To our knowledge, this is also the first report showing that the inhibition of miR-152 results functionally in global DNA hypermethylation and increased methylation levels of the TSGs GSTP1 and CDH1 in HCC cell lines. The overexpression of miR-152 in HepG2.2.15 cells reduced GDM from 6.31% to 4.08%, whereas the miR-152 inhibitor in HepG2 cells increased GDM from 4.55% to 5.88%. The GSTP1 gene has been reported to be commonly epigenetically silenced by methylation in HBV-associated HCC, and somatic GSTP1 inactivation may contribute to the pathogenesis of this malignancy.35 In our study, the GSTP1 gene was demonstrated

to be methylated in HepG2 cells, and the methylation level of its promoter that we detected was increased from 58.18% to 86.36% after transfection of the miR-152

inhibitor. CDH1 is also frequently silenced by methylation in HCC, and it has been reported that HBx can repress CB-839 in vivo CDH1 expression by inducing the hypermethylation of its promoter.36, click here 38 In the current study, the methylation level of the CDH1 promoter region, which we measured, was increased from 0% to 23.8% in HepG2 cells. From these results, we can see that the TSG methylation levels increased, regardless of the initial methylation status. The relative mRNA level measurement showed that GSTP1 expression was significantly decreased after transfection of the miR-152 inhibitor in comparison with the control group, whereas the CDH1 mRNA level was not Thiamine-diphosphate kinase significantly changed. This probably occurred because the increasing DNA methylation level of the CDH1 promoter was not sufficient to inhibit the mRNA expression. The hypermethylation of CpG islands of TSGs promotes oncogenesis not only through transcriptional inactivation of these genes but also through the following mechanisms: A signature CT mutation in cancer cells: the cytosine residues in the methylated dinucleotide CpG have a higher mutation rate than the unmethylated cytosine. The induction of chromosomal instability: aberrant DNA methylation leads to the genomic instability necessary for the development and progression of cancer, and DNA methylation

is also correlated with allelic deletions.41, 42 Moreover, HBV DNA has been shown to contain CpG islands that can be methylated in human tissue both in a nonintegrated form43 and after integration into the human genome.44 The methylation of viral CpG islands can regulate viral protein production,45 which likely reflects viral adaptation to host cells. A DNA methylation–associated blockade of viral antigen presentation could help the virus to evade our immune system. The depletion of DNMT1 and DNMT3B by siRNA or upon treatment with the DNA demethylation agent caused DNA hypomethylation of the HBV genome in HCC cells.46 In the present study, we have demonstrated that HBx can up-regulate DNMT1 activity by inhibition of miR-152. These mechanisms may also be involved in the methylation of the HBV genome and the survival of HBV in host cells.