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目的运用RNA干扰(RNAi)技术抑制雄激素非依赖前列腺癌细胞株(PC-3)中生存素的表达,检测生存素抑制对PC-3细胞的影响。方法构建负载生存素短发夹RNA(shRNA)且携带绿色荧光蛋白(GFP)的重组腺病毒,体外转染PC-3细胞。MTT法监测细胞增殖活性,流式细胞术检测凋亡率,RT-PCR和Western blot分别对生存素mRNA及其蛋白表达进行测定。结果负载生存素shRNA重组腺病毒构建成功;转染96h后,PC-3细胞增殖受到抑制,并可诱导出PC-3细胞凋亡,凋亡率达到(14.43±0.57)%,生存素mRNA及其蛋白的表达强度分别降低了(72.23±1.11)%和(71.75±2.64)%,与阴性对照组及空白对照组比较差异有统计学意义(P<0.01)。结论腺病毒载体介导的生存素shRNA可有效地抑制PC-3细胞中生存素的表达,RNAi结合腺病毒载体系统抑制生存素表达有望成为治疗雄激素非依赖前列腺癌的有效方式之一。
Objective To detect the expression of survivin in androgen-independent prostate cancer cell line (PC-3) by RNA interference (RNAi) technique and to detect the effect of survivin inhibition on PC-3 cells. Methods The recombinant adenovirus carrying survivin short hairpin RNA (shRNA) carrying green fluorescent protein (GFP) was constructed and transfected into PC-3 cells in vitro. The activity of cell proliferation was monitored by MTT assay. The apoptosis rate was detected by flow cytometry. Survivin mRNA and protein expression were determined by RT-PCR and Western blot respectively. Results Survivin shRNA recombinant adenovirus was constructed successfully. After transfection for 96h, the proliferation of PC-3 cells was inhibited and the apoptosis of PC-3 cells was induced. The apoptotic rate of PC-3 cells was (14.43 ± 0.57)%, (72.23 ± 1.11)% and (71.75 ± 2.64)% respectively, which was significantly lower than that of negative control group and blank control group (P <0.01). Conclusions Adenovirus vector-mediated survivin shRNA can effectively inhibit the expression of survivin in PC-3 cells. RNAi combined with adenoviral vector system can inhibit the expression of survivin, which is expected to be one of the effective ways to treat androgen-independent prostate cancer.