Survivin基因沉默促进胃癌细胞凋亡及其机制研究

Mechanism study of Survivin knockdown promotes apoptosis of gastric carcinoma cells

目的研究凋亡抑制蛋白人生存素(Survivin)基因mRNA、蛋白质在胃癌和癌旁组织的表达量;以及siRNA-Survivin转染人胃癌MKN-45细胞对其增殖、凋亡的影响和机制研究。方法使用实时荧光定量PCR(qRT-PCR)和免疫印迹实验(Western Blot)检测Survivin m RNA和蛋白在胃癌和癌旁组织的表达量;利用脂质体Lipofectamine 2000将siRNA-Survivin(si-Survivin),错配siRNA(si-Control)转入人胃癌MKN-45细胞中,未转染细胞为空白对照(Normal),Western Blot检测Survivin蛋白表达量;CCK8实验法检测细胞的增殖速度;流式细胞术检测凋亡率;Western Blot检测Wnt/β-catenin信号通路中的关键蛋白(β-catenin、Cyclin D1)、Caspase-3蛋白的表达量,对其增殖、凋亡机制进行研究。结果 Survivin mRNA和蛋白质在胃癌组织中的表达量显著高于癌旁组织(P <0.05);siRNA-Survivin转染人胃癌MKN-45细胞可使Survivin蛋白的表达量低于si-Control组和Normal组(P <0.05);转染后si-Survivin组细胞的增殖受到抑制,尤其是72 h抑制效果最明显;转染后si-Survivin组细胞凋亡率显著高于si-Control组和Normal组(P <0.05);沉默Survivin基因使Caspase-3蛋白表达量升高,β-catenin、Cyclin D1蛋白表达量降低。结论 SurvivinmRNA和蛋白在胃癌中的表达量高于癌旁组织;沉默Survivin基因可通过Wnt/β-catenin信号通路以及Caspase-3蛋白的表达量抑制细胞增殖、促进其凋亡。

Objective To investigate Survivin mRNA and protein expression in gastric carcinoma and the mechanism study of silencing Survivin on apoptosis of gastric carcinoma cells.Methods The mRNA and protein expression of Survivin gene in gastric carcinoma was detected by RT-PCR and Western blot assay,respectively.SiRNA-Survivin was transfected into MKN-45 cells as the si-Survivin group,along with MKN-45 cells transfected with negative control siRNA(si-Control)group and the normal control(Normal)group.The expression of Sur-vivin,caspase3,β-catenin and Cyclin D1 protein was detected by Western blot assay.CCK8 assay and flow cytom-etry were used to detect the proliferation and apoptosis of MKN-45 cells.Results Survivin mRNA expressoin in gastric carcinoma was significantly higher than that in the matched tumor-adjacent tissue(P<0.05).The Survivin protein level in the si-Survivin group was obviously lower than that in the si-Control group and in the Normal group(P<0.05,respectively).The proliferation rate of MKN-45 cells was lower and apoptosis rate was higher in the si-Survivin group than that in the si-Control group and in the Normal group(P<0.05,respectively).The protein level ofβ-catenin and Cyclin D1 in the si-Survivin group were obviously lower than those in the si-Control group and in the Normal group(P<0.05,respectively),but Caspase-3 protein was on the contrary.Conclusion Down regulation of Survivin expression can inhibit the proliferation of MKN-45 cells and induce cell apoptosis by inhibit-ing the Wnt/β-catenin signaling pathway and inducing Caspase-3 protein expression.