Aberrant promoter methylation of retinoic acid receptor- (RAR- ) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR- via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR- in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2′-deoxycytidine. As RAR- is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.


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