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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: To investigate the effect of targeting Bmi-1 on the expression of Bmi-1 in gallbladder carcinomas and to observe its effect on the apoptosis of gallbladder carcinoma cells and the up-regulation of Caspase-3 protein expression. METHODS: GBC-SD cells were successfully transfected with miRNA-Bmi-1 recombinant plasmid and then divided into 4 groups: miRNABmi-1, miRNAScramble, Lipofectamine and GBC-SD. The expression of Bmi-1 mRNA and protein in each group was detected by the method of inverted microscope. The apoptosis rate, cell cycle distribution and the expression of Caspase-3 protein were detected by flow cytometry. Results: The mRNA and protein expressions of Bmi-1 in the miRNABmi-1 group were significantly lower than those in the control group (P <0.05) by RT-PCR and Western blot. Inverted microscopy showed that the cell death in miRNABmi-1 group was more obvious than that in control group. Cell cycle analysis showed that the number of cells arrested in G0 / G1 (72.20 ± 1.71), G2 / M and S phase decreased in the miRNABmi-1 group (18.30 ± 7.21, 9.50 ± 6.01). The apoptotic index increased (49.83 ± 5.19) and the expression of Caspase-3 protein increased significantly (30.37 ± 8.10), which was significantly different from the control group (P <0.05). Conclusion: Targeting silencing of Bmi-1 can effectively inhibit the expression of Bmi-1 mRNA and protein in gallbladder carcinoma cells and induce the apoptosis of gallbladder carcinoma cells. The mechanism may be that the cell cycle of gallbladder carcinoma is arrested at G0 / G1 phase and the expression of Caspase -3 protein expression, Bmi-1 may be involved in the regulation of mitochondria-dependent apoptotic pathway.