靶向miRNA干扰Bmi-1诱导胆囊癌细胞凋亡及上调Caspase-3表达的研究

Gallbladder Cancer Cell Apoptosis and Up-regulated Expression of Caspase-3 Induced by Interference of Targeting MiRNA on Bmi-1

目的:通过靶向抑制原癌基因Bmi-1表达,观察其诱导胆囊癌细胞凋亡及上调Caspase-3蛋白表达的效应,探讨其在胆囊癌形成中的机制。方法:通过前期成功构建miRNA-Bmi-1重组质粒转染GBC-SD细胞,然后将其分为miRNABmi-1、miRNAScramble、Lipofectamine、GBC-SD 4组,转染48h后采用RT-PCR和Western blot法检测各组Bmi-1mRNA和蛋白的表达,倒置显微镜观察细胞生长情况;流式细胞技术检测细胞的凋亡率、细胞周期分布及Caspase-3蛋白表达水平。结果:RT-PCR和Western blot结果显示miRNABmi-1组细胞Bmi-1 mRNA和蛋白的表达水平明显低于对照组(P<0.05)。倒置显微镜观察显示miRNABmi-1组细胞死亡较对照组明显;细胞周期检测显示:miRNABmi-1组细胞阻滞于G0/G1(72.20±1.71),G2/M和S期细胞减少(18.30±7.21,9.50±6.01),凋亡指数增高(49.83±5.19),Caspase-3蛋白表达量明显增加(30.37±8.10),与对照组比较,具有显著性差异(P<0.05)。结论:靶向沉默Bmi-1能有效抑制胆囊癌细胞Bmi-1mRNA及蛋白表达,诱导胆囊癌细胞凋亡,其机制可能是早期使胆囊癌细胞周期阻滞于G0/G1期,并上调了Caspase-3蛋白表达,Bmi-1可能参与调控线粒体依赖的凋亡途径。

Objective ： Through targeted inhibition of oncogene Bmi-1 expression, to observe the induction of apoptosis and the effect of up-regulated Caspase-3 protein expression and to explore the mechanism of its formation in gallbladder cancer. Methods： The previously successfully constructed miRNA-Bmi-1 recombinant plasmid were transfected into GBC-SD cells and were divided into four groups of miRNABmi-1, miRNAScramble, Lipofectamine, GBC-SD. 48h after transfection, the mRNA and protein expression levels of Bmi-1 were measured by RT-PCR or Westem blot, the cell growth was observed under inverted microscope; The apoptosis rate, cell cycle distribution and Caspase-3 protein levels were determined using flow cytometry. Results： RT-PCR and Western blot showed that Bmi-1 mRNA and protein expression of the gallbladder cancer cell in miRNA Bmi-1 group were significantly lower than those in the control groups （P 〈 0.05 ）. The inverted microscope showed a significantly higher cell death rate in the miRNA Bmi-1 group compared with the control groups; Cell cycle analysis showed that, in the miRNA Bmi-1 group, the cells were restrained at GO/G1 phase （72.20% ± 1.71 ）, the cell number in G2/M and S phase decreased （18.30% ± 7.21, 9.50% ± 6.01 ） while apoptotic index increased （49.83% ± 5.19）. Caspase-3 protein expression was increased （30.37%±8.10）. Compared with the control groups, there was a significant difference （ P 〈 0.05 ）. Conclusions ： Targeting and silencing Bmi-1 can downregulate Bmi-1 mRNA and protein expression and induce the apoptosis in the gallbladder cancer cell, probably via the mechanism of restraining the early gallbladder cancer cell cycle at GO/G1 phase and upregulating Caspase-3 protein expression. Bmi-1 may be involved in regulation of mitochondrial-dependent apoptotic pathway.