RNAi抑制宫颈癌HeLa细胞survivin基因及其对顺铂敏感性影响的研究

The study of the RNA interference silencing of Survivin gene and reversal of drug resistance in cervical cancer HeLa cells.

目的:利用RNA i技术抑制宫颈癌HeLa细胞株Survivin基因,观察HeLa细胞株的细胞凋亡率、survivin蛋白表达及对顺铂(DDP)药物敏感性的变化。方法:以培养的宫颈癌HeLa细胞株为体外研究模型,设脂质体组、对照组、RNA i组进行实验。(1)半定量RT-PCR,W estern blot法检测Survivin mRNA、蛋白在各组细胞中的表达;(2)用流式细胞术分析各组细胞周期及细胞凋亡率的影响;(3)用MTT法分析DDP对各组细胞存活率作用的浓度。结果:(1)RT-PCR检测结果显示,Survivin扩增条带(206bp)的亮度差异显著,RNA i组明显弱于脂质体组及对照组。RNA i组Survivin mRNA相对含量较质脂体组明显下降,差异有统计学意义(P<0.01);W estern blot检测各实验组Survivin蛋白的表达,脂质体组与对照组Survivin蛋白条带明显存在,且基本一致,而RNA i组同样位置的条带基本消失;(2)RNA i组细胞阻滞在G0/G1期,G/M期减少,与脂质体组和对照组分别比较,差异有统计学意义(P<0.01);(3)MTT比色法检测结果显示,不同浓度顺铂处理后脂质体组、对照组、RNA i组的IC50分别为(0.298±0.035)、(0.147±0.031)、(0.012±0.001),RNA i组HeLa细胞对顺铂的药物敏感性增高。两者相比差异有统计学意义(P=0.000)。结论:RNA i明显抑制了Survivin在转录及翻译水平的表达;RNA i抑制Survivin基因表达,能明显提高宫颈癌HeLa细胞对顺铂的药物敏感性。

Objective：Through interferring the expression of Survivin with short hairpin RNA （shRNA） technology, to investigate the effect of downregulation of Survivin on cell apoptosis and chemosensitivity to cisplatin in cervical cancer HeLa cells. Methods： （ 1 ） Using HeLa cells as a model system, three groups were set up transfected with lipofectamine, RNAi control plamid and pSSURV （Survivin RNAi plasmid） ,respectively. The expression of Survivin in HeLa cells was measured at transcriptional and translational level by using RT-PCR and Western-blotting methods. （2）The effect on the cell cycle and apoptosis was analyzed with flow cytometry. （3） The viability of cells applied with different dose of DDP was determined by using the method of 3- [ 4,5-Dimethylthiazol-2-yl ] -2,5-diphenyhetrazolium bromide reduction （ MTT method）. Results： （ 1 ） RT-PCR and Western blotting demonstrated that Survivin expression was significantly decreased by transfection with RNAi targeting plasmid;the expression proportion was reduced by nearly 70%. DDP significantly strengthened the chemosensitivity of the cells with a dose-dependent manner. （2）RNAi-treated cells were arrested at G0/G1 phase, with a consequent decrease proportion in G2/M phase. It was satistifically significant from lipofectamine control and vehicle control at a level of P 〈 0. 01. （3）The cell viability of RNAi vehicle group was almost equally to that of the lipofectamine control group. On the contrary, the cell viability of pSSURV group was dramatically decreased, only almost 50% of that of control with high concentration of DDP. Conclusion： These results show that by inducing RNAi-targeting plasmid, Survivin expression can be effectively decreased at both transcriptional and translational level. The reduction of survivuin expression exerts significant effects on chemosensitivity of HeLa cells to DDP.