静脉，四周后处死裸鼠观察裸鼠肺组织转移灶的形成情况，发现稳定过表达LINC00675的胃癌细胞的肺转移能力显著低于转染空载的胃癌细胞，而干扰LINC00675的胃癌细胞的肺转移能力明显增强。另外，在裸鼠腹腔注射实验中，采用同样的胃癌细胞注射裸鼠腹腔，四周后处死裸鼠观察裸鼠肝组织转移灶的形成情况，发现稳定过表达LINC00675的胃癌细胞的肝转移能力显著低于转染空载的胃癌细胞，而干扰LINC00675的胃癌细胞的肝转移能力明显增强。这些研究均表明LINC00675发挥了抑癌的作用，可能作为胃癌药物治疗的有效靶点。61万方数据 第三军医大学博士学位论文第四章LINC00675在胃癌细胞中的定位在上述的研究中，我们明确了LINC00675在胃癌组织中低表达，且与胃癌组织大小、TNM分期、淋巴结转移、远处转移，及胃癌患者的低生存率密切相关，并揭示了LINC00675在胃癌的发展中发挥抑癌作用，而LINC00675的过表达显著抑制了胃癌细胞的增殖、克隆形成、迁移和侵袭，以及胃癌细胞在裸鼠体内成瘤、肺转移及肝转移能力。那么，LINC00675抑制胃癌细胞增殖、克隆形成、迁移和侵袭的分子机制是什么？LncRNA的作用机制十分复杂。文献表明，位于细胞质的lncRNA主要参与基因的转录后调控：①lncRNA与蛋白相互作用，包括影响蛋白的稳定性、桥接两个或以上的蛋白、封闭蛋白的活性位点等，如lncRNANKILA与NF-KappaB的相互作用封闭了IkappaB的磷酸化位点进而抑制了IKK对IkappaB的磷酸化，从而导致NF-KappaB[58]的失活，抑制乳腺癌转移；②lncRNA与RNA相互作用，包括作为竞争性内源RNA吸附miRNAs、与mRNA相互作用影响后者的稳定性等，如lncRNA-ATB竞争性结合miR-200家族上调ZEB1进而介导肝癌细胞的表皮间质转化（EMT）和侵袭，同时lncRNA-ATB与IL-11mRNA相互作用增加后者稳定性并促进IL-11自分泌增加，从而[59]激活STAT3信号通路，促进肿瘤细胞的定植。而位于细胞核的lncRNA主要参与转录调节及DNA甲基化修饰：①lncRNA与转录因子相互作用，lincRNAYY1能与YY1转录因子发生相互作用，介导YY1/PRC2复合物与启动子相互作用，促进基因的表达[60]。②lncRNA与组蛋白修饰复合物相互作用，HOXC位点转录的lncRNAHOTAIR能够靶向PRC2复合物，该复合物包含了H3K27甲基化酶EZH2，SUZ12和EED进[61]而沉默HOXD的表达；Tsai等随后发现HOTAIR的5’端与PRC2结合而3’端与LSD1[62]（KDM1/BHC110）结合，LSD1是去甲基化酶，介导H3K4me2的去甲基化。因此，本章内容分析了LINC00675在胃癌细胞中的定位及其可能作用的靶分子。4.1实验材料4.1.1实验试剂人胃癌细胞株SGC-7901本室保存原位杂交试剂盒博士德生物制品有限公司PBS博士德生物制品有限公司柠檬酸钠重庆川东化工有限公司62万方数据 第三军医大学博士学位论文双氧水重庆川东化工有限公司TritonX-100上海碧云天生物技术有限公司CY3博士德生物制品有限公司DAPI德国Roche公司4%多聚甲醛上海溶剂厂RNA无酶离心管（1.5ml）美国AXYGEN公司RNA无酶PCR离心管美国AXYGEN公司RNA无酶吸头美国AXYGEN公司4.1.2实验仪器电子分析天平300mc型，瑞典低温冰箱（4℃、-20℃）德国Siemens公司超低温冰箱（-80℃）德国Siemens公司高压灭菌锅上海压力容器设备厂分子杂交炉美国Thermo公司避光保湿盒常州市飞勒斯仪器有限公司移液器德国eppendorf公司光学显微镜及电子成像系统日本Olympus公司超净工作台（SW-CJ-1FD）苏州安泰空气技术有限公司电热恒温水箱（DK-8B型）上海精宏实验室设备有限公司电热保湿干燥箱（S202B型）重庆实验设备厂制冰机日本SANYO公司荧光倒置显微镜日本OLYMPUS公司液氮罐成都金凤液氮容器有限公司共聚焦显微镜日本OLYMPUS公司4.1.3主要试剂配制（1）DMEM双抗完全培养基：将50mL胎牛血清加入450mLDMEM培养基中，按100:1加入青霉素-链霉素溶液，混匀。（2）PBS溶液：袋装的PBS粉剂溶解于1000mL一级水中，高压灭菌后4℃保存。（3）胰酶消化液：将0.25g胰酶、0.02gEDTA溶于100mL经高压灭菌的PBS63万方数据 第三军医大学博士学位论文溶液中，采用0.22μm的过滤膜去除细菌及杂质，混匀后4℃保存。（4）细胞冻存液：按胎牛血清:DMSO=9:1的比例混匀，4℃保存。（5）DEPC水：500ml一级水中加入500μLDEPC原液，配制成DEPC水，摇床过夜后，高温高压灭活DEPC30min。（6）20×SSC溶液配制：800mLDEPC水中溶解175.3gNaCL和88.2g柠檬酸钠，加入10mol/LNaOH溶液将pH值调至7.0，加DEPC水定容至1L，分装后高压灭菌。（7）3%柠檬酸溶液配制：在100mL蒸馏水中溶解柠檬酸3g，pH2.0左右。1mL3%柠檬酸加2滴浓缩型胃蛋白酶，混匀。（8）3%H2O2：90mL蒸馏水中加入10mLH2O2，混匀。4.2实验方法4.2.1胃癌细胞铺板。（1）细胞复苏：将冻存的细胞从液氮中取出，置于37℃恒温水浴中快速解冻，1分钟内完成，采用75%乙醇对细胞冻存管进行消毒。吸取4mL完全培养基于无菌离心管，加入冻存细胞液，混匀。将离心管置于台式离心机，800rpm离心5min。弃上清液，加入1mL完全培养基重悬，备用。（2）细胞铺板：将无菌的盖玻片铺在6孔板中，每孔加入1mL细胞重悬液，置于细胞培养箱中培养。（3）当细胞汇合率达到70%时，吸弃培养基，取出盖玻片，将其贴在载玻片上，进行分子杂交。4.2.2分子杂交。（1）30%H2O21份溶于10份蒸馏水中混匀，滴在切片上反应8min，以灭活内源性酶，蒸馏水洗涤3次，每次5min。（2）暴露RNA核酸片段：在切片上滴加3%柠檬酸溶液（1Ml3%柠檬酸加入2滴浓缩型胃蛋白酶，混匀），37℃消化20min。原位杂交用PBS洗涤3次，每次5min，蒸馏水洗涤1次。（3）后固定：胃蛋白酶消化后，用1%多聚甲醛/0.1MPBS，含有1/1000DEPC室温固定10分钟，蒸馏水洗涤3次，每次4min。（4）预杂交：湿盒底部加20%甘油20mL，以保持湿度。按每张切片20μL量加入与杂交液，恒温箱40℃孵育2-4小时，去掉多余的液体，不洗涤。（5）杂交：加入100μL杂交液于载玻片上，覆盖所用样品，恒温箱40℃杂交过夜。64万方数据 第三军医大学博士学位论文（6）杂交后洗涤：37℃水温的2×SSC洗涤2次，每次15分钟。（7）杂交后洗涤：37℃孵育30min，甩去多余液体，不洗涤。（8）滴加生物素化鼠抗地高辛：37℃孵育60分钟或室温120分钟，原位杂交用PBS溶液洗涤4次，每次5min。（9）滴加SABC：37℃孵育20分钟或室温30分钟，原位杂交用PBS溶液洗涤3次，每次5min。（10）滴加生物素化过氧化物酶：37℃孵育20min或室温30min，原位杂交用PBS溶液洗涤4次，每次5min。（11）CY3显色：使用CY3显色试剂，滴加在切片上，反应5min。（12）荧光共聚焦显微镜下观察荧光的表达。4.3实验结果荧光共聚焦显微镜观察显示，LINC00675在胃癌细胞质和细胞核内均有分布，提示LINC00675可能在细胞质和细胞核中均发挥一定的功能，见图5-1图5-1LINC00675在胃癌细胞SGC-7901中的分布。4.4讨论采用荧光原位杂交实验检测LINCOO675在胃癌细胞中的定位有助于了解其在细胞中的作用机制，例如位于胞核的lncRNA可能参与转录调控，而位于胞浆的lncRNA[58]可能参与转录后调控。Liu等采用荧光原位杂交-共聚焦明确了lnccRNANKILA位于胞浆，随后证实其在胞浆中能与NF-κB/IκB复合物结合，进而抑制NF-κB信号通路。本研究发现LINC00675在胞核和胞浆中均有分布，提示LINC00675在胞浆和胞核中可能均发挥作用。本研究主要研究了LINC00675在胞浆中的作用及机制。65万方数据 第三军医大学博士学位论文第五章LINC00675调控胃癌细胞生物学行为的分子机制为阐明LINC00675调控胃癌细胞增殖、迁移和侵袭的分子机制，课题组构建了用于分离与LINC00675相互作用的复合物的载体，即pcDNA3.1-LINC00675-MS2、[59]GFP-MS2载体，该载体的使用首次在Cancercell文献上报道，其能够模拟内源性的lncRNA与其他分子的相互作用，因此得到广泛使用。将这两个载体转染至胃癌细胞中，培养48小时后，采用RIP试剂盒、GFP的抗体可以分离与LINC00675相互作用的复合物，接着采用SDS-PAGE胶、银染分离出与对照组有明显差异的蛋白条带，将该条带切下行蛋白质谱分析，再结合文献及实验结果，获得候选的相互作用的靶标。通过这一流程，课题组成功鉴定到了Vimentin蛋白与LINC00675发生相互作用。LINC00675与靶蛋白的相互作用，可能影响其表达或/和功能。课题组首先检测了LINC00675过表达的胃癌细胞中，Vimentin蛋白的表达是否存在差异，结果显示其表达没有明显变化；在蛋白修饰方面，Vimentin的多个氨基酸位点可以被磷酸化，而不同的磷酸化位点具有不一样的生物学效应。因此，课题组采用磷酸化抗体检测LINC00675过表达的胃癌细胞中Vimentin的磷酸化状态，发现VimentinSer83的磷酸化水平显著增加，而VimentinSer39的磷酸化水平没有明显变化，文献表明，VimentinSer83的磷酸化会导致Vimentin形成的微管崩解，导致细胞结构的变化，进而影响细胞的增殖、迁移和侵袭能力。本节阐明了LINC00675通过与Vimentin相互作用影响VimentinSer83的磷酸化，促进Vimentin形成的微管的崩解，进而抑制胃癌的发展。5.1实验材料5.1.1主要试剂人胃腺癌细胞株SGC-7901本实验室保存人胃腺癌细胞株MKN-45本实验室保存6孔、12孔、96孔板美国Corning公司DEPC水上海碧云天生物技术有限公司TRIzol美国Thermo公司氯仿成都科龙化工试剂长异丙醇成都科龙化工试剂长无水乙醇重庆川东化工有限公司66万方数据 第三军医大学博士学位论文胎牛血清美国Gibco公司酵母提取物上海生物工程公司胰蛋白胨上海生物工程公司琼脂糖西班牙进口分装Lipofectamin2000美国Invitrogen公司胰酶美国Hyclone公司DMEM培养基美国Gibco公司PRMI1640培养基美国Gibco公司PBS博士德生物制品有限公司逆转录试剂盒日本TaKaRa公司Real-timePCR试剂盒日本TaKaRa公司RNA无酶离心管（1.5ml）美国AXYGEN公司RNA无酶PCR离心管美国AXYGEN公司RNA无酶吸头美国AXYGEN公司BCA蛋白浓度测定试剂盒美国ThermoScientific公司质粒DNA提取试剂盒美国Omega公司丙烯酰胺/双叉稀酰胺美国ThermoScientific公司TBS博士德生物制品有限公司吐温美国Bio-Rad公司Tris-Hcl6.8上海碧云天生物技术有限公司Tris-Hcl8.8上海碧云天生物技术有限公司甘氨酸美国ThermoScientific公司NaCl成都科龙化工试剂长Tris成都科龙化工试剂长PVDF膜德国Millipore公司化学发光显色液德国Millipore公司TEMED美国ThermoScientific公司RIPA裂解液美国ThermoScientific公司蛋白酶抑制剂德国Roche公司银染试剂盒博士德生物制品有限公司SDS上样缓冲液上海碧云天生物技术有限公司67万方数据 第三军医大学博士学位论文脱脂奶粉上海碧云天生物技术有限公司BSA上海碧云天生物技术有限公司Vimentin抗体美国CST公司VimentinSer83磷酸化抗体美国CST公司VimentinSer39磷酸化抗体美国CST公司GFP抗体美国CST公司GAPDH抗体美国CST公司5.1.2主要仪器电子分析天平300mc型，瑞典低温冰箱（4℃、-20℃）德国Siemens公司超低温冰箱（-80℃）德国Siemens公司ABSystem7500荧光定量PCR仪美国ABI公司高压灭菌锅上海压力容器设备厂移液器德国eppendorf公司光学显微镜及电子成像系统日本Olympus公司超净工作台（SW-CJ-1FD）苏州安泰空气技术有限公司ND-1000美国Thermo公司电热恒温水箱（DK-8B型）上海精宏实验室设备有限公司旋涡器（QL-901）江苏海门市麒麟医用仪器厂电热保湿干燥箱（S202B型）重庆实验设备厂台式低温离心机美国Thermo公司制冰机日本SANYO公司恒温磁力搅拌器（HWCB-2型）温州市医疗电器厂荧光倒置显微镜日本OLYMPUS公司液氮罐成都金凤液氮容器有限公司DU-640紫外分光光度计美国Beckman公司制冰机日本SANYO公司TM细胞培养箱美国Sher-Lab公司TM细菌培养箱美国Sher-Lab公司垂直电泳槽美国Bio-Rad公司68万方数据 第三军医大学博士学位论文电泳仪美国Bio-Rad公司电转仪美国Bio-Rad公司化学发光仪美国Bio-Rad公司Gel-Doc2000凝胶分析仪美国Bio-Rad公司THZ-C恒温空气震荡仪湖南湘雅离心仪器厂5.1.3主要试剂的配制（1）DMEM双抗完全培养基：将50mL胎牛血清加入450mLDMEM培养基中，按100:1加入青霉素-链霉素溶液，混匀。（2）PBS溶液：袋装的PBS粉剂溶解于1000mL一级水中，高压灭菌后4℃保存。（3）胰酶消化液：将0.25g胰酶、0.02gEDTA溶于100mL经高压灭菌的PBS溶液中，采用0.22μm的过滤膜去除细菌及杂质，混匀后4℃保存。（4）细胞冻存液：按胎牛血清:DMSO=9:1的比例混匀，4℃保存。（5）75%乙醇：在750mL无水乙醇中加入250mL一级水，混匀室温保存。（6）CaCl2溶液：将0.83g氯化钙溶解于100mL一级水中，高压灭菌，4℃保存。（7）LB液体培养基：将10g胰蛋白胨、10gNaCl，5g酵母提取物溶解至1000mL一级水中，加入NaOH将pH调至7.0，高压灭菌，4℃保存。（8）LB固体培养基：将10g胰蛋白胨、10gNaCl，5g酵母提取物及15g琼脂糖溶解至1000mL一级水中，加入NaOH将pH调至7.0，高压灭菌，待冷却至约45℃时制作平板培养基。（9）DEPC水：500ml一级水中加入500μLDEPC原液，配制成DEPC水，摇床过夜后，高温高压灭活DEPC30min。（10）RIPA裂解液：100mLPBS中加入1%NP40（NonidetP-40），0.5%脱氧胆酸钠，0.1%SDS，混匀，4℃保存。（11）2×上样缓冲液：在2mLTris-Hcl（PH6.8,0.5mol/L）中加入4mLSDS、1mLβ-巯基乙醇、2mL丙三醇、1mL溴酚蓝，混匀后4℃保存。（12）二硫苏糖醇（DTT）：取3.09gDTT溶于20mL乙酸钠溶液中，过滤除菌后-20℃保存。（13）10%SDS：取5gSDS溶解于40mL一级水中，充分溶解后加入浓盐酸将pH调至7.2，定容至50mL。（14）10%过硫酸铵（AP）：取0.1gAP溶解于1mL一级水中，避光，现用现配。69万方数据 第三军医大学博士学位论文（15）Tris-Hcl（pH6.8）：取6.06gTris碱溶解于一级水中，用浓盐酸调至pH值为6.8，定容至100mL。（16）Tris-Hcl（pH6.8）：取18.17gTris碱溶解于一级水中，用浓盐酸调至pH值为8.8，定容至100mL。（17）5×电泳缓冲液：取16gTris碱、100g甘氨酸、5.3gSDS溶解于974mL一级水中，待充分溶解。（18）转膜缓冲液：取3.03gTris碱、14.41g甘氨酸加入800mL一级水，混匀后加入200mL甲醇，混匀。（19）TBST缓冲液：将1袋TBS（标注了一袋溶解于多少水中）溶解于1000mL一级水中，完全溶解后加入100μLTween-20，混匀。（20）封闭液：取0.5g脱脂奶粉溶解于10mL1×TBST中，混匀后4℃保存；取0.3gBSA溶解于10mL1×TBST中，混匀后4℃保存。5.2实验方法5.2.1细胞培养5.2.1.1细胞复苏：将冻存的细胞从液氮中取出，置于37℃恒温水浴中快速解冻，1分钟内完成，采用75%乙醇对细胞冻存管进行消毒。吸取4mL完全培养基于无菌离心管，加入冻存细胞液，混匀。将离心管置于台式离心机，800rpm离心5min。弃上清液，加入1mL完全培养基重悬，转移至细胞培养瓶中，补加3mL完全培养基，将瓶口拧松后置于细胞培养箱中培养。5.2.1.2细胞换液：当细胞培养数十小时后，观察培养基及细胞状态，如果培养基变黄或漂浮细胞较多，就需要给细胞换液。步骤是，取出细胞培养瓶，倒掉培养基，加入新鲜完全培养基，将瓶口拧松后置于细胞培养箱中培养。5.2.1.3细胞传代：当细胞汇合率达到80%左右，就需要对细胞进行换液。步骤是，取出细胞培养瓶，倒掉培养基，加入2mL无菌PBS润洗2次，倒掉PBS，加入1mL胰酶消化数分钟，显微镜下观察细胞的消化状态，如果细胞变圆变小、细胞间失去连接，或部分细胞漂浮，立即加入1mL完全培养基终止胰酶的作用，将液体转移至无菌离心管中，至于台70万方数据 第三军医大学博士学位论文式离心机，800rmp离心5min，倒掉上清液，加入2mL完全培养基重悬，分装至2个细胞培养瓶中，每瓶中补3mL完全培养基，将瓶口拧松后置于细胞培养箱中培养。5.2.2pcDNA3.1-LINC00675-MS2载体构建（1）经网站http://www.ncbi.nlm.nih.gov/nuccore/NR_036581.1获得LINC00675的cDNA序列，上海吉玛公司根据序列合成带有HandⅢ和EcoRⅠ酶切位点的DNA序列。（2）采用HandⅢ和EcoRⅠ酶切T载体，并回收鉴定（3）采用T4DNA连接酶将合成的DNA序列与酶切后的T载体进行连接，步骤是：取50ng载体和5倍摩尔量的DNA合成片段，加入双蒸水中，体积为10μL；加入10μLMbuffer，混匀；加入1μLT4DNA连接酶，充分而轻柔混匀；轻微离心后室温放置5分钟；冰上保存，带下一步转化。（4）制备感受态1）取出冻存于-70℃的DH5a，无菌接种环蘸取DH5a保种液，三线法划线接种于LB平板，37℃培养16h。2）从LB平板中挑取色泽较好的单个菌落接种于10mL的LB液体培养基中，37℃摇床培养16h。3）将过夜培养的DH5a按照1%比例接种至LB液体培养基中，37℃摇床培养至OD600为0.5时，置于冰上2min之后，4000g离心5min，弃上清。4）加入1mL预冷的0.1mol/LCaCl2重悬沉淀，置于冰水浴2min之后，4℃条件下4000g离心5min，弃上清。5）加入100µL预冷的0.1mol/LCaCl2重悬沉淀，置于冰水浴2min之后，4℃条件下4000g离心5min，弃上清，加入100µL预冷的0.1mol/LCaCl2悬浮沉淀，即为感受态。可冻存于-70℃。（5）转化1）取3管100µL感受态细胞，其中的1管加入连接产物，1管加入controlinsertDNA连接产物作为阳性对照，1管不加入载体作为阴性对照，置于冰水浴2min，擦干离心管后于42℃热休克90s，迅速置于冰水浴2min。2）每管加入900µLLB液体培养基，37℃摇床培养16h。3）5000g离心5min，吸弃500µL上清后混匀沉淀，取50µL细胞悬液均匀涂布于Amp+平板上，置于37℃浮箱培养过夜。（6）质粒提取71万方数据 第三军医大学博士学位论文1）吸取6mL菌液于离心管中，10000g离心3min，弃上清。2）加入250µL含RNaseA的SolutionⅠ，置于漩涡器上漩涡数s以充分裂解细菌。3）加入250µLSolutionⅡ，温和颠倒混匀数次至沉淀澄清，室温静止2min。4）加入350µLSolutionⅢ，立即温和颠倒混匀数次至出现白色沉淀，在室温条件下13000g离心10min。5）转移上清液于HiBind®MiniprepoColumn（I）中，在室温条件下10000g离心1min，弃掉流穿液。6）加入500µLHB溶液洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。7）加入700µLDNAWashBuffer洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。8）重复步骤7）一次。9）在室温条件下13000g离心2min，干燥吸附柱，弃掉废液收集管。10）取新的1.5mL离心管，将吸附柱套于管中，加入35µLElutionBuffer室温反应2min，13000g离心2min，洗脱质粒。（7）酶切1）准备HandⅢ和EcoRⅠ，10×Mbuffer，ddH2O，质粒。2）按照下列体系进行酶切反应：试剂名称体积（µL）HindⅢ0.5SpeⅠ0.510×Mbuffer2ddH2O12质粒0.5µg（5µL）3）反应条件：37℃条件下反应2h。（8）连接1）将pcDNA3.1-MS2载体与目的片段进行连接，2）准备目的片段，pcDNA3.1-MS2载体，Ligationsolution试剂3）连接体系如下：72万方数据 第三军医大学博士学位论文试剂名称体积（µL）目的片段0.5µg（5µL）PcDNA3.1-MS20.2µg（4µL）Ligationsolution14）反应条件：16℃连接过夜。（9）转化1）取3管100µL感受态细胞，其中的1管加入连接产物，1管加入controlinsertDNA连接产物作为阳性对照，1管不加入载体作为阴性对照，置于冰水浴2min，擦干离心管后于42℃热休克90s，迅速置于冰水浴2min。2）每管加入900µLLB液体培养基，37℃摇床培养16h。3）5000g离心5min，吸弃500µL上清后混匀沉淀，取50µL细胞悬液均匀涂布于Amp+平板上，置于37℃浮箱培养过夜。（10）质粒提取1）吸取6mL菌液于离心管中，10000g离心3min，弃上清。2）加入250µL含RNaseA的SolutionⅠ，置于漩涡器上漩涡数s以充分裂解细菌。3）加入250µLSolutionⅡ，温和颠倒混匀数次至沉淀澄清，室温静止2min。4）加入350µLSolutionⅢ，立即温和颠倒混匀数次至出现白色沉淀，在室温条件下13000g离心10min。5）转移上清液于HiBind®MiniprepoColumn（I）中，在室温条件下10000g离心1min，弃掉流穿液。6）加入500µLHB溶液洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。7）加入700µLDNAWashBuffer洗涤吸附柱，在室温条件下10000g离心1min，弃掉流穿液。8）重复步骤7）一次。9）在室温条件下13000g离心2min，干燥吸附柱，弃掉废液收集管。10）取新的1.5mL离心管，将吸附柱套于管中，加入35µLElutionBuffer室温反应2min，13000g离心2min，洗脱质粒。（11）pcDNA3.1-MS2载体转染293T细胞1）采用6孔板培养293T细胞，至细胞贴壁生长汇合率达到70%时开始转染。TM2）准备Lipofectamine2000，Opti，pcDNA3.1-MS2载体，离心管，无血清培养基。73万方数据 第三军医大学博士学位论文3）根据LipofectamineTM2000说明书推荐体系TM4）加入250µLOpti和6µLLipofectamine2000于离心管，标记为1号。5）加入250µLOpti和2µgpcDNA3.1-MS2载体于离心管，标记为2号。6）室温静止5min。7）将1号和2号离心管溶液混合，充分混匀，室温静止15-20min。8）吸弃细胞培养基，PBS润洗2次，加入1mL无血清培养基。9）加入500µL7）步骤混合液于细胞培养基中，温和摇匀，将6孔板置于细胞培养箱中培养6h。10）取出6孔板，吸弃培养基，加入DMEM完全培养，置于细胞培养箱中培养48h。5.2.3提取总RNA（1）将冻存于液氮罐中的组织样品取出并置于含1mLTrizol的组织匀浆离心管中，将离心管置于冰上冷却2min以上。（2）设置组织匀浆仪参数为6000rpm，15s，间歇30s，共3次。（3）取出离心管置于冰上冷却2min以上，重复步骤（2）。（4）按照0.2mL氯仿/1mLTrizol的比例加入适量氯仿于上述离心管，漩涡10s。室温静止10min。（5）将离心管置于低温离心机，4℃，12000g离心15min，溶液分层三相，取上层水相溶液于新的离心管中。（6）加入吸取液等量的异丙醇溶液，充分混匀，室温放置8min，沉淀总RNAs。（7）将离心管置于低温离心机，4℃，12000g离心10min，弃上清。（8）加入1mL75%乙醇溶液，颠倒混匀数次以洗涤RNA沉淀，4℃，7500rpm离心10min，弃掉乙醇，自然干燥10min。（9）每管中加入40µLDEPC水分装，-20℃保存。5.2.4QRT-PCR5.2.4.1进入NCBI网站，获得LINC00675的RNA序列，采用Primer6设计特异扩增引物，选择一条合适的引物序列，委托上海生工合成。5.2.4.2随机引物逆转录TM（1）准备PrimeScriptRTreagentKit，总RNAs，DEPC水。（2）将总RNAs稀释为每个反应总量1µg。（3）按下列体系加入反应试剂：74万方数据 第三军医大学博士学位论文试剂名称体积（µL）5×PrimeScriptbuffer2PrimeScriptEnzymeMix0.5OligodTPrimer（50µM）0.5Random6mers（100µM）2RNA（1µg）5（4）反应条件：37℃孵育15min，85℃酶灭活5s。5.2.4.3染料法扩增（1）准备2×Real-timePCRMasterMIX，LINC00675上、下游引物，ddH2O。引物名称序列LINC00675sense:5'-GCCTACTGCTCTGGATCATCTGGTA-3'LINC00675anti-sense:5'-ACCTGCGTCTCTTCTCCTCTTCC-3'β-actinsense:5′-TTCCTTCCTGGGCATGGAGTCC-3′β-actinanti-sense:5′-TGGCGTACAGGTCTTTGCGG-3′（2）以逆转率产物为模版，SYBRgreen法扩增LINC00675基因，反应体系如下：试剂名称体积（µL）2×Real-timePCRMasterMIX5上游引物（5µM）0.5下游引物（5µM）0.5ddH2O2cDNAs2（3）反应条件：95℃反应1min，47个循环于95℃，5s；60℃，5s；72℃，20s。在72℃时采集荧光，溶解曲线，从60℃升至95℃过程中，每个循环增加0.5℃，共71个循环。5.2.4.4采用ComparativeDelta-deltaCt法计算定量结果。75万方数据 第三军医大学博士学位论文5.2.5总蛋白提取（1）培养并收集胃癌细胞，采用预冷的PBS洗2次。（2）1mLRIPA溶液中加入10μLPMSF，混匀后加入细胞收集管中，用1mL移液器吹打数次裂解细胞，冰上操作。（3）将离心管置于台式离心机上，4℃、2000rpm离心10min，吸取上清液至一新的离心管中，可-80℃保存。（4）取100μL裂解蛋白，加入100μL2×上样缓冲液，混匀，100℃煮沸10min。5.2.6蛋白定量（1）BCA法测蛋白浓度，取出Pierce®BCAproteinassaykit。（2）准备标准品BSA，将标准品稀释为20-2000µg/mL含9个梯度的标准品溶液。（3）其中9个梯度分别是：25、125、250、500、750、1000、1500、2000，具体稀释如下，A-I浓度以此降低：A0µL+300标准品原液，B125µL稀释液+375µL原液，C325µL稀释液+325µL原液，D175ul稀释液+175µLB，E325µL稀释液+325µLC，F325µL+325µLE，G325µL稀释液+325µLF，H400µL稀释液+100µLG，I400µL稀释液。（4）工作液WR的准备，按照下列公式计算WR溶液所需总量：（标准品+待测样品）×（重复数）×（每个孔加入WR的体积数）=WR需求总数（5）WR=50partsofReagentA+1partofReagentB（50:1，ReagentA:B）（6）吸取10µL已知浓度标准品于96-well板。（7）吸取10µL待测样品于上述板中（8）加入200µLWR试剂于每孔，混匀或摇匀30s，37℃孵育30min，562nm波长检测。5.2.7Westernblot5.2.7.1原理：Westernblot技术采用抗原抗体特异性反应特点检测抗原或抗体的存在及表达。该技术是把总蛋白通过SDS-PAGE胶分离成不同分子量大小的组分，再将胶上的蛋白转印至PVDF膜上，加入特定的抗体与膜上的蛋白进行反应，再加入酶标二抗与一抗反应，最后加入底物显色。显色的部位与被测蛋白的分子量大小一致。5.2.7.2步骤：（1）SDS-PAGE胶电泳1）准备40%丙烯酰胺/双叉烯酰胺溶液，4×TrisHCl（8.8），4×TrisHCl（6.8），76万方数据 第三军医大学博士学位论文10%SDS，10%过硫酸铵，TEMED，电泳仪，电泳槽，电泳缓冲液，5×蛋白上样缓冲液，超纯水，预染marker。2）配制10%SDS-PAGE分离胶，配方如下（适用于bio-rad电泳槽）：10%分离胶成分试剂1块胶（ml）2块胶（ml）4块胶（ml）Ⅰ级纯净水1.32.75.330%丙烯酰胺溶液1.73.36.71.0mol/lTris.Hcl（8.8）1.93.87.610%SDS0.050.10.210%过硫酸铵0.050.10.2TEMED0.0020.0040.0083）灌注装配好的玻板至合适的高度，加入Ⅰ级纯净水压平，待分离胶凝固。4）配制两块5%SDS-PAGE浓缩胶，配方如下：5%浓缩胶1块胶（ml）2块胶（ml）4块胶（ml）Ⅰ级纯净水1.231.8453.6940%丙烯酰胺溶液0.250.3750.754×Tris.Hcl（6.8）0.50.751.510%SDS0.020.030.0610%过硫酸铵0.020.030.06TEMED0.0020.0030.0065）已用蛋白上样缓冲液处理的蛋白样品在上样之前需沸水浴min，然后按照一定的顺序上样。6）60V电泳至溴酚蓝指示剂越过浓缩胶后，将电压调至120V，直到直到溴酚蓝指示剂达到分离胶底部，关闭电源。（2）湿转1）将滤纸，SDS-PAGE胶浸泡于电转缓冲液中，PVDF膜置于无水甲醇溶液中浸泡30s后转至电转液浸泡2min。2）装配电转三明治，200mA电转90min。77万方数据 第三军医大学博士学位论文（3）封闭1）1×PBST洗膜2次，每次2min。2）将PVDF膜置于封闭液中封闭1h。（4）一抗孵育1）1×PBST洗膜3次，每次10min。2）按照目的条带大小切下PVDF膜，置于一抗孵育盒中，分别加入已稀释过的一抗，置于4℃摇床过夜。（5）二抗孵育1）回收一抗，加入1×PBST洗膜3次，每次10min，弃1×PBST溶液。2）加入封闭液稀释的二抗，室温摇床1h。3）弃掉二抗溶液，加入1×PBST洗膜3次，每次10min。（6）显色将PVDF膜置于ChemiDocTMXRS+仪器上，加入已配好的化学发光显色底物（500µLA+500µLB），设置曝光参数，运行系统检测荧光。5.2.8银染采用生工公司的低背景银染试剂盒（1）取出电泳后的聚丙烯酰胺凝胶，用蒸馏水冲洗凝胶，根据下列程序所需要的试剂量，取溶液B和D用双蒸水稀释成工作液待用。（2）加入20mL蒸馏水，置于摇床上，震荡5min。（3）倾出蒸馏水，加入含10mL50%乙醇、2.4mL12%乙酸和55μL溶液A的溶液20mL，置于摇床上，震荡60min。（4）倾出溶液，加入20mL50%的乙醇，置于摇床上，震荡5min。（5）倾出50%乙醇，加入20mL溶液B，置于摇床上，震荡1min。（6）倾出溶液B，加入20mL蒸馏水，置于摇床上，震荡1min，重复3次。（7）倾出蒸馏水，加入20mL溶液C和43μL溶液A，置于摇床上，震荡20min。（8）倾出溶液C，加入20mL蒸馏水，置于摇床上，震荡1min，重复2次。（9）倾出蒸馏水，加入20mL溶液D和28μL溶液A，缓慢摇动，直到蛋白条带具有足够的显色强度（约2-3min）。（10）加入含50%乙醇和12%乙酸的溶液20mL，置于摇床上，震荡5min。（11）倾出液体，加入20mL蒸馏水，置于摇床上，震荡5min。78万方数据 第三军医大学博士学位论文5.2.9RIP（1）采用6-well板培养SGC-7901细胞至80%汇合率。（2）采用Lip2000共转染pcDNA3.1-LINC00675-MS2和GFP-MS2载体至SGC-7901细胞中，培养48h。（3）收集细胞进行RIP实验，步骤如下：1）裂解液准备：100μLRIPLysisbuffer加入0.5μLProteaseinhibitor和0.25μLRNaseinhibitor，在冰上操作。2）裂解单层或贴壁细胞，SGC-7901细胞。①采用预冷的PBS将细胞清洗2次。②加入1mL预冷的PBS，刮取细胞并转移至一新的无酶离心管中，可对细胞计数。③1500rmp、4℃离心5min，去上清。④用细胞沉淀体积大小的RIPLysisbuffer重悬沉淀，直到细胞被裂解均匀，冰上放置5min，可冻存于-80℃。3）准备磁珠进行免疫沉淀。①完全吹散并重悬磁珠。②对各组进行标记：interest,negativecontrol,positivecontrol.③吸取50μL磁珠重悬液至每管中。④加入0.5μLRIPwashbuffer至每管中，混匀。⑤将离心管放置在磁珠分离器上，去除上清液。⑥重复④⑤步骤。⑦用100μLRIPwashbuffer重悬磁珠，加入5μg抗体，室温旋转孵育30min。⑧简单离心后将离心管放置磁珠分离器，去上清，加入0.5mLRIPwashbuffer至每管，混匀。⑨将离心管放置于磁珠分离器，去上清，重复⑧步骤。⑩加入0.5mLRIPwashbuffer至没管，混匀，置于冰上。4）RIP①准备RIPImmunoprecipitatioonbuffer.79万方数据 第三军医大学博士学位论文RIPwashbuffer860μL0.5MEDTA35μLRNaseInhibitor5μLTotal900μL②去除离心管的上清液，加入900μLRIPImmunoprecipitationbuffer至没管中。③迅速解冻RIPLysate，14000rpm、4℃离心10min。将上清液转移至已加入900μLRIPImmunoprecipitation的离心管中至总体积为1mL。④吸取10μLRIPLysate上清液至一新的离心管，并标记“input”，冻存于-80℃，在做RNApurification时取出。“input”可做RT-PCR标准曲线，亦可进行Westernblot实验。⑤将离心管旋转4℃孵育过夜。⑥将离心管离心后置于磁珠分离器，去上清。⑦加入0.5mLRIPwashbuffer至每管中，混匀。⑧将离心管放置在磁珠分离器上，去上清。⑨重复⑦⑧步骤，5次。可在最后一次时去50μL磁珠重悬液做Westernblot实验检测免疫沉淀效果，在SDS上样buffer的作用下，蛋白与磁珠在95℃孵育后能被分离，离心后磁珠被离下，上清用于Wersternblot实验。5）RNA的纯化①准备ProteinaseKbufferRIPwashbuffer117μL10%SDS15μLProteinaseK18μLTotal150μL②用150μLProteinaseKbuffer重悬4）-⑨步溶液。③解冻“input”，加入107μLRIPbuffer，15μL10%SDS，18μLProteinaseK至150μL。④将离心管在55℃孵育30min，摇动，消化蛋白。80万方数据 第三军医大学博士学位论文⑤简单离心后将离心管置于磁珠分离器，转移上清液至新的离心管。⑥加入250μLRIPwashbuffer至每管中。⑦加入400μL酚氯仿至每管中，混匀，14000rmp离心10min。⑧吸取350μL上清至新的离心管，加入400μL酚氯仿，混匀，14000rmp离心10min。⑨吸取300μL上清至新的离心管，加入50μLSaltsolutionI，15μLSaltsolutionII，5μLPrecipitateEnhacer，850μLabsoluteethanol，混匀，-80℃放置过夜。⑩14000rmp、4℃离心30min，去上清，用80%ethanol洗涤沉淀，14000rpm、4℃离心15min，去上清，干燥沉淀。加入10-20μLRNase-freewaster重悬，置于冰上。5.3结果5.3.1成功构建pcDNA3.1-LINC00675-MS2、MS2-GFP载体5.3.1为探寻LINC00675调控胃癌细胞生物学行为的分子机制，课题组构建了pcDNA3.1-LINC00675-MS2、MS2-GFP载体，通过酶切，琼脂糖凝胶电泳鉴定，发现在1000-1500bp处有一条明显的电泳带，见图6-1。81万方数据 第三军医大学博士学位论文图6-1琼脂糖凝胶电泳检测pcDNA3.1-LINC00675-MS2载体酶切后片段大小82万方数据 第三军医大学博士学位论文83万方数据 第三军医大学博士学位论文84万方数据 第三军医大学博士学位论文图6-2测序鉴定pcDNA3.1-LINC00675-MS2载体构建成功。85万方数据 第三军医大学博士学位论文5.3.2采用RIP实验分离与LINC00675相互作用的蛋白质，原理理见图6-3。图6-3RIP技术分离与LINC00675相互作用的蛋白质示意图。5.3.3过表达LINC00675-MS2组在60Kda左右存在特异的蛋白，提示该蛋白可能与LINC00675相互作用。5.3.3.1将pcDNA3.1-LINC00675-MS2、MS2-GFP质粒共转染SGC-7901细胞，培养48h以后检测细胞中LINC00675的表达，发现LINC00675显著高表达，提示转染的质粒pcDNA3.1-LINC00675-MS2过表达LINC00675-MS2序列成功，见图6-4。图6-4在SGC-7901细胞中转染pcDNA3.1-LINC00675-MS2载体后，QRT-PCR检测LINC00675-MS2的表达。86万方数据 第三军医大学博士学位论文5.3.3.2采用RIP实验分离与LINC00675结合的复合物，再通过SDS-PAGE、银染，发现GFP组在60KDa处存在特异性的蛋白，见图6-5。图6-5RIP实验分离与LINC00675结合的复合物，发现60Kda出有特异条带。87万方数据 第三军医大学博士学位论文5.3.4将上述特异性蛋白条带切下行质谱分析，发现该段含多个蛋白，见图6-6。88万方数据 第三军医大学博士学位论文89万方数据 第三军医大学博士学位论文图6-6前两幅图分别为input组和GFP组的蛋白离子流图；后面两幅图分别表示input组和GFP组的蛋白质谱鉴定的结果，发现在GFP组Vimentin具有很高的匹配分数，而Vimentin的蛋白大小是60KDa，提示LINC00675可能与Vimentin发生相互作用。5.3.5采用Vimentin的抗体验证与LINC00675结合的复合物发现GFP组60KDa处有Vimentin特异性抗体结合，见图6-7，提示LINC00675与Vimentin相互作用。图6-7Westernblot实验证实GFP组60KDa处有Vimentin特异性抗体结合。90万方数据 第三军医大学博士学位论文5.3.6采用RIP技术在胃癌细胞SGC-7901和MKN-45中验证与Vimentin结合的复合物发现LINC00675有大量富集，见图6-8。图6-8QRT-PCR检测SGC-7901和MKN-45细胞中Vimentin结合的复合物中LINC00675的富集情况。SNRNP70为U1snRNA特异性结合蛋白，通过检测U1snRNA的表达证实RIP方法的有效性，作为阳性对照；IgG组作为阴性对照。5.3.7采用竞争法分析LINC00675与Vimentin的相互作用。在胃癌细胞SGC-7901和MKN-45中共转染pcDNA3.1-LINC00675和pcDNA3.1-LINC00675-MS2，由于仅MS2环状序列与MS2蛋白相结合，MS2蛋白偶联了GFP蛋白，采用GFP的抗体拉这一复合物可以将与LINC00675结合的蛋白拉出，而没有MS2的LINC00675将会对这一反应产生干扰，表现为浓度依赖性差异，差异如图6-9所示：pcDNA3.1-LINC00675载体的浓度越高干扰RIP反应的结果越明显，被检测的vimentin蛋白量依次下降。91万方数据 第三军医大学博士学位论文图6-9采用竞争法分析LINC00675与Vimentin的相互作用。在SGC-7901和MKN-45细胞中，分别共转染不同浓度的pcDNA3.1-LINC00675和pcDNA3.1-LINC00675-MS2，以及表达GFP-MS2融合蛋白的载体，其中pcDNA3.1-LINC00675采用不同的浓度，分别是0,1,2,4μg，其转录的LINC00675与pcDNA3.1-LINC00675-MS2载体转录的LINC00675-MS2竞争结合vimentin，导致RIP实验检测结果存在浓度梯度差异。5.3.8采用Westernblot实验验证发现，LINC00675的过表达不影响Vimentin蛋白的表达，但显著促进了VimentinSer83的磷酸化水平，而非VimentinSer39的磷酸化水平，同样干扰LINC00675的表达显著抑制了VimentinSer83的磷酸化水平，见图6-10。图6-10Westernblot实验检测干扰或过表达LINC00675的SGC-7901和MKN-45细胞中Vimentin及其磷酸化（Ser83或Ser39）的表达。92万方数据 第三军医大学博士学位论文5.4讨论本研究将pcDNA3.1-LINC00675-MS2和GFP-MS2载体共转染胃癌细胞SGC-7901，采用RIP实验分离与LINC00675结合的复合物，经SDS-PAGE胶电泳分离和银染发现对照组与处理组间存在差异显著的蛋白条带，进一步切取该条带行蛋白质谱分析，发现了大约16个候选蛋白，其中vimentin蛋白具有较高的评分且与癌细胞的转移密切相关。因此，课题组采用westernblot检测了RIP实验分离的与LINC00675结合的复合物中vimentin的表达，结果表明在该复合物中的确存在vimentin蛋白。进一步采用vimentin的抗体拉vimentin的复合物并采用qRT-PCR检测LINC00675的表达，同样也能检测到LINC00675的表达，这些结果证明LINC00675与vimentin发生了相互[63,64]作用。Vimentin的表达也与胃癌细胞转移密切相关。那么，是否LINC00675调控了vimentin的表达，进而影响了胃癌细胞的转移？课题组采用westernblotting检测了干扰或过表达LINC00675的胃癌细胞中vimentin蛋白的表达，结果显示LINC00675对vimentin蛋白的表达没用显著影响。文献表明，vimentin的功能也受到其磷酸化水[65]平的影响。因此，课题组选择了两个vimentin不同位点磷酸化的抗体，其位点分别在Ser83和Ser39。Westernblotting显示LINC00675可以明显增强vimentinSer83位点[66]的磷酸化水平，而对Ser39位点的磷酸化水平没有显著影响。Zhang等研究表明过氧化物酶体增殖物激活受体（PPARα）介导vimentinSer83位点的磷酸化，导致波形蛋白细丝（vimentinfilaments）崩解，进而增加了可溶性vimentin蛋白的浓度。另外，等[67]Jeganathan研究指出intersectin-1s导致vimentinfilaments崩解，进而抑制肺癌细胞的增殖和转移。同样，课题组的研究揭示了LINC00675可导致vimentinfilaments的崩解，进而抑制胃癌细胞的增殖和转移。但是，什么蛋白激酶导致了vimentinSer83的磷酸化并不清楚，尽管PPARα被认为是介导vimentinSer83磷酸化的蛋白，但并没有被证实。本文通过RIP及蛋白质谱发现肌肉丙酮酸激酶（PKM），其能够催化膦酰基从磷酸烯醇式丙酮酸转移至ADP，进而产生ATP和丙酮酸。PKM是蛋白激酶C（PKC）[68]的水解产物，而PKC被认为是抑制胃癌侵袭的新靶点。PKC被报道能够磷酸化[65,69]vimentin蛋白多个位点，包括Ser6，Ser8，Ser9和Ser20，但没有数据表明其可以磷酸化vimentinSer83位点。另外，文献报道PKM2的高表达与多种肿瘤的不良预[70]后密切相关，但其与胃癌的相关性并不清楚，提示PKM2可能并不是促进胃癌发育的独立因子。因此，PKM2是否可以磷酸化vimentin及这一过程是否需要LINC00675的参与需进一步验证。93万方数据 第三军医大学博士学位论文第六章LINC00675调节的信号通路为明确LINC00675-Vimentin复合物在胃癌的发展中影响的信号通路，课题组将LINC00675过表达的SGC-7901和平行对照SGC-7901细胞行mRNA芯片，与对照相比，LINC00675过表达的SGC-7901细胞中大量基因表达上调或下调。通过Pathway分析发现GO富集分析发现LINC00675的表达与细胞增殖和EMT相关基因的6.1实验材料6.1.1主要试剂DEPC水上海碧云天生物技术有限公司TRIzol美国Thermo公司氯仿成都科龙化工试剂长异丙醇成都科龙化工试剂长无水乙醇重庆川东化工有限公司逆转录试剂盒日本TaKaRa公司Real-timePCR试剂盒日本TaKaRa公司PBS博士德生物制品有限公司RNA无酶离心管（1.5ml）美国AXYGEN公司RNA无酶PCR离心管美国AXYGEN公司RNA无酶吸头美国AXYGEN公司6.1.2主要仪器电子分析天平300mc型，瑞典低温冰箱（4℃、-20℃）德国Siemens公司超低温冰箱（-80℃）德国Siemens公司ABSystem7500荧光定量PCR仪美国ABI公司高压灭菌锅上海压力容器设备厂移液器德国eppendorf公司超净工作台（SW-CJ-1FD）苏州安泰空气技术有限公司ND-1000美国Thermo公司94万方数据 第三军医大学博士学位论文电热恒温水箱（DK-8B型）上海精宏实验室设备有限公司旋涡器（QL-901）江苏海门市麒麟医用仪器厂台式低温离心机美国Thermo公司制冰机日本SANYO公司恒温磁力搅拌器（HWCB-2型）温州市医疗电器厂液氮罐成都金凤液氮容器有限公司6.1.3主要溶液配制（1）DMEM双抗完全培养基：将50mL胎牛血清加入450mLDMEM培养基中，按100:1加入青霉素-链霉素溶液，混匀。（2）PBS溶液：袋装的PBS粉剂溶解于1000mL一级水中，高压灭菌后4℃保存。（3）胰酶消化液：将0.25g胰酶、0.02gEDTA溶于100mL经高压灭菌的PBS溶液中，采用0.22μm的过滤膜去除细菌及杂质，混匀后4℃保存。（4）细胞冻存液：按胎牛血清:DMSO=9:1的比例混匀，4℃保存。（5）75%乙醇：在750mL无水乙醇中加入250mL一级水，混匀室温保存。（6）DEPC水：500ml一级水中加入500μLDEPC原液，配制成DEPC水，摇床过夜后，高温高压灭活DEPC30min。6.2实验方法6.2.1mRNA芯片（1）将转染LINC00675和NC的SGC-7901细胞收集至1mLRNA无酶离心管中。（2）将TRIzol溶解物送至上海康成生物工程有限公司行mRNA芯片。（3）分析芯片结果，评价LINC00675对信号通路的影响。6.2.2提取总RNA（1）将转染了LINC00675和NC的胃癌细胞SGC-7901收集至1.5ml离心管中，加入1mlTRIzol进行裂解。（2）按照0.2mL氯仿/1mLTrizol的比例加入适量氯仿于上述离心管，漩涡10s。室温静止10min。（3）将离心管置于低温离心机，4℃，12000g离心15min，溶液分层三相，取上层水相溶液于新的离心管中。95万方数据 第三军医大学博士学位论文（4）加入吸取液等量的异丙醇溶液，充分混匀，室温放置8min，沉淀总RNAs。（5）将离心管置于低温离心机，4℃，12000g离心10min，弃上清。（6）加入1mL75%乙醇溶液，颠倒混匀数次以洗涤RNA沉淀，4℃，7500rpm离心10min，弃掉乙醇，自然干燥10min。（7）每管中加入40µLDEPC水分装，-20℃保存。6.2.3QRT-PCR6.2.3.1进入NCBI网站，获得以下mRNAs的序列，采用Primer6设计特异扩增引物，选择一条合适的引物序列，委托上海生工合成。6.2.3.2随机引物逆转录TM（1）准备PrimeScriptRTreagentKit，总RNAs，DEPC水。（2）将总RNAs稀释为每个反应总量1µg。（3）按下列体系加入反应试剂：试剂名称体积（µL）5×PrimeScriptbuffer2PrimeScriptEnzymeMix0.5OligodTPrimer（50µM）0.5Random6mers（100µM）2RNA（1µg）5（4）反应条件：37℃孵育15min，85℃酶灭活5s。6.2.3.3染料法扩增（1）准备2×Real-timePCRMasterMIX，LINC00675上、下游引物，ddH2O。引物名称序列MDM2Sense:5′-CGGATCTTGATGCTGGTGTAAGT-3′Antisense:5′-CAGGAAGCCAATTCTCACGAAGG-3′CPA4Sense:5′-TCACCACAACCTCTGCCTCCT-3′Antisense:5′-TTGAGACCAGCCTGACCAACATG-3′DKK1Sense:5′-CCTTGAACTCGGTTCTCAATTCC-3′96万方数据 第三军医大学博士学位论文Antisense:5′-CAATGGTCTGGTACTTATTCCCG-3′POP1Sense:5′-TGGTCTGTGTAGTTGTGGCTGAA-3′Antisense:5′-GCGTCTTCTCTTGTCTTCTGCTAT-3′STEAP4Sense:5′-TTGTCCTAATCATGCCATGTGTAGA-3′Antisense:5′-GTGTTCTCTTCCAGTATGTCAGTCA-3′H1F0Sense:5′-CGGCAAGAAGAAGTGACAATGAAGT-3′Antisense:5′-GGTGGTGGTGAGCATCAAGAGA-3′CGASense:5’-CACATTGTCGGTGTTTCT-3’Antisense:5’-ACCTTAGTGGAGTGGGATA-3’p21Sense:5’-AAGTCAGTTCCTTGTGGAGCC-3’Antisense:5’-GGTTCTGACGGACATCCCCA-3’LINC00675Sense:5'-GCCTACTGCTCTGGATCATCTGGTA-3'Antisense:5'-ACCTGCGTCTCTTCTCCTCTTCC-3'β-actinSense:5′-TTCCTTCCTGGGCATGGAGTCC-3′Antisense:5′-TGGCGTACAGGTCTTTGCGG-3′（2）以逆转率产物为模版，SYBRgreen法扩增LINC00675基因，反应体系如下：试剂名称体积（µL）2×Real-timePCRMasterMIX5上游引物（5µM）0.5下游引物（5µM）0.5ddH2O2cDNAs2（3）反应条件：95℃反应1min，47个循环于95℃，5s；60℃，5s；72℃，20s。97万方数据 第三军医大学博士学位论文在72℃时采集荧光，溶解曲线，从60℃升至95℃过程中，每个循环增加0.5℃，共71个循环。6.2.3.4采用ComparativeDelta-deltaCt法计算定量结果。6.3结果6.3.1提取SGC-7901细胞（过表达LINC00675和NC）中的总RNA，并分析RNA质量，如图7-1所示：图7-1琼脂糖凝胶电泳分离总RNA，发现RNA条带完整，未见杂带和明显的降解。6.3.2mRNA芯片热图，如图7-2，显示50个最大差异表达的mRNAs。98万方数据 第三军医大学博士学位论文.图7-2mRNA芯片显示50个表达差异最显著的mRNAs。6.3.3在SGC-7901细胞中，LINC00675过表达导致大量mRNAs的差异表达，如下表：GeneProbeSetIDLINC00675NClog2FoldchangeestyleSymbolTC07001582.hg.14.3346236.804741-2.4701180.1804764downSTEAP4TC22000288.hg.16.7844239.17612-2.3916970.1905581downH1F0TC06001926.hg.16.6027268.80357-2.2008440.2175104downCGATC12000822.hg.14.4444476.206466-1.7620190.2948353downTCP11L2TC18000179.hg.15.0110326.539734-1.5287020.3465891downMAPK4TC17001711.hg.14.3768025.834957-1.4581550.3639583downMMDTC05000701.hg.14.2032865.652689-1.4494030.3661729downEGR1TC03001767.hg.17.4064258.817649-1.4112240.3759926downH1FXTC03000985.hg.14.8987456.270837-1.3720920.3863306downKLHL2499万方数据 第三军医大学博士学位论文TC11000310.hg.14.5131285.869821-1.3566930.3904763downPRRG4TC13000733.hg.14.2943195.556808-1.2624890.4168242downKLF12TC08000127.hg.16.3743337.630243-1.255910.4187294downSLC7A2TC12001705.hg.14.3099685.558009-1.2480410.4210195downCPMTC0X000112.hg.16.064167.306582-1.2424220.4226625downSAT1TC06000097.hg.16.6764717.916609-1.2401380.4233322downRNF182TC11001793.hg.15.5743336.807576-1.2332430.4253602downSLC43A1TC05001411.hg.14.9711166.186603-1.2154870.4306277downSGTBTC12001033.hg.16.6574877.854473-1.1969860.4361856downFZD10TC12000509.hg.16.7222237.905893-1.183670.4402302downCOQ10ATC11000911.hg.15.9260097.096999-1.170990.4441165downC11orf54TC16000514.hg.16.1108477.278917-1.168070.4450163downCDH5TC02002445.hg.15.7978916.963643-1.1657520.4457319downNR4A2TC02000705.hg.15.40616.564429-1.1583290.4480312downZC3H6TC19000073.hg.16.5829637.739482-1.1565190.4485936downGIPC3TC17001083.hg.16.7751097.926838-1.1517290.4500855downCLDN7TC06001094.hg.14.7897945.934665-1.1448710.4522301downPLEKHG1TC17002908.hg.17.5129198.650629-1.137710.4544804downKRT17TC11002002.hg.17.1220618.247934-1.1258730.4582247downCHKATC15000431.hg.15.0353786.160576-1.1251980.4584391downC15orf65TC06000062.hg.16.7727527.892989-1.1202370.4600182downBMP6TC04001809.hg.16.5969887.713321-1.1163330.4612648downACSL1TC05002066.hg.18.2191069.322261-1.1031550.4654974downDUSP1TC17000965.hg.15.1767316.279543-1.1028120.4656081downFAM101BTC16001115.hg.14.4917465.580983-1.0892370.4700099downTOX3TC15000368.hg.14.3670355.456269-1.0892340.4700109downSEMA6DTC05000958.hg.15.2815896.369076-1.0874870.4705804downCREBRFTC01000757.hg.16.7377017.816339-1.0786380.4734756downCTHTC17001822.hg.17.4884228.566874-1.0784520.4735367downWIPI1TC01001090.hg.17.2420248.320379-1.0783550.4735685downTXNIPTC05001310.hg.14.4379015.515578-1.0776770.4737911downSEPP1TC07001039.hg.17.8009538.870192-1.0692390.4765703downINSIG1TC17000720.hg.13.4336624.495719-1.0620570.4789487downGDPD1100万方数据 第三军医大学博士学位论文TC06000162.hg.16.13417.193843-1.0597430.4797175downHIST1H3ATC09000015.hg.15.1232556.172411-1.0491560.4832508downVLDLRTC12003243.hg.14.4786055.51726-1.0386550.4867811downERBB3TC07001917.hg.15.1727226.207804-1.0350820.4879881downJHDM1DTC07000689.hg.16.1931787.218958-1.025780.4911447downHBP1TC19000805.hg.15.5541066.577788-1.0236820.4918594downERVV-2TC09001325.hg.15.973676.992608-1.0189380.4934795downNFIL3TC01002166.hg.16.8641727.881966-1.0177940.4938709downERRFI1TC02000197.hg.15.7624556.763279-1.0008240.4997145downYPEL5TC06001348.hg.17.4190768.415564-0.9964880.5012186downHIST1H4DTC12001377.hg.15.9563266.942251-0.9859250.5049019downPKP2TC20000067.hg.16.1177137.083094-0.9653810.5121431downBMP2TC03000013.hg.15.808926.772446-0.9635260.5128021downITPR1TC05001714.hg.14.516245.474724-0.9584840.5145974downLOXTC06000541.hg.18.0790919.036372-0.9572810.5150267downPIM1TC06002181.hg.16.0198646.967051-0.9471870.5186427downHIVEP2TC01006343.hg.15.2345296.178977-0.9444480.5196283downRHOUTC12000414.hg.17.4258728.370268-0.9443960.5196471downNR4A1TC01003624.hg.16.4971017.433508-0.9364070.5225326downFAM129ATC22000350.hg.16.3245637.2583-0.9337370.5235006downSEPT3TC06001077.hg.14.8910825.824617-0.9335350.5235739downUSTTC12000159.hg.16.1231857.054955-0.931770.5242148downGABARAPL1TC06001057.hg.15.532826.461506-0.9286860.5253366downSTX11TC08000310.hg.12.8828633.809675-0.9268120.5260194downC8orf4TC14000409.hg.14.572185.494824-0.9226440.5275413downARG2TC12001129.hg.15.2135616.134918-0.9213570.5280121downANO2TC16001019.hg.16.1624377.08268-0.9202430.52842downYPEL3TC11002370.hg.15.2352236.147842-0.9126190.5312199downUSP2TC07001502.hg.15.7974946.708442-0.9109480.5318355downMLXIPLTC13000738.hg.14.7814365.691916-0.910480.5320081downTBC1D4TC16000948.hg.17.2793878.186314-0.9069270.5333199downCDR2TC19001045.hg.18.2501169.155733-0.9056170.5338044downMFSD12TC01003193.hg.18.4754099.380037-0.9046280.5341704downHIST2H2BE101万方数据 第三军医大学博士学位论文TC20000245.hg.16.2905167.189177-0.8986610.5363843downACSS2TC07001618.hg.14.7788125.674709-0.8958970.537413downPDK4TC01000462.hg.15.1154636.007388-0.8919250.5388946downAGO4TC08001659.hg.17.362388.253795-0.8914150.5390851downNDRG1TC05002009.hg.14.9200055.809285-0.889280.5398835downC5orf54TC09001201.hg.16.4163257.304441-0.8881160.5403193downKLF9TC01002980.hg.17.4015448.28959-0.8880460.5403455downDRAM2TC17000481.hg.16.1630347.047506-0.8844720.5416857downGRB7TC12000099.hg.15.6587786.541743-0.8829650.5422519downENO2TC01002342.hg.15.6805316.562692-0.8821610.5425541downTCEA3TC06001510.hg.17.6008778.477515-0.8766380.5446351downIER3TC17000807.hg.17.7506098.626624-0.8760150.5448704downMAP2K6TC06000060.hg.18.3671099.243021-0.8759120.5449093downDSPTC11000924.hg.15.3628346.225049-0.8622150.5501073downAMOTL1TC01000703.hg.14.9816645.839631-0.8579670.5517295downINADLTC0X000721.hg.15.7484526.605254-0.8568020.5521752downMAMLD1TC06000224.hg.16.3173587.166612-0.8492540.5550717downHIST1H4ITC6_qbl_hap6000133.hg.18.1193128.967764-0.8484520.5553803downIER3TC6_cox_hap2000143.hg.18.099778.948221-0.8484510.5553807downIER3TC6_dbb_hap3000132.hg.18.099778.948221-0.8484510.5553807downIER3TC6_mann_hap4000121.hg.18.099778.948221-0.8484510.5553807downIER3TC6_ssto_hap7000123.hg.18.099778.948221-0.8484510.5553807downIER3TC03000127.hg.16.6500697.495454-0.8453850.5565623downNR1D2TC03001590.hg.14.3750785.218504-0.8434260.5573185downPROS1TC02001071.hg.14.4950715.335007-0.8399360.5586684downOSBPL6TC04001751.hg.15.4988286.333567-0.8347390.5606845downFBXO8TC06001089.hg.16.1703666.999869-0.8295030.5627231downULBP1TC07001614.hg.16.6149947.436637-0.8216430.5657972downSGCETC05002062.hg.15.9142586.729856-0.8155980.5681729downSH3PXD2BTC16000062.hg.17.0552947.86566-0.8103660.5702372downRAB26TC17001903.hg.14.8591635.666297-0.8071340.5715161downST6GALNAC2TC06001046.hg.18.5408519.345536-0.8046850.5724871downGPR126TC17000722.hg.14.1364984.941115-0.8046170.572514downYPEL2102万方数据 第三军医大学博士学位论文TC03000015.hg.17.1235297.926587-0.8030580.5731331downBHLHE40TC05000220.hg.14.8958815.698513-0.8026320.5733023downFSTTC18000413.hg.18.0875428.888956-0.8014140.5737865downNPC1TC16000363.hg.15.6190566.420428-0.8013720.5738032downORAI3TC17000794.hg.14.2880515.089422-0.8013710.5738036downPITPNC1TC16000190.hg.16.8594887.660198-0.800710.5740666downC16orf45TC01002608.hg.14.3221045.12166-0.7995560.574526downTESK2TC21000506.hg.18.4888259.287416-0.7985910.5749104downSIK1TC01002452.hg.15.9732086.767543-0.7943350.5766089downFABP3TC17000058.hg.16.8674977.66176-0.7942630.5766377downENO3TC10000449.hg.19.105389.897695-0.7923150.5774168downDDIT4TC12001726.hg.17.7668218.55843-0.7916090.5776994downZFC3H1TC01002954.hg.16.2783857.068417-0.7900320.5783313downSORT1TC09000398.hg.14.348615.136905-0.7882950.579028downDAPK1TC01004026.hg.16.2789027.065077-0.7861750.5798795downOPN3TC11001716.hg.17.3299718.110839-0.7808680.5820165downPHF21ATC10000801.hg.16.8343597.613687-0.7793280.5826381downDUSP5TC01002314.hg.16.4447597.221828-0.7770690.5835511downCAMK2N1TC09001220.hg.14.4192455.195934-0.7766890.5837049downNMRK1TC03000378.hg.15.7266456.501207-0.7745620.5845661downPXKTC03000766.hg.18.5547489.328382-0.7736340.5849422downATP1B3TC17001590.hg.16.7661967.538884-0.7726880.5853259downC1QL1TC10000293.hg.16.0574766.829823-0.7723470.5854643downALOX5TC10000140.hg.16.4452087.217287-0.7720790.585573downARL5BTC02001021.hg.16.1156.886099-0.7710990.5859709downCYBRD1TC01003732.hg.19.1664289.936745-0.7703170.5862886downPPP1R15BTC07001534.hg.15.7398736.509869-0.7699960.5864191downHIP1TC0X001299.hg.17.595038.363783-0.7687530.5869246downCXorf61TC09000916.hg.17.1051567.870789-0.7656330.5881952downNFIBTC01003412.hg.15.3049626.070189-0.7652270.5883608downTSTD1TC08000209.hg.16.280627.042008-0.7613880.5899285downSCARA3TC05000635.hg.16.341067.100639-0.7595790.5906687downSLC22A5TC15000930.hg.16.1853226.944293-0.7589710.5909177downMCTP2103万方数据 第三军医大学博士学位论文TC11001101.hg.15.0836665.841554-0.7578880.5913614downTMEM136TC18000436.hg.13.7056794.458989-0.753310.5932409downDSC2TC16000668.hg.14.8817745.633615-0.7518410.5938453downFOXF1TC05000389.hg.17.4240858.174644-0.7505590.5943732downJMYTC17001459.hg.16.3298877.0789-0.7490130.5950105downNR1D1TC09001223.hg.16.8440017.589096-0.7450950.5966286downRFKTC14000067.hg.14.8481635.591481-0.7433180.5973639downANGTC01003546.hg.16.042666.779514-0.7368540.6000464downKIAA0040TC02000141.hg.15.0041845.739484-0.73530.6006931downEFR3BTC06000168.hg.110.3397911.07254-0.732750.6017558downHIST1H1ETC04001595.hg.14.4886885.21864-0.7299520.602924downRNF150TC16000223.hg.13.6998654.427334-0.7274690.6039625downACSM3TC01001043.hg.16.9213547.647982-0.7266280.6043147downPHGDHTC6_apd_hap1000118.hg.15.9029226.628488-0.7255660.6047597downHLA-DMATC17001251.hg.14.6304425.355509-0.7250670.6049689downULK2TC01002984.hg.13.8483694.572361-0.7239920.6054199downOVGP1TC06001341.hg.15.2525395.975275-0.7227360.6059472downHIST1H4BTC20000088.hg.17.3056588.027287-0.7216290.6064123downBTBD3TC03000636.hg.17.4640728.185377-0.7213050.6065485downDIRC2TC03000087.hg.17.6014748.318882-0.7174080.6081892downSLC6A6TC01002590.hg.18.0292848.744711-0.7154270.6090248downSLC6A9TC02002830.hg.14.4830785.198186-0.7151080.6091595downIRS1TC03003324.hg.18.1059428.820171-0.7142290.6095308downIL20RBTC01006316.hg.15.2500185.963815-0.7137970.6097133downZBTB8ATC05000667.hg.16.1505786.86367-0.7130920.6100114downCAMLGTC09002921.hg.15.7908796.499542-0.7086630.6118869downST6GALNAC6TC14000419.hg.18.1464178.855029-0.7086120.6119086downSMOC1TC05001673.hg.15.9759276.682834-0.7069070.6126322downSTARD4TC12001783.hg.16.7017027.407949-0.7062470.6129125downSLC6A15TC20000009.hg.17.1879917.893479-0.7054880.613235downTRIB3TC6_dbb_hap3000210.hg.15.9005856.598356-0.6977710.616524downHLA-DMATC17001647.hg.17.0000177.69625-0.6962330.6171816downHOXB9TC18000103.hg.14.8877635.583579-0.6958160.61736downLAMA3104万方数据 第三军医大学博士学位论文TC09001012.hg.17.5777188.26927-0.6915520.6191874downAQP3TC06000978.hg.14.4276755.118203-0.6905280.619627downL3MBTL3TC11001122.hg.14.7617545.450264-0.688510.6204944downTC03001720.hg.16.3649197.051757-0.6868380.6212139downHEG1TC12002000.hg.17.0364157.722667-0.6862520.6214663downTBX3TC18000477.hg.13.5788784.264613-0.6857350.621689downPSTPIP2TC16001294.hg.17.6602088.34585-0.6856420.6217291downCDYL2TC01002763.hg.16.4421427.127591-0.6854490.6218123downWLSTC15000435.hg.17.401368.080782-0.6794220.6244154downTCF12TC21000487.hg.16.1666376.844618-0.6779810.6250394downTMPRSS3TC19001573.hg.16.271056.947905-0.6768550.6255274downLIPETC12001040.hg.14.5581055.234901-0.6767960.625553downGPR133TC05001297.hg.17.3313068.005472-0.6741660.6266944downTTC33TC11001881.hg.17.2091677.88275-0.6735830.6269477downSTX5TC02000762.hg.14.0099964.680959-0.6709630.6280873downINHBBTC11000066.hg.16.0521876.722748-0.6705610.6282623downKCNQ1TC17001930.hg.17.5706298.239338-0.6687090.6290694downCBX4TC12001755.hg.17.0605227.729124-0.6686020.629116downCSRP2TC19001103.hg.19.2234699.888607-0.6651380.6306284downC3TC06001035.hg.15.7340036.396627-0.6626240.6317283downHECATC17001085.hg.16.6137257.275805-0.662080.6319665downYBX2TC06001351.hg.12.7054223.366426-0.6610040.632438downHIST1H1DTC16001134.hg.16.6351927.295939-0.6607470.6325507downBBS2TC11000174.hg.16.7736037.434202-0.6605990.6326156downWEE1TC19000075.hg.15.8966556.555562-0.6589070.633358downTJP3TC19001181.hg.16.2509966.902191-0.6511950.6367527downRAB3DTC04000072.hg.15.6159396.264912-0.6489730.6377341downS100PTC17001722.hg.17.7016428.350509-0.6488670.637781downVEZF1TC19000187.hg.19.92913210.57789-0.6487580.6378292downC19orf38TC16000857.hg.18.194468.842402-0.6479420.63819downEMP2TC01006324.hg.14.8896235.537369-0.6477460.6382767downTMEM56TC17001572.hg.16.7764767.420415-0.6439390.6399633downHDAC5TC11001458.hg.13.881114.52362-0.642510.6405975downUSH1C105万方数据 第三军医大学博士学位论文TC09000988.hg.15.2387015.880032-0.6413310.6411212downMOB3BTC14001209.hg.16.1363896.777019-0.640630.6414328downSGPP1TC06000163.hg.16.2596976.898987-0.639290.6420288downHIST1H4ATC01001077.hg.16.7600147.398626-0.6386120.6423306downLOC101060698TC06004081.hg.15.6477936.285696-0.6379030.6426464downSTK19TC12001467.hg.14.9815165.618441-0.6369250.6430822downRHEBL1TC16000989.hg.17.5518048.188459-0.6366550.6432025downNUPR1TC19000228.hg.17.3474427.982935-0.6354930.6437208downJUNBTC16001137.hg.17.090417.723375-0.6329650.6448498downPLLPTC12000521.hg.16.7131267.344063-0.6309370.6457569downLRP1TC01002245.hg.16.9125147.541734-0.629220.6465259downCASP9TC04001491.hg.13.3319023.961031-0.6291290.6465666downTRAM1L1TC0X001074.hg.14.9057625.533168-0.6274060.6473393downFGD1TC17000764.hg.18.0077718.634357-0.6265860.6477073downTANC2TC09001281.hg.13.8563384.480413-0.6240750.6488357downGKAP1TC06001401.hg.15.2612255.885235-0.624010.6488649downZNF184TC14001441.hg.13.8425254.465887-0.6233620.6491564downTC2NTC6_apd_hap1000078.hg.18.5209389.144094-0.6231560.6492491downFLOT1TC17000074.hg.15.4403826.06103-0.6206480.6503787downC17orf100TC06001126.hg.18.2237838.844378-0.6205950.6504026downSNX9TC18000213.hg.16.1523986.770735-0.6183370.6514214downPMAIP1TC6_dbb_hap3000077.hg.16.1537456.769871-0.6161260.6524205downSTK19TC6_mcf_hap5000070.hg.16.1537456.769871-0.6161260.6524205downSTK19TC10000798.hg.16.106296.721931-0.6156410.6526399downMXI1TC18000047.hg.19.0572419.671648-0.6144070.6531983downRAB31TC16000771.hg.17.6587188.272768-0.614050.65336downZNF598TC18000252.hg.15.578816.190053-0.6112430.6546324downTSHZ1TC02001350.hg.17.9831328.59407-0.6109380.6547708downACSL3TC01003497.hg.17.3181497.92754-0.6093910.6554733downSLC19A2TC16000851.hg.16.5578047.166691-0.6088870.6557024downCARHSP1TC02001613.hg.14.1360614.744618-0.6085570.6558524downTTC32TC6_cox_hap2000233.hg.15.6253136.233847-0.6085340.6558628downHLA-DMATC6_mann_hap4000210.hg.15.6253136.233847-0.6085340.6558628downHLA-DMA106万方数据 第三军医大学博士学位论文TC6_mcf_hap5000231.hg.15.6253136.233847-0.6085340.6558628downHLA-DMATC6_qbl_hap6000237.hg.15.6253136.233847-0.6085340.6558628downHLA-DMATC6_ssto_hap7000220.hg.15.6253136.233847-0.6085340.6558628downHLA-DMATC09000264.hg.14.9623965.570574-0.6081780.6560247downLOC100132167TC01006333.hg.16.3431446.95062-0.6074760.656344downEFNA3TC0X001103.hg.15.3094295.916873-0.6074440.6563585downOPHN1TC14000332.hg.15.5386936.144106-0.6054130.6572832downPELI2TC04001554.hg.15.9259486.531353-0.6054050.6572868downPABPC4LTC17002850.hg.16.0212796.626561-0.6052820.6573429downVAMP2TC07001227.hg.15.409836.01499-0.605160.6573985downHOXA13TC06000758.hg.14.3344774.939252-0.6047750.6575739downSH3BGRL2TC16000461.hg.15.5980486.201706-0.6036580.6580832downGNAO1TC11000017.hg.17.7388838.342505-0.6036220.6580997downPTDSS2TC01002163.hg.13.8878474.490353-0.6025060.6586089downTNFRSF9TC02000219.hg.13.8360944.437433-0.6013390.6591419downLTBP1TC09001643.hg.16.4559317.056342-0.6004110.659566downCRATTC15000664.hg.15.7973266.397732-0.6004060.6595683downNEO1TC04001830.hg.13.7802544.380084-0.599830.6598317downTRIML2TC15000339.hg.17.0822497.681511-0.5992620.6600915downCTDSPL2TC20000395.hg.14.8867855.485936-0.5991510.6601423downSNAI1TC07003320.hg.15.8973996.495965-0.5985660.6604101downGLCCI1TC16000480.hg.18.4505399.048489-0.597950.6606921downHERPUD1TC01003663.hg.15.5958066.192547-0.5967410.661246downZBTB41TC05001601.hg.13.9437144.540139-0.5964250.6613909downKIAA0825TC06000770.hg.14.9785375.574962-0.5964250.6613909downRWDD2ATC05001031.hg.15.6962116.291984-0.5957730.6616898downN4BP3TC15002769.hg.17.5271218.122791-0.595670.6617371downCHD2TC02001948.hg.19.56208110.15735-0.5952690.661921downGFPT1TC03000803.hg.18.5281939.123108-0.5949150.6620835downHPS3TC11002366.hg.18.1778128.772416-0.5946040.6622262downMCAMTC6_ssto_hap7000120.hg.17.6523048.246002-0.5936980.6626422downPPP1R18TC01000385.hg.16.6601197.253588-0.5934690.6627474downRAB42TC17001029.hg.15.9868316.579857-0.5930260.6629509downCXCL16107万方数据 第三军医大学博士学位论文TC18000517.hg.16.6019197.1944-0.5924810.6632014downMEX3CTC06000010.hg.16.890417.48266-0.592250.6633076downFOXF2TC19000538.hg.17.5240048.114371-0.5903670.6641739downNCCRP1TC04000629.hg.13.7567634.34697-0.5902070.6642476downBBS12TC06001353.hg.18.5105729.100204-0.5896320.6645124downHIST1H3FTC09001475.hg.15.2455945.834574-0.588980.6648128downSVEP1TC02000374.hg.16.9444317.532658-0.5882270.6651599downSLC1A4TC05000384.hg.16.5538017.141583-0.5877820.6653651downSCAMP1TC11001407.hg.15.9236646.509964-0.58630.6660489downSBF2TC08000200.hg.18.8739749.459011-0.5850370.6666322downBNIP3LTC03003352.hg.16.4515755.8662890.5852861.5003364upRBM5TC01000463.hg.17.4062346.8209450.5852891.5003395upAGO1TC19000557.hg.19.663749.0783480.5853921.5004466upPSMC4TC19001218.hg.18.0314247.4450430.5863811.5014756upTRMT1TC15001703.hg.16.4694525.8829760.5864761.5015744upWDR61TC16000315.hg.18.6149658.027740.5872251.5023542upTC16002100.hg.110.8119610.224690.587271.5024011upNPIPB11TC11001364.hg.16.439625.8523440.5872761.5024073upMRPL17TC09001726.hg.17.4384546.8505460.5879081.5030656upQSOX2TC6_apd_hap1000029.hg.110.547759.959660.588091.5032553upTUBBTC6_dbb_hap3000038.hg.110.547759.959660.588091.5032553upTUBBTC6_mann_hap4000040.hg.110.547759.959660.588091.5032553upTUBBTC6_mcf_hap5000032.hg.110.547759.959660.588091.5032553upTUBBTC6_qbl_hap6000038.hg.110.547759.959660.588091.5032553upTUBBTC6_ssto_hap7000038.hg.110.547759.959660.588091.5032553upTUBBTC15001642.hg.15.3455024.7573140.5881881.5033574upCYP11A1TC17000350.hg.17.0459646.4572730.5886911.5038816upGOSR1TC19001215.hg.19.5834368.9944750.5889611.5041631upFARSATC03003371.hg.18.5935488.0045490.5889991.5042027upFAM157ATC18001001.hg.16.9367316.3472350.5894961.504721upSERPINB8TC04002917.hg.15.5656984.9756510.5900471.5052958upTLR6TC02001439.hg.16.0555235.4649210.5906021.505875upCOPS8TC05000010.hg.18.8526778.2619450.5907321.5060107upTRIP13108万方数据 第三军医大学博士学位论文TC01001518.hg.15.5687264.9779040.5908221.5061046upZBTB37TC09001674.hg.16.8200296.2291360.5908931.5061788upTTF1TC19000431.hg.16.650086.0578940.5921861.5075293upC19orf40TC11002449.hg.16.3628635.7703020.5925611.5079212upPRDM10TC09000962.hg.13.5195982.9257550.5938431.5092617upIFNETC09001671.hg.17.6009267.0069070.5940191.5094458upMED27TC19000427.hg.18.1908297.5963630.5944661.5099136upPDCD5TC02002317.hg.14.4768963.8822620.5946341.5100894upPOTEFTC07001843.hg.16.4754585.8793110.5961471.5116739upMGC27345TC11001294.hg.15.3924054.7950560.5973491.5129339upZNF195TC05003404.hg.17.2666756.6685870.5980881.5137091upBRD9TC16000568.hg.18.1957827.5960690.5997131.5154151upNIP7TC01001093.hg.16.9852056.385330.5998751.5155852upRBM8ATC14001267.hg.17.2842286.6839540.6002741.5160045upSYNJ2BP-COX16TC05000612.hg.17.0345696.433740.6008291.5165878upSLC12A2TC02002339.hg.14.1942873.5919780.6023091.5181444upPOTEITC19000937.hg.17.6044497.0013270.6031221.5190001upZNF460TC02002522.hg.15.6604765.0568820.6035941.5194972upMETTL8TC02002123.hg.17.8725497.2682070.6043421.5202852upTMEM131TC03001146.hg.17.3539476.7496050.6043421.5202852upRAD18TC01006289.hg.18.3224537.7180680.6043851.5203305upSFT2D2TC22000580.hg.16.3274665.722560.6049061.5208797upGSTT2TC20000871.hg.18.8352398.2301830.6050561.5210378upTOMM34TC06001742.hg.17.4178486.8125060.6053421.5213394upCUL7TC14000351.hg.15.9527635.3471750.6055881.5215988upKIAA0586TC12002084.hg.17.3595436.7538240.6057191.521737upMPHOSPH9TC03001465.hg.16.7358166.1295430.6062731.5223214upRFT1TC12001139.hg.18.9996268.3932210.6064051.5224607upMRPL51TC13000077.hg.18.04117.434690.606411.522466upGTF3ATC16002034.hg.19.5080258.901340.6066851.5227562upMT2ATC03001100.hg.18.3993917.7926980.6066931.5227647upPAK2TC07000786.hg.18.6271558.0203230.6068321.5229114upATP6V1FTC02001108.hg.18.2350427.6280870.6069551.5230412upWDR75109万方数据 第三军医大学博士学位论文TC11003431.hg.17.6384247.030120.6083041.524466upDPP3TC22000829.hg.17.7590977.1496870.609411.5256352upDESI1TC17001111.hg.16.6235216.0136630.6098581.526109upCTC1TC0X000470.hg.17.749837.1399430.6098871.5261397upCSTF2TC16000599.hg.16.8219546.211950.6100041.5262634upDHODHTC20001742.hg.17.14146.530640.610761.5270634upNSFL1CTC01000344.hg.17.5668036.9522840.6145191.5310475upDHDDSTC06004084.hg.17.0830316.4681750.6148561.5314051upNFYATC01001690.hg.14.0061323.3909680.6151641.5317321upLINC00260TC09000247.hg.16.3472115.7318120.6153991.5319816upLOC101060578TC16000920.hg.18.4239887.8085540.6154341.5320188upKNOP1TC17001893.hg.17.1615326.5449770.6165551.5332097upEXOC7TC06000684.hg.18.6528738.0352220.6176511.5343749upBAG2TC12000012.hg.16.3635455.7452740.6182711.5350344upWNK1TC15001786.hg.17.327686.7093050.6183751.5351451upWDR73TC01003008.hg.16.896066.277170.618891.5356932upAP4B1TC09001013.hg.17.542256.9233550.6188951.5356985upNOL6TC03001067.hg.16.8723066.2527840.6195221.5363661upOPA1TC09001539.hg.18.8660568.2463780.6196781.5365322upPSMD5TC10001739.hg.15.9223045.3017960.6205081.5374164upUROSTC01002498.hg.17.6135756.9918120.6217631.5387544upPSMB2TC17002860.hg.110.7926710.170850.621821.5388152upKPNA2TC20000473.hg.16.8175916.1957650.6218261.5388216upFAM217BTC01002769.hg.17.0462046.4217510.6244531.5416262upLRRC40TC07002034.hg.18.9568748.3319230.6249511.5421584upABCF2TC17000073.hg.16.2661595.6411030.6250561.5422707upTXNDC17TC18000088.hg.18.3560677.7302520.6258151.5430823upSNRPD1TC02002008.hg.15.400884.7746380.6262421.5435391upGCFC2TC07001530.hg.18.4566637.8296680.6269951.5443449upPOM121CTC03001731.hg.17.8391337.2119470.6271861.5445494upALG1LTC14000946.hg.18.2061397.5783590.627781.5451855upPSMB5TC01002096.hg.17.8025837.1747570.6278261.5452347upMRPL20TC08000846.hg.17.0639526.4355080.6284441.5458968upFAM203A110万方数据 第三军医大学博士学位论文TC07000430.hg.18.4093377.7805740.6287631.5462386upPOM121TC07003323.hg.16.998386.3695640.6288161.5462954upEPDR1TC12000029.hg.16.5647815.935330.6294511.5469762upITFG2TC20000289.hg.16.4338635.8035560.6303071.5478943upACTR5TC15001576.hg.17.1440596.5137390.630321.5479083upTIPINTC01002455.hg.16.3475675.7161850.6313821.5490482upPEF1TC01002660.hg.18.3459477.7141360.6318111.5495089upSELRC1TC06001130.hg.14.8580064.2259880.6320181.5497312upGTF2H5TC22000334.hg.17.057376.4253470.6320231.5497366upL3MBTL2TC01006322.hg.17.0012086.3677520.6334561.5512767upTTC4TC11000325.hg.18.9921778.3584050.6337721.5516165upNAT10TC10000340.hg.19.1295478.4955710.6339761.5518359upAGAP6TC03000051.hg.17.3360626.701390.6346721.5525847upFANCD2TC18000220.hg.16.0306815.3956660.6350151.5529539upKIAA1468TC04001686.hg.18.3808737.745450.6354231.5533931upPPIDTC01002500.hg.17.9040197.2683360.6356831.5536731upCLSPNTC16000468.hg.18.9475858.3116030.6359821.5539951upMT1ETC04001171.hg.16.622395.9861780.6362121.5542429upNFXL1TC16000564.hg.19.9964449.3594260.6370181.5551115upCIRH1ATC09000613.hg.17.6231026.9858820.637221.5553292upMRRFTC17001017.hg.17.1966616.5590690.6375921.5557303upZZEF1TC10001349.hg.17.2571896.6195550.6376341.5557756upSLC25A16TC03001702.hg.16.3752875.7373850.6379021.5560646upCCDC58TC02001386.hg.17.7977987.1596540.6381441.5563257upPSMD1TC0X001214.hg.17.8712087.2320260.6391821.5574458upTRMT2BTC0X000255.hg.17.2377126.5985050.6392071.5574728upRBM3TC06000347.hg.111.4895810.848050.641531.5599827upTUBBTC19001350.hg.15.8927765.2510620.6417141.5601816upZNF708TC01003607.hg.14.2171253.5753190.6418061.5602811upRNASELTC19000067.hg.18.1550087.5127380.642271.560783upNCLNTC11000662.hg.14.0047583.3619080.642851.5614106upTC05000400.hg.17.2231516.5795120.6436391.5622648upMSH3TC08000553.hg.16.4406855.7957940.6448911.5636211upOTUD6B111万方数据 第三军医大学博士学位论文TC12001142.hg.19.1427228.4971130.6456091.5643995upNOP2TC10001347.hg.16.6613026.0140280.6472741.566206upDNA2TC08000213.hg.18.5346687.8873290.6473391.5662766upESCO2TC02002859.hg.19.2032788.5548970.6483811.5674083upNCLTC03000429.hg.14.9399224.2909230.6489991.5680798upEBLN2TC16001088.hg.18.2451767.5959470.6492291.5683298upNETO2TC17000562.hg.18.2801687.6306220.6495461.5686745upG6PC3TC07003353.hg.18.1355227.4855360.6499861.569153upPSMA2TC12003287.hg.17.5764776.9264020.6500751.5692498upVPS33ATC16001004.hg.110.048639.3973330.6512971.5705795upNPIPL3TC19000699.hg.110.78910.13690.65211.571454upEMP3TC01000125.hg.18.1525487.5003120.6522361.5716021upUBE4BTC11002071.hg.16.7078946.0548710.6530231.5724597upCOA4TC17000098.hg.19.6830319.0299570.6530741.5725152upPOLR2ATC15001276.hg.16.3559875.7028250.6531621.5726112upLCMT2TC08000382.hg.17.2858316.6316970.6541341.573671upTGS1TC0X001419.hg.18.9410228.2864830.6545391.5741129upRBMXTC11000970.hg.17.945067.2904110.6546491.5742329upAASDHPPTTC12000890.hg.17.8651287.2101880.654941.5745505upTPCN1TC11000530.hg.16.5986565.9433580.6552981.5749412upTMEM138TC07000485.hg.18.8897738.233970.6558031.5754926upHSPB1TC14002203.hg.19.9697629.3136320.656131.5758498upSLIRPTC17001306.hg.17.4218186.7627480.659071.5790644upTLCD1TC09000929.hg.13.2628282.6034710.6593571.5793785upMGC24103TC18000003.hg.19.0156068.3551030.6605031.5806336upUSP14TC22001472.hg.17.1266476.4661030.6605441.5806785upADSLTC12002049.hg.16.9147856.2537160.6610691.5812539upPOP5TC02001177.hg.16.9701456.3088080.6613371.5815476upCASP8TC09000573.hg.18.7391688.0776440.6615241.5817526upPRPF4TC16000524.hg.17.5494486.8875180.661931.5821978upCES2TC12001240.hg.13.5862542.9233270.6629271.5832916upTAS2R46TC17000633.hg.18.2383067.5752090.6630971.5834782upATP5G1TC01003867.hg.15.535244.8711760.6640641.5845399upTAF1A112万方数据 第三军医大学博士学位论文TC11001557.hg.17.7038197.038720.6650991.5856771upCOMMD9TC01001112.hg.16.7631416.0948230.6683181.5892191upCHD1LTC05000334.hg.17.5977726.9290450.6687271.5896697upMCCC2TC14000797.hg.18.7308938.0617230.669171.5901579upEIF5TC08002603.hg.15.9414435.2718860.6695571.5905845upRRS1TC21001067.hg.15.2470654.5772490.6698161.5908701upTPTETC10001228.hg.18.5798937.9100360.6698571.5909153upAGAP4TC11002058.hg.16.7440846.0724380.6716461.5928893upCLPBTC12000991.hg.17.3005826.6256550.6749271.596516upGTF2H3TC01002603.hg.16.9874266.311540.6758861.5975776upEIF2B3TC18000270.hg.17.936767.2606940.6760661.5977769upTC10001281.hg.19.2347018.5561810.678521.600497upAGAP7TC22000922.hg.16.2704835.5912910.6791921.6012427upRABL2BTC06000114.hg.17.2599456.5806430.6793021.6013648upCAP2TC17000052.hg.19.0226218.343060.6795611.6016523upPSMB6TC02001069.hg.14.3347443.6546310.6801131.6022652upRBM45TC03000586.hg.16.8881816.2061570.6820241.604389upQTRTD1TC16002072.hg.17.9125257.2300820.6824431.6048551upSRCAPTC19002645.hg.17.4021416.7186770.6834641.6059912upGRWD1TC01003344.hg.17.4778386.7939930.6838451.6064154upGPATCH4TC13000539.hg.19.0770338.3930040.6840291.6066203upHSPH1TC06000605.hg.17.3073466.6221540.6851921.607916upPOLHTC06000530.hg.18.7441958.055280.6889151.6120707upSRSF3TC09001591.hg.17.5113836.8221210.6892621.6124585upMAPKAP1TC01000031.hg.17.1659016.4761720.6897291.6129805upB3GALT6TC07000734.hg.15.0319854.3414220.6905631.6139132upING3TC20000333.hg.16.8254926.1332860.6922061.6157522upTTPALTC12001238.hg.13.6776552.984220.6934351.6171293upTAS2R19TC10001545.hg.16.6082535.9145010.6937521.6174846upNOC3LTC09001629.hg.18.1534427.4587480.6946941.6185411upTRUB2TC08001743.hg.17.6343116.9391060.6952051.6191145upBOP1TC17000967.hg.17.4440166.7486080.6954081.6193423upGEMIN4TC12003277.hg.18.0088267.3124370.6963891.6204438upKMT2D113万方数据 第三军医大学博士学位论文TC17002873.hg.17.2102796.5136760.6966031.6206842upTMEM199TC17000048.hg.17.005946.3089540.6969861.6211145upMED11TC17000849.hg.18.9477628.2507370.6970251.6211583upMRPS7TC01003337.hg.110.152599.4510870.7015031.6261981upCCT3TC12003241.hg.19.132998.4287010.7042891.6293415upPRR13TC16000837.hg.19.1876038.4816610.7059421.6312094upGLYR1TC06000966.hg.16.5091025.8028810.7062211.6315249upCENPWTC16001020.hg.17.035326.3284780.7068421.6322273upGDPD3TC11000981.hg.17.9722797.2623310.7099481.6357452upDDX10TC09001733.hg.14.643293.9327750.7105151.6363882upSEC16ATC09001218.hg.17.4721626.7608490.7113131.6372935upC9orf41TC02000816.hg.14.2799543.5671630.7127911.6389718upPOTEJTC14000491.hg.18.4590967.7445820.7145141.6409303upAHSA1TC07000110.hg.18.0424157.3273610.7150541.6415447upBZW2TC10001240.hg.17.8763017.1611330.7151681.6416744upAGAP9TC10001416.hg.18.0643577.3483130.7160441.6426715upAGAP5TC17000991.hg.18.7779888.0619280.716061.6426897upTSR1TC0X001491.hg.17.4995936.7817440.7178491.644728upGABRETC01006304.hg.18.4650417.7455850.7194561.646561upGBATC12001635.hg.16.0768855.3546370.7222481.6497507upMETTL1TC16002092.hg.15.3400544.6137070.7263471.6544446upFLJ00104TC01000719.hg.14.732024.0040040.7280161.6563597upALG6TC10000312.hg.18.0413577.3128070.728551.6569729upAGAP9TC12001239.hg.14.9191484.1849390.7342091.6634852upTAS2R31TC14002298.hg.15.7721075.0371950.7349121.6642959upSLC38A6TC17000771.hg.18.6683367.9331580.7351781.6646028upDDX42TC12000946.hg.17.6659316.9298570.7360741.665637upDYNLL1TC05001171.hg.14.3934583.6558250.7376331.6674379upDNAH5TC02001866.hg.18.4382097.700120.7380891.667965upPNPT1TC0X001113.hg.18.3512027.6129220.738281.6681858upPDZD11TC02000608.hg.16.2193575.479810.7395471.6696515upPDCL3TC02000443.hg.16.4399195.7003480.7395711.6696793upALMS1TC12001993.hg.17.4424126.7026850.7397271.6698598upRBM19114万方数据 第三军医大学博士学位论文TC02001807.hg.17.2482366.5073770.7408591.6711706upSRBD1TC01002892.hg.16.747156.0059170.7412331.6716039upALG14TC17000104.hg.18.4737517.7320.7417511.6722042upMPDU1TC01006364.hg.18.1596867.4161790.7435071.6742408upCLCC1TC10000715.hg.19.0202528.2755680.7446841.6756072upABCC2TC17000007.hg.18.9268138.1817550.7450581.6760416upTIMM22TC0X000417.hg.16.9079746.1626510.7453231.6763495upPBDC1TC03001169.hg.16.9571816.211360.7458211.6769283upTIMP4TC01002154.hg.16.9303676.1816680.7486991.6802769upNOL9TC08000648.hg.16.5279015.77910.7488011.6803957upDCAF13TC12000932.hg.16.7695916.0199110.749681.6814198upHSPB8TC01001605.hg.14.21253.461390.751111.6830873upOCLMTC01000245.hg.17.3500776.5988340.7512431.6832425upMRTO4TC17001147.hg.18.2883217.5362650.7520561.6841913upELAC2TC05001149.hg.16.416295.6628380.7534521.6858217upFASTKD3TC16000225.hg.15.3511634.5928010.7583621.691569upLOC81691TC17001524.hg.18.3796727.6204860.7591861.6925354upKAT2ATC04000268.hg.15.9706955.211270.7594251.6928158upGUF1TC13000453.hg.16.529795.7685350.7612551.6949644upPSPC1TC15000270.hg.17.0437466.2812950.7624511.6963701upTHBS1TC15000971.hg.16.8988216.1351380.7636831.6978194upALDH1A3TC10000726.hg.15.6512424.8861370.7651051.6994937upFAM178ATC11002054.hg.16.8279536.0627610.7651921.6995962upANAPC15TC06004137.hg.16.7808736.011950.7689231.7039972upARHGAP18TC17000376.hg.19.4323548.6626340.769721.7049389upPSMD11TC05000184.hg.15.6345174.8647950.7697221.7049412upPTGER4TC06001226.hg.16.4066665.6363040.7703621.7056977upTUBB2ATC14001526.hg.19.8988589.127740.7711181.7065918upHSP90AA1TC17001813.hg.18.3189287.5462520.7726761.7084358upPSMD12TC11002268.hg.17.882767.1082860.7744741.7105663upKDELC2TC16002065.hg.19.8679149.0931190.7747951.7109469upNPIPB11TC11000332.hg.19.1034878.3240950.7793921.7164074upCD44TC09001088.hg.16.4068465.6266490.7801971.7173654upANKRD18A115万方数据 第三军医大学博士学位论文TC14001346.hg.17.9864017.2057290.7806721.7179309upVIPAS39TC09000318.hg.16.1258155.3441420.7816731.7191233upFXNTC01000448.hg.16.9873266.2049120.7824141.7200065upGJB5TC17000617.hg.17.9123357.1272130.7851221.723238upPNPOTC11000201.hg.17.4792356.6940720.7851631.723287upPARVATC02004994.hg.13.8656443.0794680.7861761.7244975upIL1RL1TC13000111.hg.15.948035.1611460.7868841.725344upBRCA2TC16000965.hg.16.3690975.580860.7882371.7269628upEARS2TC0X000322.hg.18.2874427.4976180.7898241.7288635upGNL3LTC17000474.hg.19.0542218.2634060.7908151.7300515upCDK12TC11000742.hg.17.4099546.6160970.7938571.7337033upFAM86C1TC15001189.hg.15.4237494.6286440.7951051.7352037upAVENTC01001790.hg.17.8019467.0065280.7954181.7355802upSMYD2TC10000773.hg.17.4806886.6852210.7954671.7356391upPDCD11TC02002256.hg.17.652726.8570270.7956931.735911upTMEM185BTC01001475.hg.18.1992917.4008570.7984341.7392122upBLZF1TC05001974.hg.17.8533687.0548770.7984911.739281upGEMIN5TC17001374.hg.16.5728665.7717470.8011191.7424521upPEX12TC05001342.hg.15.7803724.9781180.8022541.7438235upMOCS2TC18000180.hg.17.6759966.8726380.8033581.7451584upME2TC04001079.hg.16.6640115.8562330.8077781.7505133upGPR125TC19000076.hg.15.6171694.8057790.811391.7549014upMRPL54TC14000346.hg.17.6955886.8831330.8124551.7561974upPSMA3TC14000309.hg.17.1917456.3776820.8140631.7581559upPSMC6TC05000354.hg.17.1649086.3487590.8161491.7606999upUTP15TC12001880.hg.16.977536.1609050.8166251.7612809upGNPTABTC06004099.hg.18.4866297.6694150.8172141.7620001upLTV1TC17000103.hg.18.5849477.7657980.8191491.7643649upCD68TC03001695.hg.17.2931346.4709970.8221371.7680229upPOLQTC02001729.hg.17.3435916.5211350.8224561.7684139upFAM98ATC01000494.hg.16.5866225.7639030.8227191.7687363upKIAA0754TC07001570.hg.15.9092885.0833750.8259131.7726565upSEMA3DTC08001051.hg.17.3611626.5329330.8282291.7755045upTNFRSF10D116万方数据 第三军医大学博士学位论文TC17000437.hg.17.1744096.3452730.8291361.7766211upPIGWTC06001777.hg.17.7548186.922070.8327481.7810747upTNFRSF21TC11001723.hg.18.2242237.3848540.8393691.7892674upARHGAP1TC03001854.hg.16.9463416.1061630.8401781.790271upATRTC10000771.hg.18.0517467.208830.8429161.7936719upINATC0X001176.hg.17.5083226.6639350.8443871.7955017upTAF9BTC02000105.hg.17.0724476.2253560.8470911.7988701upGEN1TC02000435.hg.17.5767586.7250010.8517571.8046975upSPRTC11001115.hg.15.4240724.5718890.8521831.8052304upUBASH3BTC03001604.hg.17.3286516.4762060.8524451.8055583upDCBLD2TC05000159.hg.14.5282673.6738270.854441.8080568upIL7RTC01001957.hg.16.4680695.6129250.8551441.8089393upRYR2TC12000452.hg.15.5537494.6981610.8555881.8094961upHOXC8TC10000185.hg.16.8693626.0105970.8587651.8134852upPDSS1TC07000643.hg.17.803616.940010.86361.8195731upSERPINE1TC16001283.hg.17.2600416.3954490.8645921.8208247upADAT1TC03002137.hg.17.9818387.1145380.86731.8242456upLSG1TC05002092.hg.17.1521226.2839370.8681851.825365upNOP16TC20000896.hg.17.9630167.094340.8686761.8259864upNCOA5TC20000316.hg.18.3166287.4459510.8706771.8285208upSRSF6TC03000175.hg.16.7376625.8669630.8706991.8285486upMLH1TC09000874.hg.17.8168796.9451930.8716861.8298upKIAA0020TC07001844.hg.17.837346.9653250.8720151.8302174upRBM28TC01000346.hg.18.7534487.8795080.873941.8326611upHMGN2TC02000470.hg.16.6582845.7811780.8771061.8366873upPOLE4TC02001757.hg.17.9307957.0492810.8815141.8423077upSRSF7TC12000606.hg.18.3335577.448920.8846371.8463upMDM2TC17001037.hg.16.8695695.9834870.8860821.8481502upSPAG7TC02005004.hg.16.7046415.8176840.8869571.8492714upHSPE1TC09000186.hg.16.3934125.5019230.8914891.8550898upRPL36AP33TC01003044.hg.17.1592396.2675780.8916611.8553109upWARS2TC16000139.hg.18.1065147.2130970.8934171.8575705upALG1TC17002884.hg.17.576976.6825180.8944521.8589037upTAX1BP3117万方数据 第三军医大学博士学位论文TC09001675.hg.17.0842766.1883710.8959051.8607768upDDX31TC08000245.hg.16.6246565.7283350.8963211.8613134upUBXN8TC01001516.hg.18.0682447.1619340.906311.8742456upDARS2TC02000215.hg.16.5510655.6434340.9076311.8759625upTTC27TC02002051.hg.17.9152787.005710.9095681.8784829upPOLR1ATC12000487.hg.17.9508667.0405610.9103051.8794428upMETTL7BTC07001910.hg.18.0366847.1238520.9128321.8827377upZC3HAV1TC17001786.hg.18.4507497.5375360.9132131.883235upFTSJ3TC01002985.hg.18.3061087.3893330.9167751.8878904upWDR77TC07000329.hg.16.9152765.9979950.9172811.8885526upLANCL2TC02002823.hg.15.3401674.4125960.9275711.9020709upSERPINE2TC03001202.hg.16.5562565.6222950.9339611.9105142upMRPS25TC07000874.hg.13.9389553.0046050.934351.9110294upPP12708TC11001911.hg.18.4386827.5036130.9350691.9119821upSF1TC18000277.hg.17.3810466.4381130.9429331.9224326upENOSF1TC05001157.hg.17.1650146.2206550.9443591.9243337upFAM173BTC01001804.hg.17.7287156.7817680.9469471.9277888upRRP15TC16000372.hg.18.4925987.5446350.9479631.9291469upFUSTC14002324.hg.16.9000525.9480920.951961.934499upGPR135TC01001026.hg.17.5779096.6247640.9531451.9360886upTTF2TC02001789.hg.16.7868365.8274240.9594121.9445172upTHADATC19000426.hg.15.7740654.8038920.9701731.9590755upDPY19L3TC09000844.hg.110.725579.7291080.9964621.9951013upTUBB4BTC03000507.hg.17.5760146.5759531.0000612.0000846upNIT2TC11001000.hg.16.9607525.9594581.0012942.0017947upC11orf1TC19000906.hg.19.642448.638071.004372.0060673upU2AF2TC16001302.hg.17.5016196.4957741.0058452.0081193upMPHOSPH6TC12001566.hg.19.2847338.2646621.0200712.0280188upCBX5TC15001592.hg.18.1717227.1461141.0256082.0358172upCLN6TC01001582.hg.19.2089668.1771671.0317992.0445722upDHX9TC11002074.hg.17.560866.5231581.0377022.052955upUCP2TC01006365.hg.19.2128478.1673391.0455082.064093upBCAS2TC09000134.hg.19.729048.6828121.0462282.0651234upDNAJA1118万方数据 第三军医大学博士学位论文TC20000410.hg.15.9444354.8938711.0505642.0713394upMOCS3TC05001822.hg.17.375346.3236051.0517352.0730214upFAM13BTC06001239.hg.16.559995.5078411.0521492.0736164upRPP40TC02001524.hg.17.3492166.2870051.0622112.0881292upADI1TC12002058.hg.16.7295695.6657681.0638012.0904318upC12orf43TC12000500.hg.19.537898.4705361.0673542.0955864upRPS26TC05001385.hg.16.6252495.5497991.075452.1073793upPLK2TC01002246.hg.17.0770755.9963161.0807592.1151486upAGMATTC15001837.hg.19.2972948.2164451.0808492.1152805upANPEPTC14002223.hg.15.7648054.6825731.0822322.1173092upIGHD2-21TC01000842.hg.17.6291286.5411361.0879922.1257796upZNF326TC06001136.hg.14.4152433.3102111.1050322.1510365upTC01002195.hg.19.2080218.0687371.1392842.2027168upSRMTC12001243.hg.14.8554013.6986531.1567482.229543upTAS2R30TC20000341.hg.18.3987567.1900861.208672.3112447upPI3TC21000008.hg.17.5615846.3417971.2197872.3291233upLOC644450TC11001336.hg.14.6916473.4663651.2252822.3380115upOR51I1TC06000384.hg.19.8489898.5286851.3203042.4971872upHSPA1ATC6_dbb_hap3000069.hg.19.1479027.8187371.3291652.5125721upHSPA1ATC6_apd_hap1000039.hg.19.1849617.8557951.3291662.5125738upHSPA1ATC6_cox_hap2000078.hg.19.1850337.8557231.329312.5128246upHSPA1ATC6_qbl_hap6000069.hg.19.1850337.8557231.329312.5128246upHSPA1ATC6_cox_hap2000079.hg.19.2547017.885471.3692312.5833283upHSPA1BTC06000385.hg.19.7962418.4209071.3753342.5942796upHSPA1BTC6_dbb_hap3000070.hg.19.2602357.8799361.3802992.6032232upHSPA1BTC6_qbl_hap6000070.hg.19.4125198.0249291.387592.6164125upHSPA1BTC6_apd_hap1000040.hg.19.1916327.8040421.387592.6164125upHSPA1BTC12000781.hg.17.9601336.5678231.392312.6249865upUTP20TC06000886.hg.17.2476565.8548951.3927612.6258072upGTF3C6TC02001570.hg.19.8576138.4606631.396952.6334426upODC1TC12001123.hg.17.2689995.8695861.3994132.6379423upC12orf4TC06000271.hg.15.6930894.2639281.4291612.6929006upOR2B6TC11002390.hg.111.319699.8631111.4565792.7445678upHSPA8119万方数据 第三军医大学博士学位论文TC18000226.hg.15.4196413.9003481.5192932.8665054upSERPINB5TC14001181.hg.16.6070085.0144561.5925523.0158235upGPR135TC08000602.hg.16.2863794.4621271.8242523.5412336upPOP1TC10000350.hg.17.8039925.9088141.8951783.7196787upDKK1TC07000810.hg.17.0038485.0851211.9187273.7808929upCPA4120万方数据 第三军医大学博士学位论文6.3.4信号通路分析LINC00675相关的信号通路，见图7-3和7-4。DifgeneSigPathway(-LgP)024681012ProteasomeRibosomebiogenesisineukaryotesSpliceosomeRNAtransportEpstein-BarrvirusinfectionMetabolicpathwaysFanconianemiapathwayProteinprocessinginendoplasmicreticulump53signalingpathwayAntigenprocessingandpresentationN-GlycanbiosynthesisGlutathionemetabolismTastetransductionHerpessimplexinfectionPathwayPathogenicEscherichiacoliinfectionLegionellosisAminoacyl-tRNAbiosynthesisPyrimidinemetabolismPhagosomeSulfurrelaysystemInfluenzaAFolatebiosynthesisBasaltranscriptionfactorsNucleotideexcisionrepairEstrogensignalingpathwayArginineandprolinemetabolism图7-3LINC00675上调激活的信号通路。121万方数据 第三军医大学博士学位论文DifgeneSigPathway(-LgP)02468SystemiclupuserythematosusAlcoholismViralcarcinogenesisBiosynthesisofaminoacidsToxoplasmosisTGF-betasignalingpathwaySNAREinteractionsinvesiculartransportPancreaticsecretionMetabolicpathwaysOvariansteroidogenesisSerotonergicsynapsePathwaymTORsignalingpathwayGlycolysis/GluconeogenesisTightjunctionPPARsignalingpathwayAdipocytokinesignalingpathwayRNAdegradationGastricacidsecretionArrhythmogenicrightventricular…图7-4LINC00675下调激活的信号通路。122万方数据 第三军医大学博士学位论文6.3.5GO分析LINC00675富集的基因与信号通路的关系，见图7-5和7-6。DifgeneSigGO(-LgP)051015202530geneexpressionregulationofcellularaminoacidmetabolicprocessDNAdamageresponse,signaltransductionbyp53classmediator…negativeregulationofubiquitin-proteinligaseactivityinvolvedinmitotic…mRNAsplicing,viaspliceosomesmallmoleculemetabolicprocessregulationofubiquitin-proteinligaseactivityinvolvedinmitoticcellcycleSphaseofmitoticcellcycleanaphase-promotingcomplex-dependentproteasomalubiquitin-…proteinpolyubiquitinationregulationofapoptoticprocessG1/StransitionofmitoticcellcycleapoptoticprocessrRNAprocessingmitoticanaphaseresponsetounfoldedproteinDNArepairncRNAmetabolicprocesstranslationspliceosomalsnRNPassemblymRNAprocessingtranscriptionfromRNApolymeraseIIpromoterregulationofARFGTPaseactivityribosomebiogenesisterminationofRNApolymeraseItranscriptioncellularproteinmetabolicprocesstranscriptioninitiationfromRNApolymeraseIpromotersensoryperceptionoftastebiological_processubiquitin-dependentproteincatabolicprocessmicrotubule-basedprocesspositiveregulationofmRNAcatabolicprocessregulationofDNArecombinationreplicationforkprotectionnegativeregulationofgrowthtranscriptionelongationfromRNApolymeraseIpromotercellularresponsetoheatmaturationof5.8SrRNAmolybdopterincofactorbiosyntheticprocessregulationofstressfiberassemblyMo-molybdopterincofactorbiosyntheticprocessribosomeassemblyresponsetogammaradiationDNAdamagecheckpointresponsetomagnesiumionreciprocalmeioticrecombinationcellularresponsetoantibioticnucleotide-excisionrepairresponsetocaffeinepositiveregulationofmacrophageactivationG2/MtransitionofmitoticcellcycleresponsetoironionnegativeregulationofDNAdamageresponse,signaltransductionby…negativeregulationofreleaseofcytochromecfrommitochondriapositiveregulationoftranslationnegativeregulationofmulticellularorganismgrowthcellularresponsetopeptidehormonestimulusGeneontologycategoryresponsetostressmitochondrialtransportresponsetoorganiccycliccompoundoocytematurationregulationofproteincatabolicprocesscellularresponsetocadmiumionpositiveregulationofapoptoticprocessproteinN-linkedglycosylationviaasparaginemitochondrialRNA5'-endprocessingnegativeregulationofantigenprocessingandpresentationofpeptide…leftventricularcardiacmuscletissuemorphogenesisintrinsicapoptoticsignalingpathwayinresponsetoosmoticstress7-methylguanosineRNAcappingregulationofferrochelataseactivitynegativeregulationofcGMP-mediatedsignalingtRNAcatabolicprocesscerebellargranularlayermorphogenesisribonucleoproteincomplexbiogenesisputrescinebiosyntheticprocessfromornithineDNAreplication,OkazakifragmentprocessingnegativeregulationofintegrinactivationRNAimportintomitochondrionmatingplugformationcellularmacromoleculecatabolicprocessspindlemidzoneassemblyinvolvedinmeiosisseminalvesicleepitheliumdevelopmentmitochondrialasparaginyl-tRNAaminoacylationcellularresponsetochemicalstimulusribonucleoproteincompleximportintonucleuscellularresponsetoaminestimulusestablishmentofproteinlocalizationtoendoplasmicreticulummitochondrialmRNApolyadenylationpositiveregulationofcalcium-transportingATPaseactivitypositiveregulationofmiRNAcatabolicprocesscellularcomponentdisassemblyinvolvedinexecutionphaseofapoptosisfatcelldifferentiationDNAreplicationhistoneacetylationoogenesishemebiosyntheticprocesscellularresponsetofibroblastgrowthfactorstimulusregulationofalternativemRNAsplicing,viaspliceosomemismatchrepair图7-5LINC00675上调富集的基因与信号通路的关系。123万方数据 第三军医大学博士学位论文DifgeneSigGO(-LgP)024681012negativeregulationoftranscriptionfromRNApolymeraseIIpromoterpositiveregulationofapoptoticprocessGolgitoplasmamembraneproteintransportbundleofHiscelltoPurkinjemyocytecommunicationinductionofapoptosissignaltransductionfollicle-stimulatinghormonesecretionneurotrophinTRKreceptorsignalingpathwaytransportregulationofexocytosispositiveregulationoftranscriptionfromRNApolymeraseIIpromotermalegenitaliadevelopmentactincytoskeletonorganizationheartdevelopmentregulationofgeneexpressionlong-chainfattyacidimportpositiveregulationofhairfollicledevelopmentfibroblastgrowthfactorreceptorsignalingpathwaynegativeregulationofinsulinsecretioncellcycleregulationofmyotubedifferentiationnucleosomepositioningpalatedevelopmentinnateimmuneresponseregulatedsecretorypathwaylungvasculaturedevelopmentendothelialcellmorphogenesispositiveregulationofproteinoligomerizationcellcommunicationbloodvesseldevelopmentlipidbiosyntheticprocesscircadianrhythmresponsetohypoxiaenergyreservemetabolicprocessresponsetolightstimuluscellularcomponentdisassemblyinvolvedinexecutionphaseofapoptosisglutaminemetabolicprocessfatcelldifferentiationregulationofautophagyintrinsicapoptoticsignalingpathwayinresponsetoendoplasmic…transcriptioninitiationfromRNApolymeraseIIpromoterimmuneresponse-inhibitingsignaltransductionglucosaminebiosyntheticprocessriboflavinbiosyntheticprocessGeneontologycategoryglucosemediatedsignalingpathwaytaurinetransportprotein-pyridoxal-5-phosphatelinkageviapeptidyl-N6-pyridoxal…endosometransportviamultivesicularbodysortingpathwayhydroxyprolinetransportnegativeregulationofhepatocytegrowthfactorbiosyntheticprocessembryonicectodermaldigestivetractmorphogenesisquorumsensinginvolvedininteractionwithhostpositiveregulationofintestinalepithelialstructuremaintenanceendothelialcellfatespecificationlungciliatedcelldifferentiationcellularresponsetotumorcellresponsetonorepinephrinestimuluspositiveregulationofglomerularmetanephricmesangialcellproliferationpositiveregulationofspindlecheckpointnegativeregulationofendoplasmicreticulumunfoldedproteinresponseregulationoffemalegonaddevelopmentnegativeregulationofepithelialcellproliferationinvolvedinlung…cell-celljunctionorganizationchromatinorganizationpositiveregulationofepithelialtomesenchymaltransitionpositiveregulationofneurondifferentiationcellularmembranefusionnegativeregulationoffibroblastproliferationresponsetoprogesteronestimulusphotoreceptorcellmaintenanceepidermisdevelopmenthairfolliclemorphogenesispositiveregulationoftypeIIahypersensitivitynitricoxideproductioninvolvedininflammatoryresponseventricularcompactmyocardiummorphogenesisresponsetosteroldepletionproteasomalproteincatabolicprocessnegativeregulationoftriglyceridebiosyntheticprocesscysteinebiosyntheticprocessacetyl-CoAbiosyntheticprocessfromacetatelimbicsystemdevelopmentpositiveregulationofaldosteronebiosyntheticprocessglycoproteintransportnervegrowthfactorsignalingpathwayproteincomplexdisassemblyresponsetodsRNAplasmamembranetoendosometransportregulationofmembranerepolarizationregulationofgenesilencingnegativeregulationofoxidativephosphorylationpositiveregulationofintegrin-mediatedsignalingpathway图7-6LINC00675下调富集的基因与信号通路的关系。124万方数据 第三军医大学博士学位论文6.3.6富集分析LINC00675调控基因的富集区域，如7-7。图7-7富集分析LINC00675调控基因的富集区域。6.3.7qRT-PCR验证mRNA芯片的准确性，分别选择在胃癌中高表达或低表达的基因各3个，并设计引物进行验证，如图7-8所示，除CAP4的表达与芯片不一致以外，其他5个基因（DKK1,POP1,STEAP4,H1F0,CGA）的表达与芯片一致。图7-8qRT-PCR检测6个基因在过表达LINC00675的SGC-7901细胞中的表达，与NC的SGC-7901细胞比较。β-actin作为内参。125万方数据 第三军医大学博士学位论文6.3.8信号通路分析发现过表达LINC00675激活了p53信号通路，通过检测p53的靶基因p21和MDM2，发现LINC00675显著促进了p21的表达，但MDM2的表达受到抑制。图7-9qRT-PCR检测p21和MDM2在过表达LINC00675的SGC-7901细胞中的表达，与NC的SGC-7901细胞比较。β-actin作为内参。6.4讨论为了进一步了解LINC00675调控的信号通路，课题组采用mRNA芯片筛选了过表达LINC00675的SGC-7901细胞中信号通路的变化。根据芯片的结果，课题组分析[71,72]了与细胞增殖相关的信号通路，其中包括p53。尽管用于此次mRNA芯片的样本没有生物学重复，但课题组采用qRT-PCR验证了芯片数据中高表达或低表达的基因，包括CAP4，DKK1，POP1，STEAP4，H1F0和CGA。其中，除了CAP4不符合芯片数据的表达趋势之外，其余5个基因的表达趋势均与芯片数据匹配，提示芯片数据可反应绝大部分真实情况。通过信号通路分析发现LINC00675影响了p53信号通路，进一步采用qRT-PCR检测p53下游基因p21和MDM2的表达，发现LINC00675显著促进了p21mRNA的表达，但抑制了MDM2mRNA的表达。总所周知，p53可以促进MDM2的表达，而MDM2可以介导p53蛋白的降解，进而形成一个简单的负反馈调节通路。我们的研究提示LINC00675可能在另外的机制上影响了MDM2的表达，而具体的分子机制需要进一步研究。GSEA分析显示“ZWANG_TRANSIENTLY_126万方数据 第三军医大学博士学位论文[73][74]UP_BY_1ST_EGF”和“BENPORATH_ES_WITHH3K27ME3”的相关基因在LINC00675过表达的SGC-7901细胞中的富集是降低的，提示LINC00675可能阻碍高[75]分化的胃癌细胞向低分化的转变。另外，“MANALO_HYPOXIA_DN”和“GO_RIBONUCLEOPROTEINCOMPLEX”相关基因在LINC00675过表达的SGC-7901细胞中富集增加，提示LINC00675/vimentin复合物可能涉及胃癌细胞的缺氧。另外，HPARNA-seqnormaltissues分析（website:https://www.ncbi.nlm.nih.gov/geoprofiles/?term=LINC00675）显示LINC00675在正常胃组织中的表达具有较高的水平，提示LINC00675在正常胃组织中可能发挥重要作用。GEO分析显示LINC00675的表达在结直肠癌和尿道膀胱癌中呈低表达，提示LINC00675可能在癌症中普遍降低。127万方数据 第三军医大学博士学位论文全文结论1.通过基因芯片及生物信息学分析，首次鉴定lncRNALINC00675在胃癌组织中低表达且与患者不良预后密切相关。2.功能研究表明，LNC00675的过表达显著抑制胃癌细胞的增殖、迁移和侵袭能力，而抑制LINC00675的表达则显著促进了胃癌细胞的这些能力。3.机制上，LINC00675可与vimentin相互作用，促进vimentinSer39位点的磷酸化，导致vimentinfilaments的崩解。文献表明，vimentinfilaments的崩解抑制了癌细胞的增殖、侵袭和迁移。以上研究表明LINC00675作为一个肿瘤抑制子，其与vimentin的相互作用调控了胃癌细胞的增殖、侵袭和迁移，其表达与胃癌的预后密切相关且可能作为胃癌治疗的新靶点。128万方数据 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第三军医大学博士学位论文[72]ChengLL,ItahanaY,LeiZD,ChiaNY,WuY,YuY,ZhangSL,ThikeAA,PandeyA,RozenS,VoorhoevePM,YuQ,TanPH,BayBH,ItahanaK,TanP.:TP53genomicstatusregulatessensitivityofgastriccancercellstothehistonemethylationinhibitor3-deazaneplanocinA（DZNep）.ClinCancerRes2012,18(15):4201-4212.[73]ZwangY,Sas-ChenA,DrierY,ShayT,AvrahamR,LauriolaM,ShemaE,Lidor-NiliE,Jacob-HirschJ,AmariglioN,LuY,MillsGB,RechaviG,OrenM,DomanyE,YardenY.:Twophasesofmitogenicsignalingunveilrolesforp53andEGR1ineliminationofinconsistentgrowthsignals.MolCell2011,42(4):524-535.[74]Ben-PorathI,ThomsonMW,CareyVJ,GeR,BellGW,RegevA,WeinbergRA:Anembryonicstemcell-likegeneexpressionsignatureinpoorlydifferentiatedaggressivehumantumors.NatureGenet2008,40(5):499-507.[75]ManaloDJ,RowanA,LavoieT,NatarajanL,KellyBD,YeSQ,GarciaJG,SemenzaGL:TranscriptionalregulationofvascularendothelialcellresponsestohypoxiabyHIF-1.Blood2005,105(2):659-669.138万方数据 第三军医大学博士学位论文文献综述LongNon-codingRNAs:ThePromisingBiomarkersandTherapeuticTargetsinGastricCancerRunningTitle:lncRNAsinGCAbbreviations:lncRNAs,LongNon-CodingRNAs;GC,GastricCancer;ncRNAs,Non-CodingRNAs;ORF,OpenReadingFrame;PCAT-1,ProstateCancerAssociatedTranscripts1;ceRNAs,CompetingEndogenousRNAs;HOTAIR,HOXTranscriptAntisenseIntergenicRNA;PRC2,PolycombRepressiveComplex2;HCC,HepatocellularCarcinoma;HER2,HumanEpithelialGrowthFactorReceptor2;RUNX1,RuntDomainTranscriptionFactor1;MALAT1,Metastasis-AssociatedLungAdenocarcinomaTranscript1;NEAT2,Nuclear-EnrichedAbundantTranscript2;MEG3,MaternallyExpressedGene3;DMRs,DifferentiallyMethylatedRegions;DNMT1,DNAMethyltransferase1;HULC,HighlyUpregulatedinLiverCancer;GAPLINC,GastricAdenocarcinomaPredictiveLongIntergenicNoncodingRNA;GAS5,GrowthArrest-Specifictranscript5;GHET1,GastriccarcinomaHighExpressedTranscript1;CCAT1,ColonCancer-AssociatedTranscript1;FER1L4,Fer-1-Likeprotein4ABSTRACTLongnon-codingRNAs(lncRNAs)areendogenousRNAmoleculesthatarelongerthan200nucleotidesinlengthbutlackofprotein-codingpotentials.AccumulatingstudieshaveelucidatedthatlncRNAsplaycrucialrolesinvariousbiologicalprocesses.Moreover,lncRNAshavebeenreportedtobedysregulatedingastriccancer(GC)andthealteredexpressionoflncRNAshasbeenprovedtobeinvolvedinthetumorigenesisandprogressionofGC.AberrantexpressionoflncRNAsshowssignificantcorrelationwiththeprognosisinGCpatients.However,thebiologicalandmolecularmechanismsbywhichlncRNAscontributetotheoccurrenceanddevelopmentofGCremainslargelyunknown.Inthisreview,wesummarizethewell-knownlncRNAswhichhavebeenprovedtobedysregulatedinGCandhighlightthesignificanceoftheirfunctionsincancerbiology.Moreover,wealsodiscusstheknownmolecularmechanismsoflncRNAsinthe139万方数据 第三军医大学博士学位论文developmentofGCandtheirpotentialsforthediagnosis,treatmentandprognosisinGCpatients.KeyWords:longnon-codingRNAs;gastriccancer;HOXtranscriptantisenseintergenicRNA;H19;metastasis-associatedlungadenocarcinomatranscript1;maternallyexpressedgene3IntroductionTheENCODEconsortiumhasdemonstratedthatabout70%ofthehumangenomeis[1,2]transcribedintoRNA,however,only2%ofthegenomepossessesprotein-coding[3]potentials.SeverallinesofevidencesuggestthatRNAsdon’tpossesstheprotein-codingcapacity,yetitisknownasthatnon-codingRNAs(ncRNAs)accountforthemajorityof[4]transcribedproducts.Previously,ncRNAswereconsideredastranscriptional“noise”or[5]“junk”.WiththeadvanceofDNAtilingarraysanddeepsequencingtechnology,anincreasednumberofncRNAshavebeenidentifiedoverrecentyears,whichareprovedto[6,7]beofdiversebiologicalfunctions.HousekeepingncRNAsandregulatoryncRNAsareincludedinncRNAs,rRNAs,tRNAs,snRNAs,andsnoRNAsbelongtotheformerwhich[5]areconstitutivelyexpressed.Accordingtothesize,ncRNAsareclassifiedintotwocategories:shortncRNAs(<200nt),suchassiRNAs,miRNAs,piRNAs,andlongncRNAs(lncRNAs,>200nt).LncRNAsaredefinedasRNAmoleculeswhoselengthrangefrom200bpto100kbpthatlackprotein-codingpotentials.Forlackofafunctionalopenreadingframe(ORF),translationoflncRNAsisimpossibletooccur.ThevastmajorityoflncRNAsare[8,9]transcribedbyRNApolymeraseII,andsubsequenttoco-transcriptionalmodifications.[10]LncRNAsarelocatedinnucleusorcytoplasm,especiallynucleus.IncomparisonwithsmallncRNAsthatpossesshighconservatismduringspeciesevolution,lncRNAsseemtobepoorlyconserved.Accordingtotheirlocusrelativetoprotein-codinggenes,lncRNAsaredividedintofivecategories:sense,antisense,bidirectional,intronic,andintergenic.LncRNAsplaycriticalrolesinpervasivebiologicalprocesses,suchasimprinting,X-inactivation,chromosomaldosagecompensation,cellcycleregulation,apoptosis,[11-13]transcription,splicing,translationandcelldifferentiation.140万方数据 第三军医大学博士学位论文AccumulatingstudieshaveelucidatedthatthedysregulationoflncRNAsusuallycontributestodiversehumandiseases,includingAlzheimerdisease,spinocerebellarataxia,cardiovasculardisease,Prader-Willisyndrome,diabetesmellitusandparticularcancers[14-19].CurrentresearcheshaveshownthatlncRNAsexhibitapparenttissuespecificityandareupregulatedordownregulatedintumorcells,indicatingthatthedysregulationof[20]lncRNAsmightbeassociatedwithspecificcancers.Forexample,lncRNAPCAT-1(ProstateCancerAssociatedTranscripts1)isoverexpressedinprostatecancertissues,but[21]low-expressedinadjacentnormaltissues.AndPCAT-1promotescellproliferation[22]throughupregulatingcMycatpost-transcriptionallevel.Therefore,lncRNAsmightbeefficientbiomarkersofcancers.What’smore,massivestudiesalsohaveprovedthatlncRNAsplaysignificantrolesintheoncogenesis,tumorproliferation,invasion,andmetastasis.Inthisreview,wewillsummarizesomewell-knownlncRNAsassociatedGCanddiscusstheirfunctionsandmolecularmechanismsinthedevelopmentofGC(Table1).Furthermore,wealsodiscusstheirpotentialsasdiagnosticindicators,prognosticbiomarkersandnoveltherapeutictargetsinGCpatients.Table1Characteristicsandfunctionsofsomewell-knownlncRNAsinGC.LncRNAGenomeRegulationFunctioninAuthorRef.locationtumorigenesisHOTAIR12q13.13upregulatedoncogeneM.Hajjarietal.40H1911p15.5upregulatedoncogeneH.Lietal.54MALAT111q13.1upregulatedoncogeneJ.Wangetal.63HULC6p24.3upregulatedoncogeneY.Zhaoetal.76GAPLINC18upregulatedoncogeneY.Huetal.77GHET17q36.1upregulatedoncogeneF.Yangetal.84CCAT18q24.21upregulatedoncogeneF.Yangetal.86MEG314q32downregulatedtumorsuppressorM.Sunetal.39GAS51q25.1downregulatedtumorsuppressorM.Sunetal.36FER1L420q11.22downregulatedtumorsuppressorZ.Liuetal.88141万方数据 第三军医大学博士学位论文ProfilesoflncRNAsMassivestudieshaverevealedthatlncRNAsshowcrucialrolesindiversebiologicalprocesses.Thestructureofanymoleculesalwaysdecidesitsfunction.SinceprimarystructuresoflncRNAsappeartobemultifariousandpoorlyconserved,lncRNAscanfoldintocomplexbutspecificsecondarystructures.ThespatialstructuresmayprovidevariousbindingsitesforproteinsorinteractwithDNAsandRNAsdirectlythroughcomplementary[23]basepairs.LncRNAsmaycomeintobeingviafivepotentialways,includingtheinterruptionofprotein-encodinggenes’structures,theresultofchromatinremodeling,retro-transpositionduringthereplicationofnoncodinggenes,localtandemreplicon,andinsertionoftransposableelementsintogenes.RecentstudieshaverevealedthatlncRNAscanparticipateintheregulationofgeneexpressioninvariousmanners(Figure1).J.E.Wiluszandhiscolleaguessummarizedthe[7]molecularmechanismsofhowlncRNAsfunctionasbelow.(1)LncRNAscantranscriptfrompromotersthatarelocatedintheupstreamoftheprotein-codinggenes,therebyinterferingtheexpressionofdownstreamgenes.(2)ThenoncodingtranscriptaffectsthedownstreamgenesexpressionthroughinhibitingRNApolymeraseIIormediatingchromatinremodelingandhistonemodifications.(3)Complementarybase-pairingreactionofanantisensetranscriptandasensetranscriptcausetheinterferenceofthemRNAsplice,thusleadingtoadifferentsplicedtranscript.(4)Inasimilarway,thebindingofsenseandantisensetranscriptscangenerateendogenoussiRNAswiththeinvolvementofDicer.(5)LncRNAtranscriptscanregulatetheactivityofproteinsviabindingwithspecificproteins.(6)Asastructuralcomponent,anoncodingtranscriptcanallowtheformationofRNA–proteincomplex.(7)LncRNAscanchangethecytoplasmiclocationoftheproteinsbycombiningtospecificproteins.(8)LncRNAscanbeservedasprecursorsforsmallRNAs,suchasmiRNAsandpiRNAs.142万方数据 第三军医大学博士学位论文Figure1．MolecularmechanismsofhowlncRNAsfunction.(A)LncRNAscanactasceRNAsandspongemiRNAs.Throughcomplemenntarybase-pairingwithmRNAs,lncRNAscangenerateendogenoussiRNAs(B)andmediatealternativesplicing(C).LncRNAsparticipateintheregulationofgeneexpressionatchromatinmodificationlevel(E)andtranscriptionallevel(D).((F)LncRNAsserveasscaffoldsbybindingvariousproteins.TheregulationofgeneexpressionisassociatedwithlncRNAsatvariouslevels,suchaschromatinmodificationlevel,transcriptionalandpost-transcriptionallevel.Atchromatinremodelinglevel,lncRNAcanrecruitchromatinremodelingcomplexestospecificloci,thenmediatethesilenceofrelatedgenes.Forexample,lncRNAHOTAIRmayrecruitand[24]relocatePRC2toHOXDlocus,whichleadingtotheepigeneticsilenceofHOXDlocus.LncRNAXistplaysacrucialroleinXchromosomeinactivationinasimilarwaymentioned[25]above.LncRNAscanregulatetranscrriptionthroughdiversemechanissms.Functioningastranscriiptionalco-activators,lncRNAEvvf2canregulatetheactivityoftranscriptionfactor[26]bybindingtoDLX2andsubsequentlyenhancetheexpressionofDLX6.LncRNAsalsocaninterferetheexpressionofadjacentgenesbyinteractingwithRNAbindingproteins.143万方数据 第三军医大学博士学位论文Forexample,thelncRNAsaretranscribedfromtheupstreamofcyclinD1genepromoterwhichcanregulatetheactivitiesoftheRNAbindingproteinTLS,thusinturninhibitingthe[27]expressionofcyclinD1.LncRNAscanformcomplementarydoublechainswithmRNAsandmodulategeneexpressionatthepost-transcriptionallevel,includingmRNAsplicing,editing,transport,translationanddegradationaswellassmallncRNAsreservation.Inaddition,lncRNAsalsocanbeservedascompetingendogenousRNAs(ceRNAs)and[28]interactwithmiRNA.ThentheinteractionbetweenlncRNAsandmiRNAscanregulatetheexpressionoftargetgenes.Insometumorcells,lncRNAsmayinhibitthetargetgenes[29]expressionviaspongingspecificmiRNAs.K.C.WangandhiscolleaguesbrieflysummarizedthemolecularfunctionsoflncRNAsintofourarchetypes,includingsignals,[30]decoys,guidesandscaffolds.TheaberrantexpressionoflncRNAshasbeenprovedtobeassociatedwithdiversediseasesincludingcancers.Forexample,lncRNABACE1-AS,theantisensetranscriptofBACE-1,playsapivotalroleinthepathophysiologyofAlzheimer’sdisease.BACE1-AScanregulatethestabilityofBACE1mRNAandthenaffecttheexpressionofBACE1[31]protein,whichisasignificantenzymeinAlzheimer'sdisease.IthasbeendemonstratedthatmanylncRNAsappeartobedysregulatedinspecifichumantumortissues.Therefore,theabnormallncRNAsexpressionmaybeservedassensitivelydiagnosticbiomarkers,andtherapeuticstrategiesfocusedonlncRNAsaspotentialdrugtargetsareconsideredtobepossibleandpromisingforcancerpatients.DysregulationoflncRNAsinGCAlthoughtheincidenceanddeathratehavebeendramaticallydeclinedinrecentyears,[32]GCremainsoneoftheleadingcausesofcancer-relateddeaths.TheprognosisofGCpatientshasbeenimprovedasaresultofadvancementintreatmentandearlydetection.NumerousstudieshaveperformedtoexplorethemolecularmechanismsofGCandithas[33]achievedagreatprogressuptonow.However,ourunderstandingofthepathogenesisandprogressionofGCremainlimited,andthereisstilllargenumberofpatientsdieofGCbecausetheywerediagnosedatadvancedstages,whichresultinginmissingthebestopportunityfortreatment.Therefore,itisnecessarytofurtherexploresomemoleculesormechanismswhichmaybeofgreatvalueinthediagnosisandtreatmentofGC.144万方数据 第三军医大学博士学位论文TheresearchesthatfocusonlncRNAshaveemergedincreasinglysincetherevelationof[34]biologicalfunctionsoflncRNAs.ManylncRNAsaredysregulatedinGCtissuescomparedtoadjacenttissues,andfunctionascrucialmembersinthedevelopmentand[35-37]progressionofGC.AlteredexpressionofspecificlncRNAsinGCtissuesindicatesthepotentialrelationbetweenlncRNAsandGC.ItisestimatedthatmanylncRNAmayact[38,39]astumorsuppressorsoroncogenestoregulatethetumorgenesis.ThemolecularmechanismsoflncRNAsinGChavebeenrevealedtosomeextent,however,thedetailsremainunclear.Wewillsummarizeseveralwell-knownlncRNAsandtheirmolecularmechanismsinGC.HOTAIRHOXtranscriptantisenseintergenicRNA(HOTAIR)isalincRNAtranscribedfrom[40]HOXClocus,whichisupregulatedinprimarybreastcancer.Itwasinitiallydescribedto[24]playacrucialroleinthedevelopmentofbreastcancerbyR.A.Guptaetal..Recently,aberrantoverexpressionofHOTAIRhasbeenconfirmedinmanyothertumors,includinghepatocellularcarcinoma(HCC),colorectalcancer,pancreaticcancer,non-smallcelllung[41-44]cancerandalsoGC.Theup-regulationofHOTAIRrelocatespolycombrepressivecomplex2(PRC2)tospecificgenesthroughbindingwithPRC2,resultinginH3K27[24]trimethylationandsilencingoftargetgenes.What’smore,enhancedexpressionofHOTAIRhasconsideredtopromotecancermigration,invasivenessandmetastasis.RecentstudieshaveelucidatedthatHOTAIRisalsoup-regulatedingastrictumortissuescomparedwithadjacentnormalones,whichindicatesthatthedysregulatingofHOTAIRmightbeanimportantparticipateintheGCdevelopment.M.HajjariandhiscolleagueshavedemonstratedthatincreasedHOTAIRexpressionwascorrelatedwithTNM[40]stageandlymphnodemetastasisinpatientswithGC.PatientswithhighlevelsofHOTAIRexpressionhadarelativelypoorprognosis.Meanwhile,thereisapositivecorrelationbetweentheexpressionofHOTAIRandSUZ12.ItispronetometastasisamongpatientswithhighexpressionofHOTAIR.Moreover,thereisasignificantrelevance[45]betweenenhancedHOTAIRexpressionandapoorprognosisinpatients.Inaddition,depletionofHOTAIRinGCcellscouldsuppresscellproliferationandinvasion,induce[46,29]apoptosis,aswellasreverseepithelialmesenchymaltransitionprocess.145万方数据 第三军医大学博士学位论文Furthermore,HOTAIRcanfunctionasaceRNAtospongemiR-331-3p,thusderepressthemiR-331-3ptarget,humanepithelialgrowthfactorreceptor2(HER2).HER2isanoncogenethatpresentingacloserelationshipwithtumormetastasis.HOTAIRhelpsrelieftheinhibitionofHER2frommiR-331-3pbybindingmiR-331-3p,therebycontributingto[29]tumormetastasis.Inaddition,HOTAIRcanactasmodularscaffoldsthroughofferingbindingsitestotetherspecifichistonemodificationenzymes,andthuscoordinatingthe[47]patternofhistonemodificationsontargetgenes.Inshort,HOTAIRplaysacrucialroleinthemolecularetiologyofGC.Accordingtoitsfunctionsandmechanismsmentionedabove,lncRNAHOTAIRmayserveasapotentialprognosticbiomarkerandtherapeutictargetinGC.H19H19,amaternallyexpressedgene,thatislocatedinchromosome11p15.5,itisanareathatcontainsahighlyconservedclusterofimprintedgenes.Thepaternallyexpressedgene[48,49]adjacenttoH19,insulin-likegrowthfactor2isalsoanimprintedgene.TheexpressionlevelofH19ishighduringembryogenesisbutreducedinmosttissuesafterbirth,[49]exceptinadultcardiacandskeletalmuscle.TheH19genepossessesnoprotein-codingpotentialbutencodesa2.3kblncRNA.InitialstudiesproposedthatH19RNAactasa[50]tumorsuppressor,however,recentresearcheshaveelucidatedthatH19expressionis[38]markedlyincreasedinsomehumanmalignanciesandischaracterizedasanoncogene.EctopicexpressionofH19hasbeenvalidatedinvarioushumancancers,including[51-53,38]breastcancer,lungcancer,glioma,HCCandalsoGC.In2012,F.Yangetal.haveverifiedthatabnormalH19expressionpromotescellproliferation,converselysuppressionofH19leadstocellapoptosisinGCcells.HaoLiandhiscolleaguesfurthermanifestedthatoverexpressionofH19promotedtumorigenesis,cellmigration,invasionaswellas[54]metastasis.InGCcelllines,thereisapositivecorrelationbetweenc-Myconcogeneand[49]H19,andc-MyconcogenecanregulatetheH19expression.Furthermore,upregulatedH19isrelatedwiththetumorsuppressorp53andcontributestopartialp53inactivation,[38]thusdecreasingthep53-mediatedapoptosis.Inaddition,miR-675isthematureproductofH19andtheyhaveapositivecorrelationinGCcells.What’smore,awell-knowntumorsuppressor,RuntDomainTranscriptionFactor1(RUNX1)isidentifiedtobeadirecttarget146万方数据 第三军医大学博士学位论文ofmiR-675.MiR-675inducesdecreasedexpressionofRUNX1inGCcells,resultinginthecellproliferationofGC.Inotherwords,H19-derivedmiR-675regulatesGCcellgrowth[55]throughtargetingRUNX1.ThenovelpathwayH19/miR-675/RUNX1mightbeservedaseffectivetargetsforthetreatmentofGC.Moreover,H19mightperformitsfunctionsinGCdevelopmentaswellasencodemiR-675throughthedirectup-regulationofitsbindingproteinISM1andtheindirectdown-regulationofCALN1,atargetgeneofmiR-675by[54]miR-675.Therefore,theH19/ISM1andmiR-675/CALN1pathwaysmightbenovelpotentialdrugtargetsinGCtherapy.MALAT1Metastasis-associatedlungadenocarcinomatranscript1(MALAT1)thatisacancer-associatedlncRNAlocatedinnuclearspecklesandisalsocalledasnuclear-enriched[56]abundanttranscript2(NEAT2).IthasbeendeterminedthatMALAT1possesseshighconservationamongmammalsfromhumantomouse.MALAT1waswidelydetectedin[57]humantissues,suchasbrain,heart,spleen,aswellaskidney.TheexpressionofMALAT1isaberrantlyelevatedinmetastasizingnon-smallcelllungcancer.Thenup-regulationofMALAT1wasobservedinvariouscancers,includingbreastcancer[58],[59][60,61]ovariancancer,hepatocellularcarcinomaandcolorectalcancer.In2014,J.Wangetal.havedemonstratedthatMALAT1ishighlyexpressedandpromotescellproliferationanddifferentiationinGCcells.Up-regulationofMALAT1caninduceanelevatedexpressionofSF2/ASFinnucleus,amemberofserine/arginine-richproteinfamily.SF2/ASFexertssignificantrolesinmRNAstability,inflammatorydisorders[62]andcancersbyregulatingalternativesplicing.Whereas,knockdownofMALAT1decreasestheSF2/ASFexpressioninnuclearandarrestcellcycleinG0/G1phase,which[63]causesuppressionofcellproliferationinGCcells.MALAT1playscriticalrolesinthedevelopmentofGC,andmightbeservedasaneffectivediagnosticbiomarkerandnoveltherapeutictarget.MEG3Maternallyexpressedgene3(MEG3)functionsasatumorsuppressorandislocatedat[64]humanchromosome14q32.MEG3isexpressedinhumannormaltissues,the147万方数据 第三军医大学博士学位论文down-expressedofitinvarioustypesofcancersthatsuggestsMEG3mightplaya[65]significantroleinthedevelopmentandprogressionofcancer.ThelossofMEG3expressionhasbeendemonstratedinmanyhumantumors,suchasHCC,colorectalcancer,[66-70]lungcancer,bladdercancerandglioma.ThedecreasedexpressionofMEG3incancertissuesmightattributetothehypermethylationofpromoterordifferentiallymethylated[65]regions(DMRs)whichlocatedupstreamofMEG3gene.MEG3re-expressioncanrepresstheproliferationoftumorcellspartlythroughtheapoptosisinducedbyMEG3.Moreover,MEG3caninteractwithp53andmodulatetheexpressionofspecificp53target[65]genes.M.Sunetal.haveshownthattheexpressionofMEG3issignificantlyreducedinGCtissuescomparedtoadjacentnormaltissuesandpresentscloseassociationwithtumorsize,[39]TNMstagesanddepthofinvasion.Down-expressionofMEG3inGCcanpromotecellproliferation,whereasre-expressionofMEG3caninhibitcellproliferationandinducecellapoptosis.Similarly,ithasalsobeenvalidatedthatMEG3canactivatep53andexertits[39]functionasatumorsuppressorinGC.IthasbeenpreviouslyreportedthatthelevelofmiR-148aisalsomarkedlydecreasedinGCtissuesandthesilenceofmiR-148aisinducedthroughDNAhypermethylation,[71]whichismediatedviahighexpressionofDNAmethyltransferase1(DNMT1).DecreasedmiR-148aexpressioncancauseup-regulationofDNMT1,whichleadingtodown-expressionofMEG3subsequently.Conversely,enhancedmiR-148aexpressioncanincreaseMEG3expression,therebysuppressingcellproliferationofGCcells.ThesefindingsexpandourunderstandingabouthowMEG3functionsasatumorsuppressorinGCdevelopment.ThemechanismsmentionedaboveindicatethatMEG3maybeservedasanovelprognosticbiomarkerinGCpatients.HULCHighlyupregulatedinlivercancer(HULC)withthelengthof1.6knucleotidethatisconsideredasthemostup-regulatedgeneinHCC.HULCRNAfailstotranslateinto[72]proteinsandinconsequenceHULCisclassedasalncRNA.IthasbeenwellknownthatHULCplaysasignificantroleinthetumorigenesisofHCC.Inlivercancer,HULCmayfunctionasaceRNAtospongemiR-372andthusinhibittheexpressionaswellasthe148万方数据 第三军医大学博士学位论文activityofmiR-372.Thedown-expressionofmiR-372resultsinderepressionofitstargetgenePRKACBandsubsequentlyinducesphosphorylationofCREB.Inturn,phosphor-CREBmightincreasetheexpressionofHULCthroughtheactivationofPKA[73]pathway,whichindicatesanauto-regulatoryloopofHULCexpression.AlertedexpressionofHULCisnotrestrictedtoHCC,laterstudiesshowedthatHULClevelwasalsoenhancedinothercancers,suchaspancreaticcancerandcolorectal[74,75]carcinomasthatmetastasizetotheliver.Y.Zhaoetal.elucidatedthatHULCwas[76]upregulatedinGCcelllinesandtissuescomparedwithadjacentnormaltissues.Moreover,theabnormalregulationofHULCwassignificantlyassociatedwithTNMstages.IncreasedexpressionofHULCinhibitedcellapoptosisandpromotedcancercellproliferationandinvasion,whereasdepletionofHULCpresentedtheoppositeresult.ThebiologicalperformanceindicatedthatHULCmightfunctionasanoncogeneinthetumorigenesisanddevelopmentofGC.Inaddition,highlyexpressedHULCinducedautophagyandtheinhibitionofautophagycouldpromoteapoptosisinSGC7901cells.What’smore,knockdownofHULCcouldinduceaseriesofbiochemicalandmorphologicalchangeswhichsuggesteda[76]reversionofepithelial-to-mesenchymaltransition.Ingeneral,HULCactsasacrucialroleinthedevelopmentofGCandmaybeconsideredasanefficientcandidatebiomarkerforGCdiagnosisandapotentialindicatorforGCprognosis.GAPLINCGastricadenocarcinomapredictivelongintergenicnoncodingRNA(GAPLINC)isa[77]longintergenicncRNAthatisupregulatedinGC.EctopicGAPLINCexpressioncanpromotecellproliferationandmigration,anditalsocanindicateapoorprognosisinGCpatients.Therefore,GAPLINCisregardedasanovelcandidatepredictorfortheprognosisofGC.OverexpressionofGAPLINCthatcanpromotecellproliferation,whileknockdownofGAPLINCinducescellapoptosis.SinceknockdownofCD44,thepro-invasionbehaviorofGAPLINCisprovedtobecounteracted.What’smore,highexpressionofCD44canreversetheeffectofGAPLINCsuppression,whichindicatingtheGAPLINCmayregulatecellinvasionviamodulatingtheexpressionofCD44inGC.GAPLINCfunctionsasaceRNA149万方数据 第三军医大学博士学位论文throughcompetingformiR-211-3p,andthusregulatingtheexpressionofCD44oncogene,[77,78]whichisresponsibleforcancercellproliferation,migrationandangiogenesis.GAS5Growtharrest-specifictranscript5(GAS5)isspecificallyexpressedingrowth-arrested[79]cells.GAS5caninducecellapoptosisbysuppressingtheactivityofglucocorticoids[80]duringnutrientstarvation.IthasbeenestimatedthatGAS5isdown-regulatedandisassociatedwithpoorsurvivalinHCC,breastcancer,cervicalcancerandcolorectalcancer[81-84].HenceGAS5isconsideredtofunctionasatumorsuppressorinvariouscancertissues.Gas5isalsodemonstratedtobesignificantlydown-regulatedinGCtissuesandissuggested[36]asapotentialprognosticindicatorforGC.DecreasedexpressionofGAS5maypromotecellproliferation,whileforcedexpressionofGAS5caninducecaspase3-dependentapoptosisandinhibitcellproliferationofGCcells.Furthermore,theunderlyingmechanismsofGAS5-inducedcellproliferationhavealsobeeninvestigated.TheexpressionlevelofE2F1andcyclinD1showstobemarkedlydecreasedwhenre-expressingGAS5inGCcells,however,thereisnochangeforthemRNAexpressionlevelofE2F1andcyclinD1.Moreover,GAS5suppressioninducesanincreaseinE2F1andcyclinD1.Takentogether,GAS5mayplayaroleintheGCdevelopmentthrough[36]regulatingE2F1andcyclinD1atpost-transcriptionallevel.GHET1Gastriccarcinomahighexpressedtranscript1(GHET1)thatisanintergeniclncRNAlocatedonchromatin7.F.Yangetal.havereportedthatGHET1isup-regulatedinGC[85]tissuesandassociatedwithpoorprognosis,tumorsizeandinvasion.Similarly,overexpressionofGHET1promotescellproliferation,however,GHET1depletionshowsareverseeffectwhichindicatesthatGHET1playsanoncogenicroleinGC.FurtherstudieshavebeenconductedtoexplorethemolecularmechanismsofGHET1-mediatedcellproliferation.GHET1canincreasetheinteractionbetweenc-MycmRNAandIGF2BP1byphysicallybindingIGF2BP1,thusenhancethestabilityandexpressionofc-MycmRNAandpromotecellproliferationofGCcellsasaconsequence.GHET1-mediatedproliferation150万方数据 第三军医大学博士学位论文isprovedtobedependentonc-Mycinthatinhibitionofc-MycresultsinnoresponsetocellproliferationinGCcells.CCAT1Coloncancer-associatedtranscript1(CCAT1),alncRNAmarkedlyup-regulatedin[86,87]coloncancertissues,wasalsofoundtobehighlyexpressedinGCcellslater.TheaberrantexpressionofCCAT1iscloselyrelatedtoc-MycandcanpromotecellproliferationandmigrationofGC,whichsuggeststhatCCAT1mayfunctionasanoncogene.F.Yangetal.haveelucidatedthatthetranscriptionfactorc-MyccouldactivatethepromoteractivityofCCAT1andincreaseitsexpressionbydirectlybindingwiththe[87]E-boxelementintheCCAT1promoterregion.Thesedataaboveindicatethatc-MycinducedCCAT1playapivotalroleintheprogressionofGCandprovideuswithanovelcandidatetargetforthetreatmentofGCpatients.FER1L4Fer-1-likeprotein4(FER1L4)isanovellncRNAthatlocatedonhumanchromosome20.IthasbeenvalidatedthatthelevelofFER1L4ismarkedlydown-regulatedinGC[88]tissues.ThedecreasedexpressionofFER1L4showssignificantassociationwiththetumorsize,invasion,differentiation,lymphnodemetastasis,distalmetastasis,TNMstages,venousandperineuralinvasion.ThereisnodifferenceinplasmaFER1L4betweenhealthpersonsandpreoperativepatients,butthereappearsasharpdeclineinpostoperative[89]patients.ThesedataindicatethatFER1L4mightplayanimportantroleinGCdevelopmentandmightrepresentapotentialindicatorofclinicalprognosisinGC.ConclusionsIthasachievedagreatprogressinthestudiesoflncRNAssincetheywereprovedtobefunctionalinpervasivebiologicalprocesses.ItisclearthatlncRNAscanparticipateinvariousbiologicalprocessesthroughdistinctmechanisms.Forexample,lncRNAGAS5functionsasadecoyforglucocorticoidreceptor,whilelncRNAHOTAIRactsasascaffoldmoleculethroughrecruitingchromatinremoldingcomplexesPRC2toregulatetargetgenes[24,80]expression.LncRNAsplaypivotalrolesindiverseprocesses,suchaspluripotentstem151万方数据 第三军医大学博士学位论文[90,91]cellsreprogrammingandintracellulartrafficking.What’smore,manylncRNAsaredysregulatedindiversehumandiseases,includingcancerandtheyfunctionascrucialrolesinthepathogenesisandprogressionofdiseases.TheaberrantexpressionoflncRNAsappearstoaffectthetumorigenesis,proliferation,invasionandmetastasisincancertissues.However,theunderlyingmechanismsthatlncRNAsdysregulationcontributestothepathogenesisofcancerhavenotyetbeenwellcharacterizedtodate,furtherstudiesareneededtoexpandourunderstandingoflncRNAs.Atpresent,someconventionaltechniquestoscreennewlncRNAsareoftenperformedinexperiments,suchasoverexpressiontechnique,insituhybridizationtechniqueandsiRNA-mediatedgenesilencingtechnique.Therapiddevelopmentofbioinformaticstechnologyandtheariseofnewgenerationsequencingtechnology,includingmicroarrayandhighthroughputsequencingtechnologywillhelptoacceleratethedetectionandpredictionoflncRNAsmoreefficiently.SomedysregulatedlncRNAsarefoundtoplayasignificantroleinGCdevelopment.Inthisreview,wefocusedonseverallncRNAsdysregulatedinGC,elaboratedthedistinctfunctionsandknownmechanismsbywhichlncRNAsactinthecancer.AlthoughmanystudieshavepreliminarilyrevealedsomemolecularmechanismsoftheaberrantexpressionoflncRNAsinGC,furtherexplorationofexactmechanismsisstillneeded.ThealteredexpressionoflncRNAshasbeendemonstratedtobecorrelatedwithtumorgrowth,invasion,migrationandmetastasisandalsoshowimportantfunctionsinGC,suggestinglncRNAsasthepotentialtargetsforanti-tumortherapy.However,furtherstudiesareneededtotestwhethersuppressingtheexpressionoftheupregulatedlncRNAsorincreasingtheexpressionofdownregulatedlncRNAsinGCcellswouldbeappliedtoclinicalpractice.Inaddition,whetherarelationshipbetweendrugresistanceandlncRNAsexistsisstillneededtobefurtherstudied.Moreover,theabnormalexpressionoflncRNAsshowsthepositiveornegativecorrelationwiththeprognosisofpatientswithGC,providingusseveralassumptions,forexample,ifwecanusespecificlncRNAsasindicatorsfordiagnosisandthepredictionofprognosisinpatientswithGC.Nevertheless,theclinicalapplicationoflncRNAsrequiresmorein-depthexplorations.AlthoughaccumulatingstudieshaveelucidatedtheregulatoryfunctionsoflncRNAsinGC,therearestillmanyunsolvedquestionsinourcurrentunderstandingoflncRNAs.152万方数据 第三军医大学博士学位论文ThereisstillalongwayforfullyunderstandingthemechanismsoflncRNAs,andmoreexplorationsaboutlncRNAswillexpandourcomprehensionoverGC.WebelievethatfuturestudiesoflncRNAswillprovideusmoreunexpectedinsightsintotheirbiologicalfunctionsandwillfinallyallowtheirclinicalapplications.AcknowledgementsThisworkissupportedbygrantsfromtheNationalNatureScienceFoundationofChina(No.81201950).ConflictofintereststatementTheauthorsdeclarethattheyhavenocommercialorassociativeintereststhatrepresentaconflictofinterestinconnectionwiththeworksubmitted.153万方数据 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第三军医大学博士学位论文附录发表在Oncologist上的综述163万方数据 第三军医大学博士学位论文164万方数据 第三军医大学博士学位论文165万方数据 第三军医大学博士学位论文166万方数据 第三军医大学博士学位论文167万方数据 第三军医大学博士学位论文168万方数据 第三军医大学博士学位论文169万方数据 第三军医大学博士学位论文170万方数据 第三军医大学博士学位论文171万方数据 第三军医大学博士学位论文攻读博士学位期间发表的论文1.ZengS,XiaoYF,TangB,HuCJ,XieR,YangSM,LiBS:LongNoncodingRNAinDigestiveTractCancers:Function,Mechanism,andPotentialBiomarker.Theoncologist2015,20（8）:898-906.2.ShuoZeng,XiaXie,Yu-FengXiao,BoTang,Chang-JiangHu,Shu-MingWang,Yu-YunWu,HuiDong,Bo-ShengLi,Shi-MingYang.LongNoncodingRNALINC00675InteractswithVimentinandEnhancesItsPhosphorylationonSer83toSuppressGastricCancerProgression.Cancerletters201726;412:179-187.172万方数据 第三军医大学博士学位论文致谢本课题是在杨仕明教授的悉心指导下完成的。杨仕明教授知识渊博、治学严谨、工作勤奋、为人诚恳，令人敬仰，堪为科研工作者之楷模。他对科学研究一丝不苟的态度，对事业永无止境的追求，坚定不移永不放弃的精神是我永远学习的榜样，将终身鼓励我在科学的道路上勇往直前。在此谨向杨仕明教授表示衷心的感谢!衷心感谢新桥医院消化科的赵晓晏教授、董辉教授、王星研究员、凌贤龙教授、郭红教授、柏健鹰教授、李宜辉教授、张朋彬教授、李春华护士长、刘璐护士长等各位老师在学习和生活上的帮助。衷心感谢新桥医院消化科胡长江博士、唐波博士、肖煜峰博士、覃勇博士的指导和帮助。特别感谢黎伯胜、刘菁菁、刘恩、王苏敏、吴玉云、吴亚冉、何佳霖、陈思源、孙雪梅、常杏、节梦梦、杨新等在实验中给予的巨大帮助。本研究得到了国家自然科学基金资助，在此特别鸣谢国家自然科学基金委员会的支持。最后，感谢家人的关怀和鼓励，让我一直不懈地努力完成学业。173万方数据