脂质体介导的小发夹干扰RNA对卵巢癌顺铂耐药细胞中切除修复交叉互补基因1表达的影响

Effect of Lipofectamine-Mediated Small Hairpin Interference RNA on Expression of Excision Repair Cross-Complementing Group 1 in Cisplatin Resistant Ovarian Cancer Cell Lines

目的:探讨脂质体介导的小发夹干扰RNA(shRNA)对卵巢癌顺铂耐药细胞中切除修复交叉互补基因1(ERCC1)表达的影响。方法:设计合成针对ERCC1基因的小发夹干扰RNA(ERCC1-shRNA),构建携带ERCC1-shRNA的重组质粒表达载体,采用脂质体Lipofectamine 2000转染COC1/DDP细胞。分别采用逆转录-聚合酶链反应(RT-PCR)及蛋白印迹法(Western blot)检测转染后细胞中ERCC1 mRNA及蛋白的表达,流式细胞计数检测细胞周期和细胞凋亡情况。结果:转染后COC1/DDP细胞中ERCC1mRNA表达较转染前明显降低(F=203.73,P<0.01),ERCC1蛋白表达亦显著下降。RNA干扰ERCC1基因后48h,COC1/DDP细胞的凋亡率较对照组有明显升高(t=20.65,P<0.01)。结论:构建的重组质粒表达载体转染COC1/DDP细胞可明显下调ERCC1 mRNA及蛋白的表达,诱导肿瘤细胞凋亡。

Objective： To investigate the interfering effect of lipofectamine-mediated small hairpin interfering RNA （shRNA） on expression of the excision repair cross-complementing group 1 （ERCCl） gene in ovarian cancer cell lines. Methods： The small hairpin interfering RNA targeting ERCCl gene （ERCCl-shRNA） was synthesized and designed. Recombinant plasmid expression vector （p^ERCC1-shRNA） which carrying ERCCl-shRNA was constructed and transfected to COCl/DDP cells with Lipofectamine 2000. RT-PCR and Western blot analysis were used to detect the expression of ERCCl mRNA and protein after transfection. Flow cytometry was used to detect cell cycle and apoptosis. Results： RT-PCR analysis showed that the expression of ERCCl mRNA and protein were significantly down-regulated after transfection （F = 203.73,P 〈 0.01）.The apoptosis rate was significantly raised after 48 hours of transfection （t = 20.65,P 〈 0.01）. Conclusion： The recombinant plasmid expression vector which carrying ERCCl-shRNA may remarkably down-regulate the expression of ERCCl mRNA and protein after transfection, and induce the apoptosis in COCl/DDP cells.