RNAi沉默survivin和HIF-1α基因对胃癌BGC-823细胞体外增殖和凋亡的影响

Effects of silencing survivin and HIF-1α genes with RNAi on proliferation and apoptosis of human gastric cancer BGC-823 cells

目的:应用RNA干扰(RNAi)技术抑制胃癌BGC-823细胞中survivin和HIF-1α基因的表达,探讨其对胃癌BGC-823细胞增殖和凋亡的影响。方法:分别设计并合成经过化学修饰的靶向survivin和HIF1αmRNA的小干扰RNA(siRNAs),同时合成错义RNA(SCR),采用HifectinⅡ真核体外转染胃癌BGC-823细胞,将转染siRNA-survivin、siRNA-HIF-1α和SCR的各组细胞分别命名为sis组、siH组和SCR组,同时设空白对照组(无血清培养基),RT-PCR法检测各组细胞中survivin和HIF-1αmRNA表达水平。将胃癌BGC-823细胞分为单干扰组(survivin-siRNA,sis组)、联合干扰组(survivin-siRNA+HIF1α-siRNA,sis+siH组)、非靶向特异性组(SCR组)和空白对照组(无血清培养基),MTT法检测各组细胞增殖活性,Western blotting法检测各组细胞中survivin和HIF-1α蛋白表达水平,流式细胞术检测各组细胞凋亡率。结果:与空白对照组比较,sis组细胞中survivin mRNA表达水平和siH组细胞中HIF-1αmRNA表达水平明显降低(P<0.05或P<0.01)。与空白对照组比较,SCR组细胞增殖活性无明显变化(P>0.05),sis组和sis+siH组细胞增殖活性明显降低(P<0.05);与sis组比较,sis+siH组细胞增殖活性明显降低(P<0.05)。与空白对照组比较,sis+siH组细胞中surviving和HIF-1α蛋白表达水平明显降低(P<0.05或P<0.01),细胞凋亡率明显升高(P<0.01)。结论:联合靶向沉默survivin和HIF-1α基因可下调靶基因的表达,抑制胃癌BGC-823细胞增殖,促进细胞凋亡,RNAi基因沉默技术有望成为治疗胃癌的新方法。

Objective：To inhibit the expressions of survivin and HIF-1αusing RNA interference（RNAi）technique,and to explore their effects on the proliferation and apoptosis of gastric cancer BGC-823 cells.Methods：Two pairs of siRNA were designed against the human survivin and HIF-1αseparately.The human gastric cancer BGC-823 cells were transfected with siRNA-survivin,siRNA-HIF-1αand SCR and used as sis group,siH group,and scrambled siRNA（SCR）group;at the same time,blank control group（serum-free medium）was set up.RT-PCR was used to detect the expression levels of survivin and HIF-1αmRNA in the BGC-823 cells in various groups.The BGC-823 cells were transfected with siRNAs using HifectinⅡ.The BGC-823 cells were divided into sis group,sis＋siH group,SCR group and blank control group. MTT assay was performed to detect the proliferation activity of cells.The expression levels of survivin and HIF-1αprotein in the cells in various groups were detected by Western blotting method.Flow cytometry was used to detect the apoptotic rates of BGC-823 cells in various groups.Results：Compared with blank control group,the expression level of survivin mRNA in sis group was significantly desreased（P〈0.05）,and the expression level of HIF-1α mRNA in siH group was significantly desreased（P〈0.05）.Compared with blank control group,the proliferation ability of cells in SCR group had no significant change（P〈0.05）,and the proliferation abilities of cells in sis group and sis＋siH group were significantly decreased（P〈0.05）;compared with sis group,the proliferation ability of cells in sis＋siH group was significantly decreased（P〈0.05）.Compared with blank control group,the expression levels of survivin and HIF-1αin sis＋siH group were significantly decreased（P〈0.05 or P〈0.01）,and the apoptotic rates of BGC-823 cells were significantly increased（P〈0.01）.Conclusion：The combined targeting silencing surviving and HIF-1αgenes can down-regulate the expressions of targeted genes,inhibit the proliferation of gastric cancer BGC-823 cells,and promote the apopotosis,indicating RNAi gene silencing may be a new method to treat gastric cancer.