Effect of the BBC3 Gene on the Proliferation and Apoptosis of Hepatocellular Carcinoma Cells Through p53-Regulated Signaling

Abstract

We examined the effect of the BBC3 gene on hyperplasia and apoptosis in HepG2 cells and its underlying mechanism. Quantitative RT-PCR was used to determine the level of BBC3 expression in HL-7702 normal human liver cells and four different hepatocellular carcinoma cell lines (HepG2, HuH-7, HCCLM3 and MHCC97H). Transfection was performed with Lipofectamine 2000 reagent and the transfectants were divided into three groups: pcDNA-BBC3 group (transfected BBC3 over-expressing plasmid), pcDNA-NC group (transfected empty plasmid), and a Ctrl group (not transfected). Quantitative RT-PCR and western blot analysis were used to measure BBC3 expression. The CCK-8 assay was used to determine the effect of BBC3 on HepG2 cell proliferation. Flow cytometry was used for testing the effect of overexpressing BBC3 on apoptosis in HepG2 cells. The levels of cleaved-Caspase-3 (C-Caspase-3), cleaved-Caspase-9 (C-Caspase-9), and proteins associated with the p53 signaling pathway were assessed by western blot analysis. The level of BBC3 mRNA in HL-7702 normal human liver cells was significantly higher compared with that in human hepatocellular carcinoma cells including HepG2, HuH-7, HCCLM3 and MHCC97H (P < 0.05). The lowest level of BBC3 mRNA was observed in HepG2 cells. The level of BBC3 mRNA and protein in HepG2 cells were significantly higher compared with that of the pcDNA-NC group following transfection with a BBC3 overexpressing plasmid. HepG2 cell proliferation in the pcDNA-NC group was higher compared with that of the pcDNA-BBC3-transfected group (P < 0.05). The apoptotic rate and levels of cleaved-Caspase-3, cleaved-Caspase-9, p53, phospho-p53, and p21 protein in cells were higher compared with that of the pcDNA-NC group. No change was observed in the pcDNA-NC and Ctrl groups. The BBC3 gene was down-regulated in hepatocellular carcinoma cells. HepG2 cell proliferation can be inhibited and HepG2 cell apoptosis can be induced by the overexpression of BBC3 through activation of the p53 signaling pathway.