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作者姓名：赵亮论文题目：人结直肠癌发生和转移相关的蛋白质组学分析及候选蛋白LASP-1功能的初步研究作者简介：赵亮，男，1981年08月出生，2007年09月师从于南方医科大学丁彦青教授，于2009年06月获博士学位。中文摘要结直肠癌是常见的恶性肿瘤之一，在我国的发病率呈逐年上升的趋势。近些年来，虽然随着诊疗新技术的应用和化疗药物的不断更新，结直肠癌患者的治疗得到了极大地改善，但患者的生存率并没有显著地提高，主要原因是多数结直肠癌患者在行根治性手术前已出现了微转移。因此，筛选和鉴定在肿瘤发生及发展中起重要作用的关键分子，寻找早期诊断的生物标志物和抗肿瘤转移的药物靶点，阐明肿瘤发生及转移机制，是提高结直肠癌患者生存率和治愈率的关键问题，也是当前肿瘤研究的热点问题。肿瘤转移是一个多步骤、多基因参与的复杂过程，肿瘤转移的全过程受以下几个因素的调控：1、在转移启动初期E-cadherin、Ig超家族、选择素、整合素等参与肿瘤细胞间的脱离以及肿瘤细胞与胞外基质的粘附；2、肿瘤细胞或间质细胞分泌的蛋白水解酶及其抑制剂，在降解胞外基质和血管的基底膜过程中发挥重要作用；3、在运动因子、生长因子、归巢因子等的作用下，肿瘤细胞迁移到转移靶器官；4、血管增生因子VEGF、bFGF、IL-8、PDGF等促进新生血管的形成，伴随着肿瘤细胞的增生，形成新的转移灶，完成转移过程。鉴于目前对肿瘤转移机制的研究往往针对单个或少数几个分子以及关注基因水平的改变，缺乏系统性和综合性的分析，因此在所有这些调控因素中，究竟哪些分子在肿瘤转移中发挥关键作用，仍有待阐明。近年来开展的蛋白质组学技术为揭示肿瘤转移机制带来了新的希望。在前期研究中，我们以人结直肠癌高、低转移细胞株为研究对象，采用高通量的双向凝胶电泳联合质谱鉴定的比较蛋白质组学策略，鉴定了11个与结直肠癌转移相关的候选蛋白，其中SW620细胞株表达上调的蛋白质有磷酸甘油酸变位酶1，磷脂酰乙醇胺结合蛋白和高迁移率族蛋白B-1，而热休克蛋白27，膜联蛋白I，甲硫腺苷磷酸化酶，切丝蛋白1和表皮型脂肪酸结合蛋白在SW620中表达下调。大多数差异蛋白功能涉及肿瘤细胞生长、运动、粘附、凋亡等过程，研究结果为阐明结直肠癌转移机制及寻找预测结直肠癌转移的潜在标志物提供了理论依据。 然而一种标志物仅能在细胞株中检测得到，那么它在早期诊断及预后判断方面的临床价值无法得到体现。因此本研究采用高通量的蛋白质组学技术，在组织水平通过比较结直肠癌发生、发展不同阶段临床标本的蛋白质表达谱，寻找结直肠癌发生及转移相关蛋白，为阐明结直肠癌发生、转移机理，寻找肿瘤早期诊断和治疗的靶标提供重要理论依据。1、人结直肠癌组织的蛋白质组双向凝胶电泳分析为了更好的理解结直肠癌细胞的恶性转化和转移机理，寻找结直肠癌预后的标志分子，我们运用双向凝胶电泳技术对结直肠癌和配对正常结直肠粘膜组织进行了差异表达双向凝胶电泳分析，软件分析normal和CRC蛋白质表达谱发现36个蛋白质斑点在两组凝胶中有显著性差异表达(p<0.05)，其中在CRC组中表达升高的蛋白质斑点有17个，在CRC组中表达下调的蛋白质斑点有12个；定性差异表达分析(量相差10倍以上)有5个仅在CRC组中表达的蛋白质点，有3个仅在normal组中检测到。结肠癌组织根据是否伴发转移分为不伴发转移的结直肠癌(Non-metastasticCRC，nmCRC)和伴发转移的结直肠癌(MetastaticCRC，mCRC)，通过比较它们全蛋白质表达谱差异，有13个蛋白质斑点在mCRC组中表达升高，其中4个蛋白质斑点仅在mCRC组中检测到。2、双向凝胶电泳差异表达蛋白的质谱鉴定和免疫印迹验证以结直肠正常粘膜和肿瘤组织为研究对象，采用2-DE分离技术结合质谱鉴定了22个结直肠癌相关蛋白，其中9个蛋白质斑点在CRC组织中表达下调，它们分别被鉴定为热休克蛋白70、细胞角蛋白20、丙酮酸激酶同工酶、转胶蛋白。13个蛋白质斑点在CRC组织中表达上调，它们分别被鉴定为纤维蛋白原、Maspin、LIM和SH3蛋白1、钙粒蛋白B、RhoGDP解离抑制因子α、磷酸甘油酸变位酶1、mRNA结合蛋白AUF1、热休克蛋白90、钙磷脂结合蛋白III和蛋白酶体α亚基3。以不同转移潜能的结直肠癌组织标本为研究对象，蛋白质组学技术筛选得到了11个结直肠癌转移相关蛋白，它们分别被鉴定为热休克蛋白90、转胶蛋白、RhoGDP解离抑制因子α、原肌球蛋白、膜联蛋白V、CC趋化因子受体5、谷胱甘肽S-转移酶、IgE依赖组胺释放因子、热休克蛋白27和乳酸酰谷胱苷肽裂解酶。我们还采用迹免疫印迹技术对差异蛋白在蛋白水平进行验证，并探讨了候选差异蛋白在肿瘤发生、发展中的作用。筛选得到的差异蛋白绝大部分蛋白都与肿瘤恶性转化和转移等行为相关，涉及肿瘤细胞生长、运动、粘附、凋亡等过程，研究结果为阐明结直肠癌发生、发展的机制及肿瘤早期诊断、寻找预测结直肠癌转移的潜在标志物提供了理论依据。3、差异候选蛋白的表达和功能验证 疾病蛋白质组学和基因组学研究的能够发现一些新的标记分子和有效的药物作用靶标，给肿瘤预后判断研究带来新的希望。本研究前期应用比较蛋白质组学方法筛选出一批结直肠癌发生、发展相关蛋白质，对于这些蛋白的确切生物学和临床意义尚有待于进一步的验证和证明，在此我们选择其中感兴趣的结直肠癌发生相关蛋白transgelin和转移相关蛋白RhoGDI，通过生物学和临床研究以期初步探讨它们在结直肠癌发生发展中的作用，同时也进一步验证前期蛋白质组学筛选研究结果的可靠性，为后续针对专门靶点进行的研究奠定基础。转胶蛋白(Transgelin)，因分子量大小为22kDa而命名为SM22α，是我们采用蛋白质组学技术筛选得到的新的肿瘤抑制蛋白，其在肿瘤方面的研究甚少。本研究证实transgelin在结直肠癌组织中表达缺失，而去甲基化药物5-aza-dC处理能使transglein表达恢复，提示启动子甲基化是结直肠癌transgelin表达调控机制之一。免疫组织化学提示transgelin表达缺失不仅是结直肠癌恶性转化的重要标志物，还是结直肠癌患者不良预后的独立预测指标。RhoGDP解离抑制因子α(RhoGDPdissociationinhibitoralpha,RhoGDIαorRhoGDI)是我们采用蛋白质组学技术筛选获得的结直肠转移相关的蛋白，RhoGDI在伴有转移的结直肠癌转移组中高表达，但其在结直肠癌转移中的作用机制尚未明确。已知RhoGDI是RhoGTP酶功能的关键调节因子，而后者则在细胞骨架重组调控方面起重要作用。本研究证实RhoGDI在高转移潜能的细胞和结直肠癌组织中表达上调，RhoGDI过表达与肿瘤的侵袭表型相关。通过基因转染内源性低表达RhoGDI的结直肠癌HT29细胞能促进细胞的体外增殖和运动潜能。免疫组织化学检测RhoGDI过表达与肿瘤浸润、淋巴结转移和临床分期相关，并提示患者的不良预后。遗憾的是，该研究中多因素分析并不支持RhoGDI作为独立的结直肠癌患者预后指标，因此还需要在更大的样本量群体中进一步验证RhoGDI过表达的预后价值。4、候选蛋白LASP-1在结直肠癌发生、发展中的作用LASP-1cDNA(No.X82456)最初是由乳腺癌转移性淋巴结(Metastaticaxillarylymphnodes,MLN)cDNA文库中筛选克隆得到，因此命名为MLN50。MLN50基因染色体定位于17q11-q21.3，该区域还包括c-erbB-2和BRCA1癌基因，并在20-30%的乳腺癌病例中发生变异。MLN50mRNA全长约4,000个核苷酸组成，所编码蛋白由261个氨基酸组成，在它的氨基末端含有LIM(Lin-11,Isl-1andMec-3)功能域，羧基末端包含一个Src同源结构域3(Srchomology3,SH3)。鉴于MLN50的结构域构成而将其确定为一种新的LIM蛋白亚家族，其特点在于既含有LIM，又含有SH3结构域。因此MLN50也被命名为LIM和SH3蛋白1(LIMandSH3protein,LASP-1)。 我们采用蛋白质组学技术筛选并鉴定LASP-1在结直肠癌组织中表达上调，目前尚无其在结直肠癌发生、发展中的作用及机制的报道。通过对16例结直肠癌组织的免疫印迹检测证实LASP-1在结直肠癌组织中高表达，进一步采用免疫组织化学的方法检测126例临床结直肠癌组织样本中LASP-1的表达情况，统计分析表明LASP-1过表达与结直肠癌患者的淋巴结转移和临床分期密切相关，生存分析LASP-1过表达提示患者的不良预后，但是多因素分析并不支持LASP-1作为独立的预后指标预测患者临床预后。为进一步研究LASP-1在结直肠癌转移中的作用，我们分别采用基因转染和RNA干扰策略，将LASP-1基因稳定转染到低内源性表达LASP-1的SW480细胞株，同时将特异性针对LASP-1的siRNA转染到高内源性表达LASP-1的SW620细胞株，分析LASP-1对结直肠癌细胞生物学行为的影响。MTT法观察LASP-1能明显加快细胞的增殖速度，流式细胞仪分析细胞周期提示LASP-1可以通过促进细胞进入增殖期而提高细胞的体外增殖及克隆形成能力，AnnexinV-FITC凋亡染色表明LASP-1与细胞凋亡无显著性相关。通过侵袭小室和划痕实验检测LASP-1能增强细胞的侵袭和运动潜能，免疫荧光提示LASP-1可能参与肌动蛋白等细胞骨架的重构过程而改变细胞的运动潜能，从而导致肿瘤发生转移。5、LASP-1参与结直肠癌发生、转移分子机制的初步探讨结直肠癌的发生与转移的分子基础是一个立体、整体水平上的蛋白质间相互作用的结果，本研究以LASP-1为研究对象，期望通过高通量的“组学”技术，分别采用双向荧光差异凝胶电泳(2-DFluorescenceDifferenceGelElectrophoresis,2-DDIGE)技术筛选LASP-1相关信号通路的关键分子，采用FLAGPull-down技术筛选LASP-1相互作用蛋白，为深入研究结直肠癌的发生、发展机制提供新的思路，寻找治疗结直肠癌的靶点和筛选能够预测结直肠癌转移及预后的分子标志物。应用2-DDIGE联合质谱的蛋白质组学研究策略，获得50种LASP-1作用相关蛋白，其中与LASP-1作用正相关的蛋白21种，与LASP-1作用负相关的蛋白29种，对部分差异蛋白进行免疫印迹验证。为理解这些差异蛋白的功能，采用GOfact软件平台进行在线聚类分析。对47种蛋白所参与的生物过程进行注释，它们分别涉及细胞通信、细胞周期、细胞增殖、骨架蛋白重构、形态改变等过程；对45种蛋白所参与构成的细胞组件进行注释，它们在细胞内定位于胞浆、细胞骨架、细胞核、细胞膜、细胞器、蛋白复合物等部位；对49种蛋白质所介导的分子功能进行注释，它们分别发挥离子结合、核酸结合、蛋白结合、催化活性、酶调节活性、结构分子激活、转运蛋白活性等生物学作用。上述分析均表明，LASP-1蛋白能通过对细胞内关键蛋白的调控，改变细胞生物学行为，从而介导结直肠癌的发生及发展。通过FLAGPull-down的筛选策略，获得两种与LASP-1相互作用蛋白，78kDa葡萄糖调控蛋白(78kDaglucose-regulatedproteinprecursor,GRP78)和热休克同源蛋白71(heatshockcognate71kDaprotein,HSC71)，它们均为热休克蛋白家族成员，作为“分子伴侣”参与蛋白的折叠和转运过程。近年来不断有研究证实其与各种恶性肿瘤发生发展过程的密切相关。本研究结果提示它们可能与LASP-1存在相互作用，二者可能共同参与结直肠上皮细胞的恶性转化及演进过程，为进一步深入研究结直肠癌发生发展的机理提供了新的思路。 结论：1、采用双向凝胶电泳联合质谱的蛋白质组学研究策略，筛选出结直肠癌发生相关蛋白22个，结直肠癌转移相关蛋白11个；2、转胶蛋白(Transgelin)是本研究筛选获得的肿瘤抑制蛋白，transgelin表达缺失可能是结直肠癌恶性转化的重要标志物，也是预测结直肠癌患者预后不良的独立指标，启动子甲基化可能是结直肠癌transgelin表达调控的重要机制之一；3、RhoGDP解离抑制因子α(RhoGDIα/RhoGDI)是本研究筛选获得的结直肠癌转移相关蛋白，RhoGDI能促进结直肠癌细胞的体外增殖和运动潜能，RhoGDI过表达与患者结直肠癌浸润、淋巴结转移、临床分期及不良预后相关；4、LIM和SH3蛋白1(LASP-1)可以提高肿瘤细胞的增殖和克隆形成能力，增强肿瘤细胞的侵袭和运动潜能，LASP-1过表达与结直肠癌患者的淋巴结转移、临床分期及不良预后密切相关；5、LASP-1可以调节肿瘤细胞内生物过程、细胞组件和分子功能相关蛋白的表达，同时可能与78kDa葡萄糖调控蛋白(GRP78)和热休克同源蛋白71(HSC71)发生相互作用，改变细胞的生物学行为，促进结直肠癌的发生及发展。关键词：蛋白质组；双向凝胶电泳；结直肠癌；转移；预后；LIM和SH3蛋白1Proteomicanalysisofgenesis-andmetastasis-associatedproteinsandpreliminaryfunctionalstudiesofLASP-1incolorectalcancerZhaoLiangABSTRACTColorectalcancer(CRC)isthesecondworldwideleadingcauseofcancerdeathintheworld.InChina,CRCoccupiesthefifthpositioninthemortalitiescausedbycancer,anditsincidenceratestillcontinuestoincrease.DespitesignificantimprovementinthetreatmentofCRCoverthelastdecades,thankstotheintroductionofnewsurgicaltechniques,improvedradiotherapytechniques,andtheuseofchemotherapy,theoverallsurvivalrateofpatientswithCRChasnotchanged markedly.Oneofthemajorfactorsforthepooroutcomeismetastasis.Sofar,however,verylittleisknownaboutthemechanismsunderlyingmetastasis.Oncethekeyfactorsintumorprogressionwerescreenedandidentified,thedrugtargetsanddiagnosismarkerscouldbeobtainedaccordingly.Metastasisisacomplexmultistepmalignantprocess.Ithasbeenwidelyreportedthatmanymoleculeswereinvolvedinthecomplexprocessoftumorinvasionandmetastasis:1)E-cadherin,immunoglobinsuperfamily,selectinandintegrinhavebeensuggestedtotakepartinthedetachmentoftumorcellsfromtheprimarysiteandinteractionoftumorcellswiththesurroundingextracellularmatrix;2)Matrix-degradingenzymesandtheirinhibitorssecretedbytumorcellsormesenchymalcellshavebeenindicatedtobeinvolvedindegradationofextracellularmatrixandvascularbasement;3)Somegrowthfactorsandmovememtfactorshavebeenimplicatedtoplayroleinmigrationoftumorcellsintosecondarysites;4)Angiogeneticfactors,suchasVEGF,bFGF,IL-8andPDGF,havebeendemonstratedtobeimportantinneoangiogenesisanddistantmetastasis.Atpresent,mostoftheresearchassociatedwithmetastasissofarhasbeenfocusedonthegeneticchangesofrelatedmoleculesorsingleorfewproteinswithoutsystematicstudy.Butlittleisknownaboutthekeyfactorstotriggertumorigeniccellstoinitiatefurtherinvasionandmetastasisfacingwithsomanyregulatorsuptonow.Inthepreviousstudy,onthebasisoftheseconsiderations,proteomicstrategy,combinedwithtwo-dimensionalelectrophoresis(2-DE)separationandmassspectrometry(MS)identificationwithadvantageofhighresolution,highreproducibilitywasusedtoseparateandidentifydifferentiallyexpressedproteinsbetweenhighlyandlowlymetastaticsubpopulations.Inthisstudywehopedtofindoutaseriesofproteinclusterdeeplyinvolvedinmetastasisprocessandaddressedthequestionwhethertherewerenewproteinstobeassociatedwithmetastasis,moreover,toclarifythemetastasis-associatedfunctionmediatedbynewcandidateproteins.Finally,11metastasis-associatedproteinswereseperatedandidentifiedbycomparativeproteometechniqueusingestablishedmetastaticmodel,moreimportantly,themetastasis-associatedclinicalcharacterizationmediatedbythecandidateproteinswasfurthercharacterized.Oftheidentifiedproteins,theexpressionsofphosphoglyceratemutase1，phosphatidylethanolaminebindingproteinandhigh-mobilitygroupbox1wereelevatedinSW620cells.However,heatshockprotein27,annexinI,methylthioadenosinephosphorylase,cofilin-1andepidermalfattyacidbindingproteinweredown-regulatedinSW620cells.Mostofthecandidateproteinshavebeenevidencedtobesomehowassociatedwithvariousaspectsoftumormetastasissuchascellgrowth,motility,invasion,adhesion,apoptosisandtumorimmunity,etc.Theseresultsprovidethebasisforsearching forpotentialmarkersforCRCprogressionandgivesomecluestoelucidatethemechanismofCRCmetastasis.However,ifamarkercanbedetectedonlyinsurgicalspecimens,itsclinicalsignificanceislimited,especiallyforearlydiagnosisorprognosis.Onthebasisoftheseconsiderations,inthestudy,proteomicstrategy,combinedwith2-DEseparationandMSidentificationwithadvantageofhighresolution,highreproducibilitywasusedtocompareproteinprofilingofclinicaltissuesamplesfrompatientswithdifferentstage.Wehopedtofindoutaseriesofproteinclusterdeeplyinvolvedincarcinogenesisandmetastasisprocessandaddressedthequestionwhethertherewerenewproteinstobeassociatedwithgenesisandmetastasisoftumor,moreover,toclarifythegenesisandmetastasis-associatedfunctionmediatedbynewcandidateproteins.Themajorresultsareasfollows:1.2-DEanalysisofhumancolorectalcancertissueTobetterunderstandthemechanismunderlyingtheCRCgenesisormetastasisandtosearchpotentialmarkersforCRCprognosis,differentialproteomicanalysisonCRCandpairednormalmucosatissuewasconductedusing2-DEanalysis.Byimageanalysis,theexpressionlevelsof36proteinspotswerefoundtobesignificantlychangedbetweennormalmucosaandCRCtissues(p<0.05).Amongthem,theexpressionlevelsof17proteinspotswereincreasedinCRCtissuesandtheexpressionlevelsof12proteinspotsweredecreasedinCRCtissues.QualitativeanalysisshowedthatfiveproteinspotswereonlydetectedinCRCtissueandthreeproteinspotswereabsentinCRCtissue.ComparedwithproteinprofilingofnmCRCandmCRCtissues,upregulatedexpressionof13proteinspotswerefoundinmCRCtissues.Amongthem,fourproteinspotswereonlydetectedinnCRCtissues,butnotinnmCRCtisses.2.FurtherMSidentificationandimmunoblotverificationofdifferentlyexpressionproteinsWhencomparedwithnormalmucosaandCRCtissues,2-DEfollowedbymatrix-assistedlaserdesorption/timeofflightmassspectrometry(MALDI-TOFMS)wasutilizedtoidentify22CRCgenesis-associatedprotein.Oftheidentifiedproteins,theexpressionofnineproteins,suchasheatshock70kDaprotein1B,cytokeratin20,pyruvatekinaseisozymesM1/M2,transgelin,weredown-regulatedinCRCtissues.However,13proteins,suchasfibrinogen,maspin、LIMandSH3protein1,calgranulinB,RhoGDPdissociationinhibitoralphah,phosphoglyceratemutase1,A+U-richelementRNA-bindingfactor,heatshockprotein90,annexinA3,proteasomesubunitalphatype3,wereup-regulatedinCRCtissues. Whencomparedwithnon-metastaticCRCandmetastaticCRCtissues,11CRCmetastasis-associatedproteinswereidentifiedasheatshockprotein90β,transgelin,RhoGDPdissociationinhibitoralpha,RhoGDIα,tropomyosin,annexinV,ccchemokinereceptor5,glutathione-transferase,IgE-dependenthistamine-releasingfactor,heatshockprotein27andlactoylglutathionelyase.Immunoblottinganalysiswasusedtofurtherverifycandidateproteinsinordertoensurethereliabilityoftheproteomeresults.Theverificationindicatedthatmostofthedifferencesofproteinexpressiondisapalyedby2-DEwasconvincing.Furthermore,thefunctionalimplicationsduringtumorgenesisandprogressionofalterationinthelevelsofthesecandidateproteinswerediscussed.Mostofcandidateproteinshavebeenevidencedtobesomehowassociatedwithvariousaspectsoftumormalignancyandmetastasissuchascellgrowth,motility,invasion,adhesion,apoptosisandtumorimmunity,etc.TheseresultsprovidethebasisforsearchforpotentialmarkersforCRCearlydiagnosisandprognosisandgivesomecluestoelucidatethemechanismofCRCgenesisandmetastasis.3.ExpressionalandfunctionalverificationofcandidateproteinsDiseaseproteomicandgenomicapproachesmaybehelpustofindsomenovelbiomarkersordrugtarget,whichmaybeusedtopredictclinicalprognosisofpatients.Inourpreviousstudy,wescreenedaseriesofCRCgenesisandprogression-associatedproteins.Furtherconfirmationandverificationwereneededtoclarifytheirprecisebiologicalandclinicalsignificance.Amongthecandidateprotein,weselectedinterestingCRCgenesis-associatedproteintransgelinandmetastasis-associatedproteinRhoGDItoinvestigatetheirfunctioninCRCdevelopmentandprogression.Transgelin,a22-kDaproteinalsocalledSM22,wasidentifiedasanoveltumorsuppressorprotein,butlittleisknownaboutthisproteinintumorssofar.Aremarkablereducedexpressionoftransgelinwasfoundincolorectalcancersamplescomparedwithnormalcolorectalmucosa.Theeffectof5-aza-2’-deoxycytidineasademethylationagentwouldobviouslyrestoretheoriginalexpressionleveloftransgelin,implicatingDNAhypermethylationoftransgelinplaysanimportantroleintheregulationoftransgelintranscriptionincolorectalcancer.Asacontrol,theinvestigationatcelllinelevelconfirmsthattransgelinproteincomesfromepitheliumbutnotmesenchymalcells.Further,immunohistochemicalstainingfortransgelinwasperformedonparaffinsectionsof62and126casesofnormalcolorectalmucosaandcolorectalcancerspecimens,respectively.Ascomparedtonormalcolorectaltissue,weobservedasignificantlylowertransgelinexpressionincolorectal cancersamples.Survivalanalysisdemonstratedthatpatientswithouttransgelinexpressionhadshorteroverallsurvival,whereaspatientswithtransgelinexpressionhadbettersurvival.Multivariateanalysisshowedthatnegativetransgelinexpressionwasanindependentprognosticindicatorforpatient’ssurvival.Ourresultssuggestthattransgelinasasuppressormayserveasimportantbiomarkersofmalignancy.Lossoftransgelininvolvesgenepromoterhypermethylationandiscloselyassociatedwithpooroverallsurvivalincolorectalcancerpatients.TheGDPdissociationinhibitors(GDIs)arepivotalregulatorsofRhoGTPases,whichareessentialfortumorprogression,particularlyintheareaofmetastasis.OnememberofGDIswasidentifiedasRhoGDI(RhoGDP-dissociationinhibitoralpha,RhoGDIαorRhoGDI),butlittleisknownaboutthisproteinintumors.Inthisstudy,weusedcomparativeproteomicanalysistoshowthatRhoGDIismarkedlyup-regulatedinmetastaticCRC.TheelevatedlevelofRhoGDIproteininmetastaticCRCwasconfirmedbyWesternblotatthetissueandcelllevels.Further,weanalyzedRhoGDIproteinexpressionin126clinicopathologicallycharacterizedCRCcasesbyimmunohistochemistry.StatisticalanalysisshowedthatthereweresignificantdifferencesofRhoGDIoverexpressioninpatientscategorizedaccordingtotumorinvasion,lymphnodemetastasisandclinicalstage.AtrendwasalsoidentifiedbetweenhighexpressionofRhoGDIandshorteroverallsurvival.Unfortunately,multivariateanalysisdidnotsupportthatRhoGDIwasanindependentprognosticindicatorforpatient’ssurvival.Inthepresentwork,wealsoanalyzedtheeffectofRhoGDIonCRCcellline.Genetransfection-mediatedoverexpressionofRhoGDIinHT29cells,containingalowdetectablelevelofendogenousRhoGDI,resultedinasignificantincreaseincellproliferationandmotilityinvitro.ThesedatasuggestthatRhoGDImaypromoteCRCprogressionandmetastasisbystimulatingtumorcellgrowthandmigration.4.TheroleofLASP-1proteininCRCdevelopmentandprogressionTheLASP-1genewasinitiallyidentifiedtogetherwiththreeothergenesfromacDNAlibraryofmetastaticaxillarylymphnodes(MLN)fromhumanbreastcancerandthereforecalledMLN50.Allfourgenesweremappedtochromosomalregion17q11-q21.3,aregionknowntocontainthec-erbB-2andtheBRCA1oncogeneandtobealteredin20–30%ofallbreastcancers.Northernblotanalysisrevealedthattheapproximately4.0kblongmRNAofMLN50isubiquitouslyexpressedatbasallevelsinnormaltissueandoverexpressedin8%ofalltestedhumanbreastcancertissues(5of61).SequenceanalysisshowedthatMLN50encodedaputativeproteinof261residuescontainingaLIMmotifatitsaminoterminusandasrchomology3(SH3)domainatitsC-terminalpart.ThisdomainorganizationdefinedanewLIMproteinsubfamilycharacterizedbythecombinedpresence ofLIMandSH3domains.MLN50wastermedaccordingly:LIMandSH3Protein1–inshortLASP-1.IncreasedexpressionofLASP-1proteinwasscreenedandidentifiedinCRCtissuebyproteomicapproaches.Sofar,nopaperwasinvolvedinitsfunctionandmechanisminCRCgenesisanddevelopment.WesternblotanalysisdemonstratedLASP-1overexpressioninCRCtissue.FurtherimmunohistochemicalassaydetectedLASP-1expressionin126casesofparaffininbeddingCRCspecimens.StatisticanalysisindicatedthatLASP-1overexpressionwascloselyrelatedtolymphnodestatusandclinicalstageofCRCpatients.AtrendwasalsoidentifiedbetweenhighexpressionofLASP-1andshorteroverallsurvival.Unfortunately,multivariateanalysisdidnotsupportthatLASP-1wasanindependentprognosticindicatorforpatient’ssurvival.GenetransfectionandRNAinterferencewereusedtofurtherstudytheroleofLASP-1inCRCmetastasis.LASP-1genewasstablytransfectedtoSW480cellswithendogenouslowLASP-1expression.Ontheotherhand,target-specificsiRNAoligowastransfectedtoSW620cellswithendogenoushighLASP-1expression.Changesofcellularbiologicalbehaviorswereanalyzed.InvitrogrowthofcellswasincreasedaftertransfectionofLASP-1genebyMTTassay.FlowcytometryindicatedthatLASP-1promotedcellintoperiodofproliferation,whichachieveenhancementofcellproliferationinvitroandabilitytoformclonies.AnnexinV-FITCstainshowedthattherewasnosignificantrelationshipbetweenLASP-1andcellapoptosis.LASP-1increasedpotentialofcellinvasionandmigrationinvitrodeterminedbytranswellassayandwound-healingassay.FurtherimmunofluorescencesuggestedthatLASP-1maybechangecellmotilitybyparticipatinginreconstitutionofcytoskeleton,suchasactin,andcausetumormetastasis.5.PreliminarilyexploringthemolecularmechanismunderlyingLASP-1-mediatedCRCgenesisandmetastasisHowdidLASP-1promoteCRCgenesisandmetastasis?Toreviewliteratures,sofar,noresearchwasinvolvedinthemolecularmechanismofLASP-1.Asallknow,protein-proteininteractionnetworksatthewholelevelleadtotumorgenesisandprogression.Therefore,forfurtherinvestigatingmolecularmechanismunderlyingCRCgenesisandprogression,highthroughputportomictechniques,including2-DFluorescenceDifferenceGelElectrophoresis(2-DDIGE)andFLAGPull-down,wereusedtoscreentoLASP-1-relatedsignalmoleculesandinteractingproteins.Onthebasisofthem,thedurgtargetsandprognosticmarkerscouldbeobtainedaccordingly.Firstly,throughprotomicstrategybasedon2-DDIGE,wescreenedandobtained50LASP-1-associatedproteins.Amongthem,21proteinswerepositivelyrelatedtoLASP-1 expressionand29proteinswerenegativelyrelatedtoLASP-1expression.ImmunoblottingdetectionwasusedtoconfirmreliabilityofthedifferentiallyexpressedLASP-1-associatedproteins.Tounderstandtheoverallfunctionoftheseidentifiedproteinsandsupplyinformationfundamentalforfurtherbioinformaticsexploration,weanalyzedallidentifiedproteinsusingtheonlineGOfactinterface.Atotalof47proteinswereannotatedasbeingidentifiedwithaspecificbiologicalprocess.Weidentifiedthemwithrolesincellcommunication,cellcycle,cellproliferation,cytoskeletonorganization,morphogenesis,etal.Atotalof45proteinswereannotatedasbeingassociatedwiththecellularcomponent.Thereproteinsmostlyoccurincytoplasm,cytoskeleton,nucleus,plasmamembrane,intracellularorganelle,proteincomplex,etal.Atotalof49proteinswereannotatedasbeingidentifiedwithmolecularfunction.Weidentifiedthemwithrolesinionbinding,nucleicacidbinding,proteinbinding,catalyticactivity,enzymeregulatoractivity,transporteractivity,etal.Aboveallmentioned,LASP-1proteinmayregulatemanyimportantintercellularproteinexpressions,whichcouldchangecellularbiologicalbehaviorsandcauseCRCprogression.Atthesametime,throughscreeningstrategybasedonFLAGPull-down,weobtainedtwoLASP-1-interactedproteins,identifiedas78kDaglucose-regulatedproteinprecursor(GRP78)andheatshockcognate71kDaprotein(HSC71),respectively.Asmembersofheatshockproteinfamily,theyplaytheroleofmolecularchaperonesinproteinfoldingandtransportprocess.Recently,increasedresearchesdemonstratedtheircloserelationshipwithtumorgenesisandprogression.Ourstudysuggestedthatintercellularprotein-proteininteractionsexistedbetweenGRP78orHSC71andLASP-1protein,whichmaybecontributetomalignanttransformationandprogressionofcolorectalepithelialcells.FurthervalidationstudieswouldprovidecluesforelucidatemolecularmechanismofCRCgenesisandprogression.Conclusion:1.Wehavescreenedandidentified22CRCgenesis-associatedproteinsand11CRCmetastasis-associatedproteinsusingproteomicstrategybasedon2-DEanalysis.2.Transgelinwasscreenedastumorsuppressorproteinbyproteomicapproaches.Transgelinasasuppressormayserveasimportantbiomarkersofmalignancy.Negativetransgelinexpressionwasanindependentprognosticindicatorforpatient’soutcome.DNAhypermethylationplaysanimportantroleintheregulationoftransgelintranscriptioninCRC.3.RhoGDP-dissociationinhibitoralpha(RhoGDIαorRhoGDI)wasscreenedasCRC metastasis-associatedproteinbyproteomicapproaches.RhoGDImaypromoteCRCprogressionandmetastasisbystimulatingtumorcellgrowthandmigration.ThereweresignificantdifferencesofRhoGDIoverexpressioninpatientscategorizedaccordingtotumorinvasion,lymphnodemetastasis,clinicalstageandshorteroverallsurvival.4.IncreasedexpressionofLIMandSH3Protein1(LASP-1)proteinwasscreenedandidentifiedinCRCtissuebyproteomicapproaches.LASP-1promotedabilityofcellproliferationandclonyformation,andalsoincreasedpotentialofcellinvasionandmigration.LASP-1overexpressionwasrelatedwithlymphnodemetastasis,clinicalstageandpoorprognosisofpatientswithCRC.5.LASP-1proteinmayregulatemanyimportantintercellularproteins,whichinvolvedinallkindsofdifferentbiologicalprocess,cellularcomponentandmolecularfunction.Ontheotherhand,LASP-1maybeinteractotherproteins,suchasGRP78andHSC71.BoththeabovemechanismscouldchangecellularbiologicalbehaviorsandjointlycontributetoCRCgenesisandprogression.Keywords:Proteome;Two-dimensionalgelelectrophoresis;Colorectalcarcinoma;Metastasis;Prognosis;LIMandSH3protein1