Hepatitis B virus X protein promotes P3 transcript expression of the insulin‐like growth factor 2 gene via inducing hypomethylation of P3 promoter in hepatocellular carcinoma

Background & Aims Hepatitis B virus (HBV) X protein (HBx) contributes to hepatocarcinogenesis. The overexpression of transcripts from P3 and P4 promoters of the insulin-like growth factor 2 (IGF2) gene is observed in hepatocellular carcinoma (HCC). Here, we aimed to explore the involvement of HBx in P3-driven mRNA overexpression and underlying epigenetic mechanism. Methods P3 mRNA, P3 methylation status, HBx mRNA and HBx protein were analysed in human HCC samples with and without HBV infection using quantitative RT-PCR, bisulphite sequencing and Western blotting. The effects of HBx on P3 mRNA expression, and P3 transcriptional activity and methylation were further evaluated in HCC cell lines. Results P3 mRNA level was higher and P3 methylation level was lower in HBV-positive HCC specimens compared with those of HBV-negative HCC specimens. P3 transcript abundance was positively correlated with HBx expression and negatively correlated with P3 methylation in HCC specimens. The stable expression of HBx upregulated P3 mRNA expression and reduced P3 methylation level in HepG2-HBx cells. The transient expression of HBx stimulated P3 promoter activity and decreased P3 methylation level of P3 promoter-luciferase construct in a dose-dependent manner in HepG2 and Huh-7 cells. Furthermore, HBx mRNA expression was found to be independent predictive factors for both shorter disease-free survival time and shorter overall survival time of HCC patients. Conclusion HBx may promote IGF2-P3 transcript expression by inducing hypomethylation of P3 promoter and may be associated with an inferior clinical outcome of HBV-related HCC patients. This study provides useful information for understanding the mechanism of HBx-mediated HCC.