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目的:研究Bestrophin 3高表达对人肝癌细胞株HepG2凋亡能力的影响,并初步探讨其作用机制。方法:将Bestrophin 3腺病毒以感染复数(multiplicity of infection,MOI)10、20、40、80转染HepG2细胞株,Western blotting检测转染细胞内Bestrophin 3的表达,确定最佳MOI值。设对照组(未转染病毒)、LacZ组(转染携带LacZ的对照腺病毒载体)、Ad-Best3组(以最佳MOI值转染Bestrophin 3腺病毒),CCK-8法和流式细胞术分别检测Bestrophin 3过表达对HepG2细胞增殖、凋亡的影响,Western blotting检测Bestrophin 3过表达对HepG2细胞内Bcl-2、Bax以及细胞色素C表达的影响,JC-1染色观察Bestrophin 3过表达对HepG2细胞线粒体膜电位的影响。结果:Bestrophin 3腺病毒转染HepG2细胞的最佳MOI为40,转染后细胞过表达Bestrophin 3。Ad-Best3组的细胞存活率显著低于对照组和Lac Z组[(79.37±1.76)%vs(98.67±3.02)%、(99.67±3.25)%,均P<0.05],而其细胞凋亡率显著升高[(29.47±2.37)%vs(5.47±0.37)%,(4.95±0.44)%,均P<0.05]。AdBest3组HepG2细胞内Bcl-2的蛋白表达显著减少,Bax的蛋白表达显著增加,导致Bcl-2/Bax比值显著降低(0.32±0.047 vs1.00±0.00,P<0.05);Ad-Best3组HepG2细胞的线粒体膜电位显著降低[(0.64±0.09)%vs(1.00±0.00)%,P<0.05],同时线粒体中细胞色素C明显减少(P<0.05),而细胞质中细胞色素C水平显著升高(P<0.05)。结论:过表达Bestrophin 3可能通过促使线粒体释放细胞色素C从而促进肝癌细胞株HepG2的凋亡。
OBJECTIVE: To study the effect of high expression of Bestrophin 3 on the apoptosis of human hepatocellular carcinoma cell line HepG2 and to explore its mechanism. Methods: HepG2 cells were transfected with 10, 20, 40, and 80 multiplicity of infection (MSI) of the Bestrophin 3 adenovirus. The expression of Bestrophin 3 in the transfected cells was detected by Western blotting to determine the optimal MOI. The control group (untransfected virus), LacZ group (control adenoviral vector transfected with LacZ), Ad-Best3 group (Bestrophin 3 adenovirus transfected at optimal MOI), CCK-8 method and flow cytometry The effects of Bestrophin 3 overexpression on the proliferation and apoptosis of HepG2 cells were detected by Western blotting. The expression of Bcl-2, Bax and cytochrome C in HepG2 cells was detected by Western blotting. Bestrophin 3 overexpression was observed by JC-1 staining Effect on Mitochondrial Membrane Potential in HepG2 Cells. Results: The optimal MOI of HepG2 cells transfected with Bestrophin 3 adenovirus was 40, and the cells were overexpressing Bestrophin 3 after transfection. Cell survival rate in Ad-Best3 group was significantly lower than that in control group and LacZ group [(79.37 ± 1.76)% vs (98.67 ± 3.02)%, (99.67 ± 3.25)%, both P <0.05] (29.47 ± 2.37)% vs (5.47 ± 0.37)%, (4.95 ± 0.44)%, all P <0.05. The protein expression of Bcl-2 in HepG2 cells was significantly decreased and the protein expression of Bax was significantly increased in AdBest3 group (0.32 ± 0.047 vs 1.00 ± 0.00, P <0.05). The Ad-Best3 HepG2 The mitochondrial membrane potential was significantly decreased in the mitochondria ([(0.64 ± 0.09)% vs (1.00 ± 0.00)%, P <0.05], while mitochondrial cytochrome C was significantly decreased (P <0.05) High (P <0.05). Conclusion: Overexpression of Bestrophin 3 may promote the apoptosis of hepatocellular carcinoma cell line HepG2 by inducing mitochondria to release cytochrome C.