RNA干扰靶向沉默EGFR基因对卵巢癌耐药细胞凋亡影响的实验观察

Effect of RNA-mediated EGFR gene on resistance to cisplatin of multidrug-resistant ovarian cancer cells

目的:探讨RNA干扰表皮生长因子受体(EGFR)基因对卵巢癌顺铂耐药细胞株SKOV3/DDP凋亡的影响。方法:利用基因重组技术构建携带EGFR小发夹干扰RNA(pEGFR-shRNA)的重组质粒表达载体,脂质体法转染SKOV3/DDP细胞。实验分组:对照组(不进行干扰)、S1组(转染非特异性质粒)、S2组(转染特异性质粒)。用逆转录聚合酶链反应(RT-PCR)和免疫细胞化学法(ICC)检测转染后细胞内EGFRmRNA和蛋白的表达,流式细胞仪(FCM)分析细胞周期和凋亡率。结果:与对照组相比,S2组细胞EGFRmRNA的表达明显受抑制,抑制率达57%(P<0.01)。蛋白质的表达水平显著降低(P<0.01)。流式细胞仪检测结果显示,顺铂作用24h后,特异性转染组细胞周期分布发生明显改变,G0/G1期细胞比例增多,而S期细胞比例减少,凋亡率显著升高(P<0.01)。结论:RNA干扰EGFR基因通过抑制SKOV3/DDP细胞中EGFR基因的表达,恢复细胞对顺铂的敏感性。

OBJECTIVE：To investigate the effect of RNA-mediated EGFR gene on reversing cisplatin resistance of multidrug-resisitant ovarian cancer cell line SKOV3/DDP.METHODS：pEGFR-shRNA was synthesized in vitro by the genetic recombination and tansfected into SKOV3/DDP by liposome.There weref control group（no transfection）,S1 group C transfetion with non-specificity plasmid）and S2 group（transfection with specificity plasmid）.RT-PCR and immunocytochemistry（ICC）were performed respectively to detect the inhibitive effect of RNA interference on the mRNA level and protein level.The apopototic rates and cycle of SKOV3/DDP cells were detected by flow cytometry after having been treated with cisplatin.RESULTS：Compared with the control group,the expression of EGFR mRNA of sequence specific pEGFR-shRNA was down-regulated and the inhibition ratio was 57%（P〈0.01）,and the protein expression was also significantly reduced（P〈0.01）.The flow cytometric analysis showed that cell cycle distribution was changed（cells in G0/G1 increased,and decreased in S）and the ratio of apoptosis significantly increased after transfection（P〈0.01）.CONCLUSION：pEGFR-shRNA can rdeuce the expression of EGFR gene in SKOV3/DDP and reverse cisplation resisitance.