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目的探讨靶向survivin的siRNA转染肝癌细胞阻抑survivin表达,及其对肝癌细胞凋亡、增殖与细胞化疗敏感性的影响。方法设计合成特异性靶向survivin的siRNA序列。肝癌细胞株HepG2接种于6孔培养板内,分为5组:空白对照组、阴性对照组和低、中、高浓度转染组(分别给予50,100和200nmol.L-1siRNA转染)。作用36h后收集各组细胞。Westernblot法检测各组细胞survivin蛋白表达情况,逆转录-聚合酶链反应(RT-PCR)检测survivinmRNA表达水平,流式细胞术检测各组细胞增殖和凋亡指数,四氮唑盐(MTT)法检测氟尿嘧啶(5-FU)和顺铂(DDP)对各组细胞的生长抑制率。结果各浓度siRNA转染组细胞survivin蛋白和mRNA表达有不同程度减弱。各浓度siRNA转染组细胞凋亡指数明显高于对照组(P<0.05),高浓度转染组最明显(P<0.05)。各浓度siRNA转染组细胞增殖指数明显低于各对照组(P<0.05),高浓度转染组最明显(P<0.05)。等浓度化疗药物5-FU和DDP对各浓度siRNA转染组细胞的抑制率明显高于对照组(P<0.05),高浓度转染组最明显(P<0.05)。结论不同浓度survivinsiRNA转染能下调survivin蛋白和mRNA表达,诱导肝癌细胞凋亡,抑制细胞增殖,增加肝癌细胞对化疗药物的敏感性。
OBJECTIVE: To investigate the effect of siRNA targeting survivin on the expression of survivin in hepatoma cells and its effect on the apoptosis, proliferation and chemosensitivity of hepatocellular carcinoma cells. Methods siRNA sequences targeting survivin were designed and synthesized. HepG2 cells were inoculated into 6-well plates and divided into 5 groups: blank control group, negative control group and low, medium and high concentration transfection groups (transfected with 50, 100 and 200 nmol.L-1 siRNA respectively). After 36h, the cells in each group were collected. The expression of survivin protein in each group was detected by Western blot, the expression of survivin mRNA was detected by RT-PCR, the proliferation and apoptosis index were detected by flow cytometry, The inhibitory rates of 5-fluorouracil (5-FU) and cisplatin (DDP) on the growth of each group were detected. Results The expression of survivin protein and mRNA in siRNA transfected cells decreased to different extents. The apoptosis index of siRNA transfected group was significantly higher than that of control group (P <0.05), and the highest concentration of transfected group was the most significant (P <0.05). The proliferation index of siRNA transfection group was significantly lower than that of control group (P <0.05), and the transfection group of high concentration was the most obvious (P <0.05). The inhibitory rates of 5-FU and DDP at the same concentrations in siRNA transfected cells were significantly higher than those in control cells (P <0.05), and those in high concentration transfected cells were the highest (P <0.05). Conclusion Different concentrations of survivinsiRNA transfection can downregulate the expression of survivin protein and mRNA, induce apoptosis of hepatoma cells, inhibit cell proliferation and increase the sensitivity of hepatoma cells to chemotherapeutic drugs.