miR-125a表达对肝癌HepG2细胞增殖、凋亡及细胞周期的影响分析

Influence of miR-125a on the proliferation,apoptosis and cell cycle of human hepatoma Hep G2 cells

目的探讨microRNA-125a(miR-125a)对肝癌Hep G2细胞增殖、凋亡及细胞周期的影响。方法采用实时荧光定量PCR(q PCR)法检测人肝癌Hep G2细胞及人正常肝细胞7702中的miR-125a水平,同时采用真核表达载体p Genesil-1质粒制备过表达miR-125a的重组质粒p Genesil-miR-125a及表达随机序列的阴性对照p Genesil-NC,根据实验设计将Hep G2细胞分为3组:空白对照组、阴性对照组和过表达组,其中空白对照组未进行转染,阴性对照组和过表达组分别成功转染p Genesil-NC和p Genesil-miR-125a,待3组转染24、48、72及96 h后采用q PCR法检测各组的miR-125a水平,噻唑盐比色法检测各组转染24、48、72及96 h的细胞增殖率,分别采用Hoechst染色和流式细胞术检测转染24、48 h后的凋亡指数和caspase-3活化率来评价凋亡情况,流式细胞仪Annexin V-FITC/PI双染流式细胞术检测各组转染48 h后的细胞周期,同时采用免疫印迹法检测转染48 h后对凋亡相关基因(Bcl-2、Bax和Cleaved caspase-3)表达的影响。结果 Hep G2细胞中miR-125a水平为(0.24±0.06),低于正常肝细胞7702细胞(P<0.05);与阴性表达组相比,过表达组转染24、48、72及96 h的miR-125a水平升高,增殖率降低,差异有统计学意义(P<0.05);与其余两组相比,过表达组转染后的凋亡指数、caspase-3活化率、G0/G1期细胞比例及Bax和Cleaved caspase-3水平均升高,S和G2/M期细胞比例及Bcl-2水平均降低,以上差异有统计学意义(P<0.05)。结论肝癌Hep G2细胞中miR-125a低表达,且过表达miR-125a可抑制增殖、诱导凋亡及G0/G1期细胞阻滞,在肝癌的发生发展中可能起到抑癌基因的作用。

Objective To explore the influence of microRNA-125a（ miR-125a） on the proliferation,apoptosis and cell cycle of human hepatoma Hep G2 cells. Methods The real-time fluorescence quantitative PCR（ q PCR） was employed to measure the miR-125 a level in human hepatocellular carcinoma Hep G2 cell and normal liver 7702 cells. The eukaryotic expression plasmid vector p Genesil-1 was used to construct recombinant plasmid p Genesil-miR-125 a. Meanwhile,a control plasmid p Genesil-control with random sequence was constructed. According to the experimental design,the Hep G2 cells were divided into 3 groups： blank control group（ Hep G2 cells without transfection）,negative control group（ Hep G2 cells transfected with p Genesil-NC） and over-expression group（ Hep G2 cells transfected with p Genesil-miR-125a）. The q PCR was used to investigate the miR-125 a level of three groups at 24,48,72 and 96 h after transfection. Tetrazolium salt（ MTT） colorimetric assay was used to assess the proliferative responses of Hep G2 at 24,48,72 and 96 h after transfection. The Hoechst staining and flow cytometry were used to evaluate the apoptosis index and caspase-3activation rate to evaluate the apoptosis at 24 and 48 h after transfection. The cell cycle at 48 h after transfection was determined by Annexin V-FITC / PI double staining with the use of flow cytometry analysis. Western blotting was employed to analyze the protein levels of apoptosis related genes（ Bcl-2,Bax and Cleaved caspase-3） at 48 h after transfection. Results The level of miR-125 a in Hep G2 cell was（ 0. 24 ± 0. 06）,lower than that of 7702 cell（ P〈0. 05）. MiR-125 a level increased but proliferation rate decreased at 24,48,72 and96 h after transfection in over-expression group versus negative control group with statistically significant difference（ P〈0. 05）. In contrast to other two groups,there were elevated apoptosis index,caspase-3 activation rate,percentage of G0/ G1 and protein level of Bax and Cleaved caspase-3 but decreased percentage of S and G2/ M phase and protein level of Bcl-2 with statistically significant difference（ P〈0. 05）. Conclusion There is low expression of miR-125 a in Hep G2 cells,and the over-expression of miR-125 a can inhibit the proliferation,induce cell apoptosis and G0/ G1 arrest,playing as tumor suppressor gene in the development of hepatocellular carcinoma.