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

中国生物工程杂志

2013, Vol. 33

Issue (12): 1-8

研究报告

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

魏东¹, 邹浩¹, 王琳¹, 王文举², 骆志玲³, 张小文¹

1. 昆明医科大学第二附属医院昆明 650101;
2. 昆明医科大学附属延安医院中心实验室昆明 650051;
3. 昆明医科大学第一附属医院昆明 650031

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

WEI Dong¹, ZOU Hao¹, WANG Lin¹, WANG Wen-ju², Luo Zhi-ling³, ZHANG Xiao-wen¹

1. Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Kunming Medical University, Kunming 650101, China;
2. Department of Research Laboratory Center, Yan'an Affiliated Hospital of Kunming Medical University, Kunming 650051, China;
3. The First Affiliated Hospital of Kunming Medical University, Kunming 650031, China

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摘要： 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-1mRNA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。

关键词： GBC-SD; Caspase-3蛋白; 细胞凋亡; 细胞周期; 原癌基因Bmi-1

Abstract: 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 Western 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 G0/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 G0/G1 phase and up-regulating Caspase-3 protein expression. Bmi-1 may be involved in regulation of mitochondrial-dependent apoptotic pathway.

Key words: Oncogene Bmi-1 GBC-SD Apoptosis Cell cycle Caspase-3 protein

收稿日期: 2013-10-08 出版日期: 2013-12-25

ZTFLH:

R735.8

基金资助: 云南省科技计划项目（NO：2011FZ124）；云南省科技厅-昆明医科大学应用基础研究联合专项（2012FB050）资助项目

通讯作者: 张小文，E-mail：zhangxiaowenlu@hotmail.com E-mail: zhangxiaowenlu@hotmail.com

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引用本文:

魏东, 邹浩, 王琳, 王文举, 骆志玲, 张小文. 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[J]. 中国生物工程杂志, 2013, 33(12): 1-8.

WEI Dong, ZOU Hao, WANG Lin, WANG Wen-ju, Luo Zhi-ling, ZHANG Xiao-wen. Gallbladder Cancer Cell Apoptosis and Up-regulated Expression of Caspase-3 Induced by Interference of Targeting MiRNA on Bmi-1. China Biotechnology, 2013, 33(12): 1-8.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/1

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