Bestrophin 3高表达促进人肝癌细胞株HepG2的凋亡

Enforced expression of Bestrophin 3 promotes apoptosis of hepatocellular carcinoma cell HepG2

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 investigate the effect and mechanism of Bestrophin 3 on the apoptosis of human hepatocellular carcinoma cell HepG2. Methods: HepG2 cells were transfected with various multiplicity of infection( MOI = 10,20,40 and 80) of adenovirus expressing Bestrophin 3,LacZ,or control. The infection efficiency of the adenovirus was measured by Western blotting. Proliferation and apoptosis rates of the transfected cells were measured with CCK-8 assay and flow cytometry. Effects of Bestrophin 3 on the expression of Bcl-2 and Bax and the release of cytochrome C( Cyt C) were determined by using immunoblotting. Influence of Bestrophin 3 overexpression on mitochondrial membrane potential was assessed by JC-1 and fluorescence microscopy. Results: The expression of Bestrophin 3 reached the maximal level after adenoviral infection at 40 MOI. Compared with the control and Lac Z-expressing groups,the cell viability of the Bestrophin 3-expressing group was significantly reduced [( 79. 37 ± 1. 76) % vs( 98. 67 ± 3. 02) % and( 99. 67 ± 3. 25) %,P <0. 05],whereas the apoptosis rate was significantly increased [( 29. 47 ± 2. 37) % vs( 5. 47 ± 0. 37) % and( 4. 95 ±0. 44) %,P < 0. 05]. In the cells infected with the adenovirus expressing Bestrophin 3,there were decreased level of Bcl-2 and increased level of Bax,leading to a significant reduction of Bcl-2 / Bax ratio. In HepG2 cells overexpressing Bestrophin 3,the mitochondrial membrane potential was also significantly lower compared with that of controls( 1. 00 ± 0. 00) %vs 0. 64 ± 0. 09%,P < 0. 05),and Cyt C content was reduced in mitochondria and elevated in cytoplasm( all P < 0. 05).Conclusion: Enforced expression of Bestrophin 3 increases the apoptosis of HepG2 through reducing Bcl-2 / Bax ratio and promoting cytochrome C release from the mitochondria.