Livin异构体特异性shRNA对宫颈癌Hela细胞凋亡的影响

Apoptosis of cervical cancer cell line Hela induced by Livin isoform-specific shRNA

目的:应用构建成功的Livin异构体(BIRC71,BIRC72)短发夹RNA(short hairpin HNA,shRNA)真核表达载体转染Hela细胞,探讨其对Livin基因的沉默效应及诱导Hela细胞凋亡的作用。方法:采用脂质体转染法转染Hela细胞,荧光实时定量PCR、Western blotting检测转染前后宫颈癌Hela细胞Livin基因拷贝数和蛋白表达的变化,将shRNA表达载体流式细胞术检测不同时间(24、48、72h)的细胞凋亡率。结果:真核表达载体的转染效率48 h较24、72 h高;转染pGenesil-1- BIRC71、pGenesil-1-BIRC72后Hela细胞中Livin拷贝数明显减少(P<0.05),蛋白质表达水平显著降低(P<0.05);流式细胞术检测24、48、72 h凋亡率,转染Livin shRNA组细胞的凋亡率显著高于对照组(P<0.05),随时间延长(24→72 h)凋亡率相应增加。结论:靶向Livin的shRNA真核表达载体可以有效特异地阻断Livin基因的表达。

Objective： To transfect a recombinant short hairpin RNA （shRNA） expression vector targeting Livin gene isoform （ B1RCT1, BIRC72 ） into the cervical cancer cell line （ Hela cell ）, in an attempt to observe RNAi-mediated silencing on Livin gene and the induction of Hela apoptosis. Methods： Hela cells were transfected with the recombinant plasmid pGenesil-1-BIRC71, pGenesil-l-BIRC72 and pGenesil-1-HK via LipofectamineTM 2000. The expression levels of Livin was determined in Hela cells before and after transfection by fluorescence quantitative real-time PCR and Western blotting. The apoptosis rate of cells was determined by FCM 24,48 and 72 h after transfection. Results： The transfection efficiency at 48 h was higher than those at 24 and 72 h. After transfection with pGenesil-l-BIRC71 and pGenesil-l-BIRC72, gene and protein levels of Livin were significantly reduced （ P 〈 0.05 ）. The apoptosis rate of Livin shRNA transfection group was significantly higher than that of the control group, and increased with the prolongation of time （24, 48, and 72 h ; P 〈0.05 ）. Conclusion： pGenesil-1-B1RC71 and pGenesil-1-BIRC72 can effectively reduce Livin gene expression and induce apoptosis of Hela cells.