Aim: To investigate the potential roles of enhancer of zeste homolog2 (EZH2), Bmi-1 and miR-203 in cell proliferation and invasion in hepatocellular carcinoma (HCC) cell line Hep3B.
Methods: A total of 73 patients who underwent surgical resection at Fuzong Clinical Medical College of Fujian Medical University were enrolled in this study. Hep3B cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C. Vectors that containing cDNA of the EZH2 gene or miR-203 targeted shRNA plasmid were constructed, and then transfected into Hep3B cells. The mRNA expression of miR-203, EZH2, and Bmi-1 was analyzed using quantitative real-time polymerase chain reaction analysis, and the protein levels of EZH2 and Bmi-1 were detected by Western blot analysis. Effect of EZH2 or miR-203 on cell proliferation was observed by methyl thiazolyl tetrazolium assay, and cell apoptosis was assessed using flow cytometry. Besides, effect of EZH2 or miR-203 on tumor cell invasion was detected using Transwell assay.
Results: The mRNA levels of EZH2 and Bmi-1 in HCC tissues and in Hep3B cells were significantly higher compared with those in normal samples (P < 0.01), while miR-203 level was significantly lower in HCC tissues (P < 0.01). Hep3B cells transfected with EZH2-shRNA or miR-203-shRNA showed lower expression levels of EZH2 and Bmi-1 (P < 0.05). Compared with controls, Hep3B cells transfected with EZH2-shRNA had relative slow cell proliferation, indicating that low expression of EZH2 and Bmi-1 and overexpression of miR-203 could inhibit Hep3B cell proliferation (P < 0.05). The average apoptosis rate of Hep3B cells transfected with EZH2-shRNA vector was about 18.631%, while that of Hep3B cells transfected with shRNA vector was about 5.33%, suggesting that EZH2 was down-regulated by transfecting with EZH2-shRNA, and the down-regulated EZH2 contributed to the cell apoptosis. Low expression of EZH2 and Bmi-1 and overexpression of miR-203 could reduce Hep3B cell invasion (P < 0.05).
Conclusion: Our study suggests that EZH2 and Bmi-1 are up-regulated while miR-203 is down-regulated in Hep3B cells. MiR-203 may contribute to the metastasis and enhance apoptosis of HCC cells by regulating EZH2 and Bmi-1. Our study may provide a theoretical basis for metastasis of HCC and targeted therapy of HCC.
Keywords: Bmi-1; EZH2; Hep3B cell line; Hepatocellular carcinoma; Invasion; Proliferation; miR-203.