Abstract:

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.