miRNA-548ah promotes the replication and expression of hepatitis B virus by targeting histone deacetylase 4.

AIM: Many studies have shown that some microRNAs (miRNAs) play an important role in the pathogenesis of chronic hepatitis B (CHB) infection. In this study, we aimed to explore the role and molecular mechanism of miRNA-548ah in the replication and expression of the hepatitis B virus (HBV). MAIN METHODS: Overexpression and knockdown of miRNA-548ah were performed in three hepatoma cell lines with HBV replication and in a murine HBV model injected with adenovirus HBV vector. The effect of miRNA-548ah on its target gene, histone deacetylase (HDAC) 4, were confirmed in in vitro studies and further investigated in liver tissues from CHB patients. KEY FINDINGS: miRNA-548ah significantly increased the expression of HBV in hepatoma cell lines and in a HBV mouse model. The expression level of covalently closed circular DNA (cccDNA) in the miRNA-548ah mimics group was significantly higher than the negative control group and significantly lower in the miRNA-548ah inhibitor group. The HBV core antigen promotes the expression of miRNA-548ah in hepatocytes. Finally, we observed a negative correlation between the expression of miRNA-548ah and HDAC4 in the liver tissue of patients with CHB. SIGNIFICANCE: miRNA-548ah promoted the replication and expression of HBV through the regulation of the target gene, HDAC4. Inhibition of HDAC4 by miRNA-548ah might influence the deacetylation state of histones binding to cccDNA, thereby enhancing the replication of cccDNA. The HBV core antigen might increase the expression of miRNA-548ah. These results may provide new potential molecular targets for the prevention and treatment of CHB.

Effect of microRNA-548ah targeting histone deacetylase-4 on the replication and expression of hepatitis B vi rus / 中华传染病杂志

Objective To investigate the effect of microRNA (miRNA )‐548ah targeting histone deacetylase‐4 (HDAC4) on the replication and expression of hepatitis B virus (HBV) .Methods HepG2 , 2 ,15 cells were transfected by mimics and inhibitors . The expressions of miRNA‐548ah and HDAC4 before and after transfection were detected by fluorescent quantitative polymerase chain reaction (PCR) . The expression of HDAC4 protein in HepG2 ,2 ,15 cells was detected by Western blotting .The target gene of miRNA‐548ah was analyzed by bioinformatics methods .3′UTR dual‐luciferase expression vector containing candidate HDAC4 target genes was built to test the luciferase activity .The levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in the supernatant of cultured HepG2 ,2 ,15 cells were detected by enzyme‐linked immunosorbent assay (ELISA) .The level of HBV DNA in the supernatant of HepG2 ,2 ,15 cells was detected by fluorescent quantitative PCR .The t‐test was used for comparison between two groups ,SNK‐q tests were used for multiple groups comparisons .Results The expressions of miRNA‐548ah in HepG2 ,2 ,15 and HepG2 cells were 5 .74 ± 0 .02 and 2 .96 ± 0 .40 , respectively (t= 11 .89 ,P< 0 .01) ,and the expressions of HDAC4 mRNA were 9 .38 ± 0 .39 and 18 .13 ±0 .34 ,respectively (t = 29 .39 , P < 0 .01) . The expression of miRNA‐548ah in HepG2 ,2 ,15 cells was inhibited by transfection of miRNA‐548ah inhibitors (1 .01 ± 0 .13 ,t= 15 .48 , P< 0 .01) .Compared with control group ,the levels of HBsAg ([6 .45 ± 0 .46 ] IU/mL vs [2 .60 ± 0 .20 ] IU/mL , t = 7 .48 , P <0 .01) ,HBeAg ([5 .49 ± 0 .27] NCU/mL vs [4 .15 ± 0 .34 ] NCU/mL , t = 3 .10 , P < 0 .05 ) and HBV DNA ([3 .93 ± 0 .06] lg copy/mL vs[2 .04 ± 0 .07] lg copy/mL ,t = 18 .89 , P< 0 .01) in the supernatant of cultured HepG2 ,2 ,15 cells significantly decreased in inhibitor group . The expression of HDAC4 in HepG2 ,2 ,15 cells significantly decreased after transfection of miRNA‐548ah mimics (2 .98 ± 0 .94) ,but significantly increased after transfection of miRNA‐548ah inhibitors (23 .77 ± 6 .74 ) , with statistical significance (F= 9 .34 , P< 0 .01) .The expression of HDAC4 protein was also significantly inhibited after transfection of miRNA‐548ah mimics (0 .53 ± 0 .14 vs 0 .23 ± 0 .02 , t = 3 .58 , P = 0 .02) .The activity of luciferase was significantly inhibited by transfection of miRNA‐548ah mimics (7 .62 ± 0 .45 vs 6 .65 ±0 .27 ,t = 3 .18 , P = 0 .03 ) .Conclusion miRNA‐548ah may promote the replication and expression of HBV through the regulation of target gene HDAC4 .