siRNA沉默HPV18-E7基因表达对人宫颈癌HeLa细胞凋亡和增殖的影响  

Effect of Apoptosis and Cell Proliferation Directed by siRNA Interference Targeting HPV18-E7 Gene in Human Cervical Cancer (HeLa) Cells

作　　者：付海丹 宋毓平 张庆华[2] 田训[2] 陈宏伟[2] 李贺梅[2] 曾真[2] 李瑞[2] 

机构地区：[1]湖北省武汉市东湖医院,武汉430074 [2]湖北省武汉市中心医院妇产科,武汉430014

出　　处：《病毒学报》2017年第4期535-540,共6页Chinese Journal of Virology

基　　金：武汉市卫生局临床医学科研项目资助(项目号:WX11B05);题目:多细胞球体模型分析缺氧诱导因子(HIF-1α)在宫颈癌发展演进进程中~~

摘　　要：研究小干扰RNA(siRNA)沉默18型人乳头瘤病毒(Human papillomavirus 18,HPV18)E7基因对人宫颈癌HeLa细胞凋亡和增殖的影响。培养人宫颈癌HeLa细胞株,构建靶向E7基因的两条siRNA,转染HeLa细胞株(实验组:分为siE7-1组和siE7-2组),以不做任何处理的HeLa细胞作为空白对照组(NC),转染无功能siRNA的HeLa细胞作为阴性对照组(Scramble,SCR)。转染48h后,采用实时荧光定量PCR(RT-PCR)检测转染E7siRNA后HeLa细胞mRNA含量变化;采用蛋白免疫印迹法(Western blot)检测E7蛋白表达量变化;流式细胞术Flow cytometry(FCM)检测HeLa细胞凋亡情况;克隆形成和CCK-8检测HeLa细胞生长增殖情况。与NC组相比,siE7-1组和siE7-2组E7基因mRNA表达明显下调,差异有统计学意义(P<0.01);转染48h后,与NC组相比,siE7-1和siE7-2实验组HeLa细胞内HPV18-E7蛋白表达水平明显下降(P<0.05);转染48h后,siE7-1组和siE7-2组的细胞凋亡率分别为17.78%和36.44%,与NC组相比明显增多,差异有统计学意义(P<0.01),细胞凋亡结果提示siRNA沉默E7促进HeLa细胞凋亡;CCK-8实验结果显示siE7-1组和siE7-2组细胞生长增殖抑制,差异有统计学意义(P<0.01);克隆形成实验结果与CCK-8实验结果一致,siE7-1组和siE7-2组HeLa细胞形成的克隆数明显少于NC组,细胞生长增殖抑制(P<0.01)。研究结果表明,沉默E7基因促进人宫颈癌HeLa细胞凋亡,抑制增殖,为宫颈癌治疗提供潜在靶点。We wished to investigate the effect of silencing of the human papillomavirus 18（HPV18）E7gene on the apoptosis and proliferation of a human cervical cancer cell（HeLa）line by small interfering RNAs（siRNAs）.Two HPV18-E7gene-specific siRNAs were designed and transfected into HeLa cells,and denoted＂siE7-1group＂and＂siE7-2group＂.Non-transfected HeLa cells were used as the＂normal control＂group（NC）.Non-functional scramble siRNA was transfected using the same method and named as the＂SCR group＂.Reverse transcription-polymerase chain reaction and western blotting were used to detect expression of the E7 gene at mRNA and protein levels.Flow cytometry was used to detect apoptosis after silencing of the E7 gene.A colony-formation assay and CCK-8assay were used to monitor cellular proliferation.Expression at mRNA and protein levels of the E7 gene in siE7-1and siE7-2groups decreased significantly compared with that of the NC group（P0.01）.Percent apoptosis in siE7-1and siE7-2groups was17.78% and 36.44%at 48 hafter transfection,which was significantly different compared with that of the NC group（P0.01）,thereby implying apoptosis induction after silencing of the E7 gene.Results of the CCK-8assay and colony formation assay were consistent with each other,and showed proliferation suppression in siE7-1and siE7-2groups compared with the NC group（P0.01）.These data suggest that siRNA interference of the E7 gene can induce apoptosis and inhibit the proliferation of HeLa cells.Therefore,the E7 gene could be a potential target site in the treatment of cervical cancer.

关 键 词：宫颈癌 HPV18-E7基因 凋亡 增殖 基因沉默 

分 类 号：R373.9[医药卫生—病原生物学]