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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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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.
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Received: 08 October 2013
Published: 25 December 2013
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