Effect of miRNA-146a on life cycle of hepatitis B virus in vitro / 第三军医大学学报

ABSTRACT

Objective To determine the effect of microRNA-146a (miR-146a) on the life cycle of hepatitis B virus (HBV) and investigate the underlying mechanisms.Methods The miRNA expression profiles were compared by miRNA array between HepG2 and HepG2.2.15 cells.Then miR-146a was chosen as objective,and its expression level was further confirmed by RT-PCR.After miR-146a mimic and inhibitor were transfected into HepG2.2.15 cells respectively,the quantification of HBV replication was determined by RT-PCR,and the levels of HBsAg and HBeAg in the supernatant were measured by ELISA,and the expression of HS3ST3B1 at mRNA and protein levels were tested by RT-PCR and Western blotting.Dualluciferase reporter assay was used to detect the interaction between miR-146a and potential target HS3ST3B1.Results The expression levels of totally 72 miRNAs were changed in HepG2.2.15 cells,with 27 upregulated and 45 down-regulated.RT-PCR showed the expression level of miR-146a was significantly higher in HepG2.2.15 cells than HepG2 cells (1.55-± 0.13 vs 1.00 ± 0.01,P ＜ 0.05).Transfection of miR-146a mimic into HepG2.2.15 cells resulted in significantly increased HBV replication and levels of HBsAg and HBeAg (P ＜ 0.05),while the transfection of its inhibited caused opposite results (P ＜ 0.05).Bioinformatic analysis showed that HS3ST3B1 was a potential target of miR-146a.The reporter luciferase reporter system indicated that the reported fluorescence intensity of HS3ST3B1 wild type vector was significantly lower than that of the control group (P ＜ 0.05),but showed no significant difference between HS3ST3B1 mutant vector and control group (P ＞0.05).The mRNA level of HS3ST3B1 was not significantly changed in HepG2.2.15 cells transfected with miR-146a mimic (P ＞ 0.05),but its protein level was significantly decreased (P ＜ 0.05).Conclusions miR-146a affects the life cycle of HBV,which may be through suppressing the translation of HBV inhibitory factor HS3ST3B1 3'UTR.