The Cooperative Complex Of Argonaute-2/MicroRNA-146a Regulates Hepatitis B Virus Replication Through FEN1

Objective:To investigate the effect of miR-146a on the replication of Hepatitis B virus and its molecular mechanism.Methods:The expression of miR-146a was tested in HBV stable replication cell line and its parent cell line HepG2 by qRT-PCR.The miR-146a mimic and inhibitor were transfected into the HepG2.2.15 for 48 hours.Then,the HBV-DNA copies were tested with qRT-PCR and the expression of FEN 1 was detected by western bolt.Furthermore the copies of HBV-DNA and the level of miR-146a were examined by qRT-PCR in HepG2.2.15 transfected with FEN1 plasmid.The potential target gene of miR-146a are IRAK1/TRAF6 by bioinformatics analysis,adaptors in NF-?B pathway,and their levels were examined by qRT-PCR.To test the role of Ago2 in miR-146a mediating HBV replication,the Ago2 siRNA and miR-146a mimic were transfected respectively into HepG2.2.15 for 48 hours,the expression level of Ago2 protein was detected by western bolt and qRT-PCR.After Ago2 RIP(RNA Binding Protein Immunoprecipitation),the level of miR-146a was detected by qRT-PCR.After Ago2 siRNA then added into exogenous miR-146a into HepG2.2.15,the expression level of FEN 1 and HBV-DNA copies were measured.Results:We found that level of miR-146a was significantly up-regulated in HepG2.2.15 cells(11.755±0.069)than that in HepG2(1.000±0.038)(P<0.05).Furthermore,HBV-DNA copies and FEN1 were significantly increased and decreased,respectively,in HepG2.2.15 cells transfected with miR-146a mimic and inhibitor for 48h,[(3.215± 0.001);(2.623±0.083)][(1.678±0.131);(0.344±0.045)]compared with the control group(2.813±0.015);(1.017±0.194)(P<0.05).After transfection FEN1 plasmid,HBV-DNA Copies(5.712±0.371)is significantly higher than the control group(2.661±0.009)(P<0.05),and the level of miR-146a(3.431±0.004)is significantly higher than the control group(1.023±0.224)(P<0.05).The expression level of IRAK1/TRAF6 are significantly lower and higher[(0.114±0.013);(0.390±0.014);(1.222±0.073);(2.1451±0.271)]than the control group[(1.000±0.038);(1.007±0.119)](P<0.05)after transfection miR-146a mimic and inhibitor into HepG2.2.15.After Ago2 siRNA,the level of miR-146a(0.105±0.002)is significantly decreased than the control group(1.000±0.041)(P<0.05)from Ago2 protein RIP.After transfection Ago2 siRNA then added into exogenous miR-146a into HepG2.2.15,The expression level of FEN1 is significantly reduced(0.485±0.100)than the control group(1.000±0.023)(P<0.05),and the HBV-DNA copies is significantly lower(3.230±0.047)than the control group(3.789±0.041)(P<0.05).Conclusion:Ago2 cooperates with miR-146a to regulate the transcription the expression level of FEN1 protein through the downstream target gene IRAK1/TRAF6,then promoting HBV replication.