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

ABSTRACT

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 .