00024, Wilcoxon signed-rank test; Fig. 4A). Next, we asked whether DNMT1 mRNA expression was inversely correlated with the levels of miR-152 in HBV-related HCC tissues. Twenty HCCs were analyzed for the expression levels of DNMT1 mRNAs and for miR-152 expression by real-time PCR. A statistically significant inverse correlation was observed between DNMT1 mRNA and miR-152 (n = 20, r = −0.462, P = 0.02, Pearson’s correlation; Fig. 4B). These data showed the reciprocal regulation of Idelalisib manufacturer the tumor suppressor miR-152 and its target DNMT1 in human HCCs and suggested that miR-152 may play a causal role in DNA methylation leading to liver cancer in chronic hepatitis B patients. Because
miR-152 was frequently down-regulated in HBV-positive HCC tissues in comparison with the adjacent noncancerous liver specimens, whereas DNMT1 expression appeared to be inversely correlated, we wondered if the reduction of miR-152 expression selleck chemicals could be driven by the increase in DNMT1 expression. We enhanced or inhibited DNMT1 expression
by transfecting the DNMT1 expression vector (pcDNA3.1-DNMT1) or DNMT1 siRNA into HepG2 and Huh-7 cells with the pcDNA3.1 vector and control siRNA, respectively, as the negative controls (Fig. 5A,B). After 48 hours of transfection, we measured the miR-152 expression levels of these cells and found that none of them were significantly changed in comparison with the negative controls (Fig. 5C,D). We investigated whether the enforced expression of miR-152 could also functionally result in DNA hypermethylation. GDM was measured with an LC-MS/MS method in HepG2 and HepG2.2.15 cell lines after 72 hours of transfection with miR-152 inhibitor or miR-152 mimics, respectively. The miRNA negative control and inhibitor negative control served as negative controls for the mimics and inhibitor, respectively. We observed a reduction of 6.31% to 4.08% in GDM for the HepG2.2.15 cell
line treated with miR-152 mimics in comparison with the negative controls Aprepitant and an increase of 4.55% to 5.88% in GDM for the HepG2 cell line treated with miR-152 inhibitors (Fig. 6A). Several genes have been found to be silenced by DNA methylation induced by HBx via interactions with de novo DNMTs in HBV-HCC, including GSTP1 and CDH1. To assess whether inhibiting the expression of miR-152 could also lead to hypermethylation and silencing of these genes in HCC, we measured the DNA methylation levels of CDH1 and GSTP1 by bisulfate sequencing in the HepG2 cell line after transfection with the miR-152 inhibitor or miRNA inhibitor negative control. The GSTP1 DNA methylation level was increased in HepG2 cells transfected with the miR-152 inhibitor in comparison with the miRNA inhibitor negative control (mean of 85.8% versus 57.4%, P < 0.005, Wilcoxon rank test; Fig. 6C) Likewise, the CDH1 DNA methylation level was increased in HepG2 cells transfected with the miR-152 inhibitor in comparison with the control (mean of 23.8% versus 0%, P < 0.