The Mechanism Of Inhibition Of HCV Replication By IFN-?1 Through Downregualtion Of Autophagy-realted Gene ATG5 And Gabarap Expression

Objective To investigate the effect of type ? interferon(IFN-?1)on autophagy in hepatocytes and to explore the molecular mechanism(s)by which IFN-?1 interrupts autophagy pathway and thereby inhibits HCV infection/replication.Methods In this study,a hepatoma cell line,Huh7 and in vitro research were used.To observe the effect of IFN-?1 on HCV replication,the expression of HCV RNA and HCV core were detected by real-time RT-PCR and western blot,respectviely,under the condition of IFN-?1 treatment in Huh7 cells.To analyze the effect of IFN-?1 on autophagy,Huh7 cells,HCV-infected cells or tunicamycin-treated Huh7 cells were treated with IFN-?1.The expression of LC3 gene,the marker of autophagy was detected by real-time RT-PCR at mRNA level,and the expression of LC3B-II protein as well as the ratio of LC3B-II/LC3B-I were detected by Western Blot.In addition,the autophagic activity was measured by amounts of GFP-LC3 dot under the fluorescent microscopy and formation of autophagosomes under transmission electron microscopy(TEM).The key autophagy-related genes regulated by IFN-?1 were screened by autophagy PCR Array and further confirmed by real-time RT-PCR and Western Blot.Next,over-expression of the seleted autophagy-related geneswas used to verify their role in IFN-?1-modulated inhibition of HCV.Finally,bioinformatics tools were used to predict miRNAs that could regulate the screened autophagy genes.And then these miRNAs were investigated whether their expression was regulated by IFN-?1 treatment.Furthermore,we used miRNA inhibitors to identify the role of these miRNAs in mediation of screened autophagy genes expression as well as HCV replication.Results1.IFN-?1 inhibits HCV replication in Huh7 cells.The level of HCV RNA in the Huh7 cells treated with IFN-?1 was lower than the control group.The level of HCV core protein in IFN-?1-treated cells was also reduced compared to the control group.2.IFN-?1 inhibits the autophagy in Huh7 cells.(1)Without pre-incubation of autophagy inhibitor bafilomycin A1,when Huh7 cells,HCV-infected cells or tunicamycin-treated cells were treated with IFN-?1 respectviely,IFN-?1 treatment does not affect the expression of autophagy-related gene LC3 at mRNA level,however,at the protein level,IFN-?1 could significantly inhibit the expression of LC3B-II and LC3B-II /LC3B-I in the three groups.(2)With pre-incubation of autophagy inhibitor bafilomycin A1,when Huh7 cells,HCV-infected cells or tunicamycin-treated cells were treated with IFN-?1respectviely,IFN-?1 treatment does not affect the expression of autophagy-related gene LC3 at mRNA level.However,at the protein level,IFN-?1 could also inhibit the expression of LC3B-II and LC3B-II / LC3B-I in the three groups.(3)Under the condition of pre-incubation with bafilomycin A1,the autophagic activity was also measured by the formation of autophagosomesunder transmission electron microscopy(TEM).The closed double-membraned vesicles,resembling autophagic vesicles,were detected in different groups.In control cells(without IFN-?1 treatment,HCV infection,and tunicamycin treatment)or IFN-?1-treated cells,only small amount of double-membraned vesicles were observed.Under the conditions of HCV infection or tunicamycin treatment,much more double-membraned vesicles were observed.However,when HCV-infection cells or tunicamycin-treated cells were treated by IFN-?1simultaneously,the significant diminution of the double-membraned vesicles were observed.(4)Under the condition of pre-incubation with bafilomycin A1,the autophagic activity was measured by the amonuts of GFP-LC3 dots under the fluorescent microscopy.In control cells(without IFN-?1 treatment,HCV infection,and tunicamycin treatment)or IFN-?1-treated cells,only small amount of GFP-LC3-positive dots were observed.Under the conditions of HCV infection or tunicamycin treatment,much more GFP-LC3-positive dots were observed.However,when HCV-infection cells or tunicamycin-treated cells were treated by IFN-?1 simultaneously,the significant diminution of GFP-LC3-positive dots were observed.3.The effect of IFN-?1 on the expression of autophagy-related genes.(1)The status of a number of autophagy-related genes by IFN-?1 treatment were screened using Autophagy PCR Array in HCV-uninfected or HCV-infected cells.The screening results displayed that the expression of ATG5,GABARAP,BAX and MTOR was significantly decreased(> 2-fold)in IFN-?1-treated cells.(2)In confirmation experiments,in HCV-uninfected Huh7 cells,the expression of ATG5 and GABARAP was significantly decreased after IFN-?1treatment at mRNA level,but no significant changes in BAX and MTOR geneexpression were observed.Western blot further confirmed that the expression of ATG5 and GABARAP was significantly decreased after IFN-?1 treatment.(3)In confirmation experiments,in HCV-infected Huh7 cells,the expression of ATG5 and GABARAP was significantly decreased after IFN-?1treatment at mRNA level,but no significant changes in BAX and MTOR gene expression were observed.Western blot further confirmed that the expression of ATG5 and GABARAP was significantly decreased after IFN-?1 treatment.4.Overexpression of ATG5 and/or GABARAP weakens the inhibitory effects of IFN-?1 on HCV.(1)The expression levels of HCV RNA and HCV core protein were increased compared with the control cells when the plasmids ATG5 and GABARAP were transfected alone or together in IFN-?1-untreated Huh7 cells.(2)The expression levels of HCV RNA and HCV core protein were increased compared with the control cells,when the plasmids ATG5 and GABARAP were transfected alone or together in IFN-?1-treated Huh7 cells.5.IFN-?1 regulates ATG5 and GABARAP expression through inducing miR-181 a and mi R-214 expression(1)In total,11 miRNAs that probably targeting ATG5,and 7 mi RNAs that probably targeting GABARAP were found through bioinformatics tools.(2)Among the predicted miRNAs,IFN-?1 treatment significantly increased the expression of miR-181 a and mi R-214(> 2-fold)in HCV-infected or uninfected Huh7 cells.(3)Inhibition of miR-181 a and miR-214 could lead to the increase of HCV replication evidenced by increased levels of HCV RNA and viral protein,along with the changes of ATG5 and GABARAP expression.Conclusion IFN-?1 could inhibit the expression of ATG5 and GABARAPby inducing the expression of miR-181 a and mi R-214,leading to the inhibition of autophagy activity of Huh7 cells,which contributes to IFN-?1 antiviral activity against HCV infection.This study reveals a novel mechanism of anti-HCV function of IFN-?1.The results also provide a theoretical basis for inhibition HCV replication by supression of cellular autophagy as well as improvement of the effectiveness of IFN therapy in the context of autophagy.