RNA干扰survivin基因对卵巢癌细胞生长凋亡及药物敏感性的影响

目的 探讨下调survivin基因对卵巢癌顺铂(DDP)耐药细胞株SKOV3/DDP细胞生长、凋亡及药物敏感性的影响.方法 构建survivin基因的小干扰RNA(siRNA)表达载体pSilencer-survivin,用脂质体方法转染SKOV3/DDP细胞株,另设未转染组和转染pSilencer-control组作为对照.显微镜下观察转染前后细胞的变化,逆转录聚合酶链反应(RT-PCR)和Western blot分别检测survivinmRNA及蛋白的表达,二苯基溴化四氮唑蓝(MTT)法检测细胞生长情况,流式细胞仪检测细胞凋亡及周期的变化.各组细胞加入DDP,检测其对DDP药物敏感性的影响.结果 survivin siRNA可明显下调SKOV3/DDP细胞中survivin mRNA及蛋白表达水平.SKOV3/DDP细胞生长受到明显抑制,生长曲线低平.转染48 h后,转染pSilencer-survivin组细胞凋亡率为19.1%,明显高于末转染组(2.6%)和转染pSilencer-control组(3.5%),G1/G0期细胞所占的比例增高,而G2/M期细胞所占的比例降低.转染pSilencer-survivin组细胞的IC50明显降低,为1.16 μg/mi.结论 survivin siRNA可下调SKOV3/DDP细胞中survivin的表达,使细胞生长减慢,凋亡增加,对DDP的药物敏感性增强.

Effects of survivin siRNA on growth, apoptosis and chemosensitivity of ovarian cancer cells SKOV3/DDP

Objective To explore a new approach in gene therapy of ovarian cancer, we used RNAi to inhibit survivin gene expression, and explore the effect of survivin and neu RNAi on growth, apoptosis and chemosensitivity of ovarian cancer cell line SKOV3/DDP cells. Methods Expression vector of survivin gene-targeting siRNA was constructed using pSilencer 1.0-U6 vector containing U6 promotor (pSilencer-survivin) and transfected into SKOV3/DDP cells by lipofectamine. The untransfected group and pSilencer-control group were used as control. The expressions of survivin mRNA and protein were identified by RT-PCR and Western blot assay. The proliferation of SKOV3/DDP cells was determined by MTF assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. Cisplatin (DDP) resistance experiment was performed in SKOV3/DDP cells with RNAi. Results Survivin RNAi plasmid knocked-down survivin expression in SKOV3/DDP cells obviously, arrested the cells at G1/G0 phase, inhibited the cell proliferation and promoted cell apoptosis. The IC50 of DDP to SKOV3/DDP cells transfected with survivin siRNA was dropped. Conclusion Survivin RNAi can partly suppress the expression of survivin in SKOV3/ DDP cells, inhibit the cell proliferation and promote cell apoptosis. Survivin RNAi can enhance the cell sensitivity to apoptosis induced by cisplatin, which implies that survivin RNAi may partly reverse the drug resistance of ovarian cancer. RNAi could be a new approach for gene therapy of cancer.