Effect Of KRT8on The Replication Of Hepatitis B Virus In Vitro

Object To investigate the effect of host protein KRT8on HBV DNAreplication.Methods Hepatitis B virus (HBV)-transfected humanhepatoblastoma cell line HepG2.2.15were cultured in vitro.(1) We usedthree fragments of KRT8specific siRNA to silence KRT8gene expression.The experiment was divided into six groups: siRNA-1group, siRNA-2group, siRNA-3group, negative control group, lipofectamin control groupand blank control group.48hours after the transfection, the cells wereharvested for detecting the level of KRT8gene expression and the culturesupernatants were collected for measure the level of HBV DNA replication.(2) We used plasmid vector to up regulate KRT8gene expression, andtransfected them to HepG2.2.15cells. The experiment was divided into twogroups: plasmid group and blank control group.48hours after thetransfection, the cells were harvested for detecting the level of KRT8geneexpression and the culture supernatants were collected for measure the levelof HBV DNA replication.(3) Lamivudine was selected to inhibit HBV DNAreplication, and was co-cultured with HepG2.2.15cells.72hours after co-cultured, the cells were harvested for detecting the level of KRT8geneexpression and the culture supernatants were collected for measurequantitative of HBV DNA. RT-PCR was chosed to detect the level of KRT8gene expression, and fluorescence quantitative PCR was used to detect thelevel of HBV DNA replication.Results (1)48hours post-transfection siRNA, the KRT8mRNAexpression level was significant decreased in siRNA-1group, siRNA-2group and siRNA-3group (RQ<0.5). And compared with blank controlgroup, the inhibition efficiency were90%,89%and80%respectively(P<0.01). In the meantime, the level of HBV DNA were dropped in the threesiRNA groups, and compared with blank control group, the drop rate were36%,34%and38%respectively (P<0.05).(2)48hours post-transfectionplasmid, the KTR8mRNA expression was significant up regulated inplasmid group (RQ>2),40.32fold compared with blank control group(P<0.01). At the same time, the level of HBV DNA in the culturesupernatants was significant increased in plasmid group,28times of blankcontrol group (P<0.01).(3)72hours after lamivudine and HepG2.2.15cellsco-cultured, the HBV DNA level was significant decreased in thelamivudine group,66%declined compared with control group (P<0.01).Meanwhile, the KRT8mRNA expression level was little declined inlamivudine group (RQ=0.758±0.414), although24%decreased comparedwith blank control group, there was no significant difference between lamivudine group and blank control group (P>0.05).Conclusions HBV DNA replication could be inhibited bysuppressing KRT8mRNA expression.