Molecular Mechanism Of Hepatitis B Virus X Regulating The Activity Of EZH2 Promoter

Hepatitis B Virus X protein (HBx), which is expressed after the infection of Hepatitis B Virus (HBV), is regarded to have various functions and play a crucial role in the development of hepatocellular carcinoma (HCC). Enhancer of zeste homolog 2 (EZH2), which is frequently over-expressed in HCC tissues, is a histone lysine methyltransferase and associated with the hepatocarcinogenesis. However, the exact mechanism of EZH2 over-expression in HCC had not been determined. In this study, the molecular mechanism of mEZH2 expression regulated by hepatitis B virus X protein was investigated in AML12 cells.Firstly, Western Blotting analysis demonstrated that the expression level of mEZH2 protein in AML12 cells could be up-regulated by HBx in a dose-dependent manner.To investigate whether HBx up-regulating the expression of mEZH2 through activating its promoter, we amplified 2500-bp regulating sequence upstream the first exon of mEZH2 gene from AML12 genomic DNA. Analyzed by luciferase reporter system, we found that the activity of mEZH2 promoter was significantly increased in AML12 cells co-transfected with HBx expression plasmid. In addition, we expressed fusion protein HBx-EGFP-TLM, which contained a translocation motif TLM. TLM translocation motif, which was derived from the PreS2-domain of Hepatitis B Virus surface antigens, could lead HBx into the cytoplasm. Luciferase Reporter Assay revealed the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. Both of these two results indicated that HBx could up-regulate the activity of mEZH2 promoter.In order to research the mechanism of HBx activation to mEZH2 promoter, we truncated mEZH2 promoter gradually. Luciferase Reporter Assay revealed that deleting the promoter from -486 to -214 or -50 to +1 gave a distinguished decline of HBx activation to mEZH2 promoter. The -486/-214 and -50/+1 regions were then analyzed in TRANSFAC 6.0 database and typical E2F1, Sp1 and TBP binding sites were found. Mutation of these binding sites would induce distinct reduction of HBx activation to mEZH2 promoter. Further research indicated knocking down E2F1 expression by RNAi would lead to a dramatic decrease of HBx activation to mEZH2 promoter and mEZH2 expression in AML12 and HepG2 cells. These results indicated that HBx might up-regulate mEZH2 expression by means of trans-activating the mEZH2 promoter through E2F1 transcription factor.In conclusion, our work suggested that HBx could trans-activate the activity of EZH2 promoter through E2F1 transcription factor and further up-regulate the expression level of EZH2 protein. These results would provide new epigenetic evidence for the cancerogenic effect of HBV X protein.