RNA干扰抑制FUBP1基因表达对人胃癌细胞系SGC7901生物学功能的影响

1·中文论著摘要·RNA干扰抑制FUBP1基因表达对人胃癌细胞系SGC7901生物学功能的影响南方医科大学刘一柏目的构建针对靶向基因FUBP1（farupstreamelementbindingprotein1，FUBP1）的慢病毒载体LV-FUBP1-RNAi，转染胃癌SGC-7901细胞，探讨应用RNA干扰技术抑制FUBP1基因的表达对人胃癌细胞增殖、细胞迁移、细胞周期和凋亡等生物学功能的影响，初步评价FUBP1基因沉默在胃癌基因治疗中的应用价值。方法构建三组不同RNA干扰序列的慢病毒载体LV-FUBP1-RNAi及阴性对照序列的慢病毒载体Negative-RNAi，通过慢病毒转染预实验选择合适的感染复数分别转染胃癌细胞SGC7901后利用荧光定量逆转录-聚合酶链反应(QRT-PCR)检测FUBP1在mRNA的表达水平，筛选出有效的RNA干扰序列用于后续实验。将实验分为FUBP1-RNAi组、Negative-RNAi阴性对照组（标记为NC）和空白对照组（标记为CON），应用QRT-PCR及蛋白免疫印迹（Westernblot）检测FUBP1在mRNA和蛋白质水平的表达量。应用CCK-8法检测各组细胞增殖、Transwell及划痕实验检测各组细胞迁移能力、流式细胞仪检测各组细胞周期分布情况及细胞凋亡。结果成功构建了三组不同RNA干扰序列的慢病毒载体FUBP1-RNAi-KD1、FUBP1-RNAi-KD2、FUBP1-RNAi-KD3；通过慢病毒转染预实验选择MOI=30的感染复数成功转染各组胃癌SGC7901细胞并筛选出有效的RNA干扰序列FUBP1-RNAi-KD2（相对于NC组FUBP1抑制效率高达70.4%）；QRT-PCR结2果显示：相对于NC组，RNA干扰组（FUBP1-RNAi-KD2）FUBP1mRNA的表达量明显下调，抑制率达70.4%（P0.05）；Westernblot结果显示：相对于NC组，RNA干扰组FUBP1蛋白质的表达量明显下调，抑制率达74.4%（P0.05）；CCK-8结果显示：相对于NC组和CON组，RNA干扰组连续5天胃癌细胞增殖倍数降低（P0.05）；PI-FACS细胞周期检测结果显示：相对于NC组和CON组，RNA干扰组S期细胞比例明显升高（达60.74%，P0.05），G0/G1期和G2/M期细胞比例分别降低达30.48%（P0.05）、8.78%（P0.05）；细胞凋亡、Transwell及划痕实验结果显示：通过RNA干扰技术沉默FUBP1基因后对胃癌SGC7901细胞凋亡和迁移无明显影响（P0.05）。结论慢病毒载体FUBP1-RNAi-KD2介导的FUBP1基因沉默能够有效的抑制FUBP1在人胃癌SGC7901细胞系内mRNA和蛋白质水平的表达量；通过RNA干扰技术沉默FUBP1基因的表达能够抑制胃癌细胞SGC7901的增殖，影响细胞周期的分布，将细胞周期阻滞在S期，抑制细胞生长；但是对细胞凋亡及迁移能力无明显影响；通过RNA干扰技术特异而有效的沉默FUBP1基因在胃癌的基因治疗中具有一定的价值。关键词RNA干扰；慢病毒转染；远端上游元件结合蛋白1；胃癌；基因治疗3·英文论著摘要·TheeffectofRNAinterferenceinhibitingFUBP1geneexpressiononthebiologicalfunctionofhumangastriccancercelllineSGC7901ObjectiveThispaperaimedtoconstructFUBP1-RNAilentiviralvectortotransfectintohumangastriccancercelllineSGC7901andinvestigatetheeffectofinhibitingFUBP1geneexpressiononthebiologicalfunctionofgastriccancercellproliferation,metastasis,cellcycleandapoptosis.Throughthisstudy,wecanevaluatethevalueofFUBP1genesilencinginthetreatmentofgastriccancer.MethodsTodesignFUBP1-RNAilentiviralvectorwiththreedifferentkindsofRNAinterferencesequencesandNegative-RNAilentiviralvectorwithnegativecontrolsequence.ThenchoicethereasonableMOIviapreliminaryexperimentoflentiviraltransfectiontotransfectintohumangastriccancerSGC7901.Thequantitativereversetranscription-polymerasechainreaction(QRT-PCR)wasusedtodetectthemRNAexpressionlevelofFUBP1toselecttheeffectiveRNAinterferencesequencewhichwasusedinthefollowingexperiment.Wedividetheexperimentintothreegroups,whichwereFUBP1-RNAinterferencegroup,Negative-RNAicontrolgroupandblankcontrolgroup.TheQRT-RCRandWesternblottingwereusedtodetectthemRNAandproteinexpressionlevelofFUBP1.CCK-8wasusedtodetectcellproliferationofeachgroup.TranswellandScratchTestwereusedtodetecttheacticityofcellmigration.PI-FACSwereusedtodetectCellcycledistributionandapoptosisofeachgroup.Results4LentiviralvectorwiththreedifferentkindsofRNAinterferencesequencesweresuccessfullyconstructed,includingFUBP1-RNAi-KD1,FUBP1-RNAi-KD2andFUBP1-RNAi-KD3.ThenchoiceMOI=30viapreliminaryexperimentoflentiviraltransfectiontotransfectintohumangastriccancerSGC7901andselectFUBP1-RNAi-KD2astheeffectiveRNAinterferencesequence(inhibitionrateofFUBP1wasupto70.4%WhichcomparedwithNegative-RNAigroup).ComparedwithNegative-RNAigroup,RT-PCRandWesternblottingresultsshowedthatthemRNAandproteinexpressionlevelofFUBP1geneinFUBP1-RNAi-KD2groupwererespectivelyreducedby77.6%(P0.05)and74.4%(P0.05).TheCCK-8testshowedthatgastriccancercellproliferationfoldinfivedaysofFUBP1-RNAi-KD2groupweredecreased(P0.05)Whichcomparedwithnegativecontrolgroupandblackcontrolgroup.FlowcytometryresultsshowedthatthenumberofgastriccancercellinSphaseofFUBP1-RNAi-KD2groupwasupto60.74%(P0.05)WhichcomparedwithNegative-RNAigroupandblackcontrolgroup,whilethenumberofgastriccancercellinG0/G1phaseandG2/Mphasewererespectivelydecreasedto30.48%(P0.05)and8.78%(P0.05).Onthecontrary,silencingofFUBP1geneviaRNAinterferencetechnologyhadnosignificanteffectonapoptosisandmigrationofgastriccancerSGC7901cells(P0.05).ConclusionsSilencingofFUBP1geneviaFUBP1-RNAilentiviralvectorcaneffectivelysuppressthemRNAandproteinexpressionlevelofFUBP1ingastriccancercellSGC7901.SilencingofFUBP1genecaneffectivelyinhibitgastriccancercellproliferation,affectdistributionofcellcycle,arrestcellcycleinSphasetosuppresscellgrowth.Whilethereisonsignificanteffectonapoptosisandmigration.ThespecificandeffectivesilencingofFUBP1geneviaRNAinterferencetechniquehasacertainvalueinthegenetherapyofgastriccancer.Keywords5RNAinterference;lentiviraltransfection;FUBP1;gastriccancer;genetherapy6·英文缩略语表·英文缩写英文全称中文译名FUBPFarupstreamelementbindingprotein远端上游元件结合蛋白RNARibonucleicacid核糖核酸RNAiRNAinterferenceRNA干扰dsRNADoublestrandedRNA双链RNAsiRNAsmallinterferingRNA小干扰RNARISCRNAindecedsilencingcomplexRNA诱导的沉默复合物FUSEFarupstreamelement远端上游元件TFIIHTranscription/repairfactorIIH转录修复因子IIHFBSFetalbovineserum胎牛血清PBSPhosphatebuffersaline磷酸盐缓冲液DMSODimethylsulfoxide二甲基亚砜RT-PCRReversetranscription-polymerasechainreaction逆转录-聚合酶链反应WBWesternBlot免疫印迹法RnaseRibonuclease核糖核酸酶DEPCDiethypyrocarbonate焦炭酸二乙酯PAGEPolyacrylamidegelelectrophoresis聚丙烯酰胺凝胶电泳法SDSSodiumdodecylsulfonate十二烷基磺酸钠TBSTTris-BufferedSalineTween-20缓冲液PVDFPolyvinylidenefluoride聚偏二氟乙烯ECLEle