miR-125a表达对肝癌HepG2细胞增殖、凋亡及细胞周期的影响分析

目的 探讨microRNA-125a（miR-125a）对肝癌HepG2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（qPCR）法检测人肝癌HepG2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pGenesil-1质粒制备过表达miR-125a的重组质粒pGenesil-miR-125a及表达随机序列的阴性对照pGenesil-NC，根据实验设计将HepG2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pGenesil-NC和pGenesil-miR-125a，待3组转染24、48、72及96 h后采用qPCR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 HepG2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）。

Influence of miR-125a on the proliferation,apoptosis and cell cycle of human hepatoma HepG2 cells

Objective To explore the influence of microRNA-125a（miR-125a） on the proliferation， apoptosis and cell cycle of human hepatoma HepG2 cells. Methods The real-time fluorescence quantitative PCR（qPCR） was employed to measure the miR-125a level in human hepatocellular carcinoma HepG2 cell and normal liver 7702 cells. The eukaryotic expression plasmid vector pGenesil-1 was used to construct recombinant plasmid pGenesil-miR-125a. Meanwhile， a control plasmid pGenesil-control with random sequence was constructed. According to the experimental design， the HepG2 cells were divided into 3 groups： blank control group（HepG2 cells without transfection）， negative control group（HepG2 cells transfected with pGenesil-NC） and over-expression group（HepG2 cells transfected with pGenesil-miR-125a）. The qPCR was used to investigate the miR-125a 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 HepG2 at 24，48，72 and 96 h after transfection. The Hoechst staining and flow cytometry were used to evaluate the apoptosis index and caspase-3 activation 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-125a in HepG2 cell was（0.24±0.06）， lower than that of 7702 cell（P<0.05）. MiR-125a level increased but proliferation rate decreased at 24， 48， 72 and 96 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-125a in HepG2 cells， and the over-expression of miR-125a can inhibit the proliferation， induce cell apoptosis and G0/G1 arrest，playing as tumor suppressor gene in the development of hepatocellular carcinoma.