靶向sirna阻断nf-kb信号通路对神经母细胞瘤侵袭转移的影响

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1、靶向siRNA阻断NF—KB信号通路对神经母细胞瘤侵袭转移的影响中文摘要目的探讨重组慢病毒介导siRNA(小干扰RNA)靶向阻断NF—KB信号通路对人神经母细胞瘤QDD删，细胞侵袭转移的影响。方法根据人p65基因序列，设计针对基因不同靶点的siRNAl、siRNA2、siRNA3、siRNA44条siRNA序列及其对照序列NC，慢病毒介导转染QDDQ—NM。，采用实时荧光定量PCR(RT—PCR)和Westernblot法从设计的4条干扰序列中筛选出siRNAI序列在转染72h干扰效果最好。在转染72h后，采用RT-PCR和Westernblot法检测NF—KB下游基

2、因CXCR4、VEGFmRNA转录和蛋白表达情况；Transwell小室检测各组间瘤细胞的转移侵袭能力。结果转染72h后，PT—PCR显示，siRNAI—NF—KBCXCR4、VEGFmRNA表达分别为空白对照组0．66倍、0．76倍，差异有统计学意义(P<0．05)；Westernblot显示，siRNA卜NF—KBCXCR4、vEGF蛋白表达分别为空白对照组0．67倍、0．76倍，差异有统计学意义(P<0．05)；Transwell小室显示，I(实验组)、II(空载对照组)、HI(空白对照组)穿过Matrigel胶的细胞数分别为34．6000±3．9749；45．

3、6000±2。3021；54．8000±2．3874，各组问差异有统计学意义(P

4、ToexploretheeffectsoftheNF-kappaBpathwayblockedbysmallinterferingRNA(siRNA)mediatedbYlentivirusontheinvasionandmetastasisofhumanneuroblastomacancercellQDDQ—NMl．MethodsAccordingtohumanp65genesequences，4siRNAstargetingdifferenttargetsofp65andNCcontrolsequencesweresynthesizedandtmusfectedi

5、ntoQDDQ删1．Andthebestsequenceoftransfectioninterferenceeffectwasscreenedbyreal-timefluorescentquantitativereversetranscription-polymerasechainreaction(RT-PCR)andWesternblot．TheinterferingeffectofsiRNA1wasoptimalamong4siRNAsafter72h．Atthistime，theexpressingofNF-r33downstreamgenesCXCR4andV

6、EGFatmRNAandproteinlevelswerealsodetected．ThecelluarinvasionandmetastasiscapabilityofdifferentgroupswereevaluatedbyTranswellassay．ResultsAt72hpost-transfection,comparedwithcontrolgroups，RT-PCRshowedthattheexpressionsofCXCR4andVEGFmRNAtargetingsiRNA1respectivelydecreased(0．66times，0．76ti

7、mesP<0．05)．AndWesternblotindicatedthattheexpressionofCXCR4andVEGFproteinrespectivelydecreased(O．67times，0．76times，P<0．05)；TranswelldemonstratedthatthenumberofcellspassingthroughMatrigelrubberofsiRNA1interference，transfectioncontrolandcontrolgroupswere34．6000士3．9749：45．6000士2．30