Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas

Aim:The aim of the present study was to examine DNA methylation and histone modification changes in hepatocellular carcinomas (HCC). Methods: DNA methylation in the P16, RASSF1a, progesterone receptor (PGR) and estrogen receptor alpha (ER alpha) promoters was determined by quantitative bisulfite-pyrosequencing technique in HCC patients. Histone H3-lysine (K) 4, H3-K9 and H3-K27 modifications in all these four genes were examined by chromatin immunoprecipitation (ChIP) assay in HCC cell lines. Expression of two DNA methyltransferases (DNMT1 and DNMT3b) and three histone methyltransferases (SUV39H1, G9a and EZH2) in HCC patients was measured by real-time polymerase chain reaction. Results: Aberrant DNA methylation was detected in all the HCC. Patients with DNA methylation in the RASSF1a, PGR andER alpha promoters in cancers also had substantial DNA methylation in their non-cancerous liver tissues, whereas DNA methylation in the P16 promoter was cancer specific. Epigenetic states in HCC cell lines showed that silencing of P16 and RASSF1a depended on DNA methylation and histone H3-K9 methylation. However, silencing of the PGR and ER alpha genes was more closely related to H3-K27 methylation rather than DNA methylation. Consistent with the alteration of histone status, higher expression of G9a and EZH2 was found in HCC than in non-cancerous liver tissues (P < 0.01). Conclusion: These data suggest that multiple epigenetic silencing mechanisms are inappropriately active in HCC cells.