| Objective Alcohol treatment could promote hepatitis C virus(HCV)replication in liver cells.However,the mechanism(s)has not yet been fully clarified.Our priliminary data have shown that alcohol treatment could upregulate the expression of activated STAT inhibitor protein y(PIASy)in Huh7 cells.In this study,we further explored the underlying mechanism of the effect of alcohol-induced PIASy on cellular autophagy as well as HCV replication.We expect to provide a new mechanism of alcohol promoting HCV replication.Methods In this study,a human hepatoma cell line Huh7(a hepatoma cell line that supports HCV JFH-1 infection and replication)was used for the in vitro research of the effect of alcohol on HCV replication.Firstly,the HCV-infected Huh7 cells were treated with different concentrations of alcohol for different times.Real-time PCR(RT-PCR)and Western blot were used to detect the levels of HCV RNA and HCV core protein,respectively,to observe the effect of alcohol on the HCV replication.Next,under the condition of alcohol treatment,RT-PCRand WB were used to detect the levels of the PIASy mRNA and PIASy protein,to verify the effect of alcohol on PIASy expression.Simultaneously,under the condition of alcohol treatment,PIASy was overexpressed or slicenced-expressed in Huh7 cells.The levels of HCV core protein were tested by WB,to analyse the role of PIASy in alcohol promotiong HCV replication.Thirdly,to detect the effect of alcohol on cellular autophagy,under the condition of alcohol treatment,the levels of LC3 mRNA and LC3B-I,LC3B-II and p62 proteins were analysed by RT-PCR and WB.Furthemore,GFP-LC3-harboring lentivirus-transfected Huh7 cells were also used for evaluating the effect of alcohol on cellular autophagy.The numbers of GFP-LC3 dots were determined using fluorescent microscopy.The effect of alcohol treatment on autophagy flux was also observed by treatment of chloroquine(CQ),an inhibitor of autophagy fusion.WB and fluorescent microscopy were used to determine the levels of the LC3B-I,LC3B-II proteins and the numbers of GFP-LC3 dots.Go further,PIASy was overexpressed or silenced-expressed in alcohol-treated Huh7 cells.The levels of LC3-I,LC3-II and p62 proteins were detect to observe the role of PIASy in alcohol-inducing cellular autophagy.In addition,the PIASy overexpressed or silenced-expressed Huh7 cells were treated with 3-methyladenine(3-MA),an inhibitor of early stage of autophagy,and the levels of LC3B-I,LC3B-II and HCV core protein were detected by WB,to observe the effect of inhibitory of cellular autophagy on the action of PIASy on HCV replication.Finally,the PIASy overexpressed or silenced-expressed Huh7 cells were treated with CQ,and the levels of LC3-I,LC3-II and the numbers of GFP-LC3 dots were determined by WB and fluorescent microscopy,to verify the effect of PIASy on autophagy flux.Then the SUMO1,SUMO2/3 antibodies,Beclin1,ATG3,ATG7 and Atg 5-12 antibodies were used to detect the effect of PIASy on the modification of SUMO pathways as well as the regulation of autophagy-related factors,to explore the underlying mechanism(s)of PIASy-inducing autophagy in Huh7 cells.Results1.Alcohol promotes HCV replication in Huh7 cellsThe HCV-infected Huh7 cells were treated with different concentrations of alcohol for different times.The level of HCV RNA and HCV core were significantly increased in alcohol-treated Huh7 cells(p<0.05)in a dose-and time-dependent manner.These data indicated that alcohol treatment could promote HCV replication in Huh7 cells.2.Alcohol promotes HCV replication via up-regulationg of PIASy expression in Huh7 cells.(1)Alcohol increases the expression of PIASy in Huh7 cellsThe HCV JFH-1-infected Huh7 cells were treated with or without different concentrations of alcohol for different times.The levels of PIASy mRNA and PIASy protein were significantly up-regulated in alcohol-treated cells in a doseand time-dependent manner(p<0.05).However,alcohol treatment had little effect on the expression of other proteins of PIAS family(p > 0.05).These data suggested that alcohol only specifically induces PIASy expression in Huh7 cells,among the members of PIAS family.(2)Up-regulation of PIASy involves in alcohol promoting HCV replicationOverexpression or silencing expression of PIASy were used to investigate the role of PIASy in alcohol promoting HCV replication.Overexpression of PIASy increased HCV core protein level(p<0.05),and also enhanced the level of alcohol-p-regulated HCV core protein(p<0.05).Silencing PIASy expression down-regulated HCV core protein level(p<0.05),and also decreased the level of alcohol-up-regulated HCV core protein(p<0.05).3.Alcohol induces autophagy through up-regulation of PIASy expression(1)Alcohol treatment induces autophagy in Huh7 cells.Huh7 cells were treated with or without different concentrations of alcohol for different times.Alcohol treatment did not significantly affect the level of LC3 mRNA in Huh7 cells(p > 0.05),but significantly up-regulated the protein level of LC3B-II,promoted the conversion of LC3B-I to LC3B-II,and down-regulated the level of p62 protein(p < 0.05).Furthermore,the numbers of GFP-LC3 dots around the nucleus were significantly increased(p < 0.05)in alcohol treatment group.These data indicated that alcohol could induce autophagy in Huh7 cells.Under the condition of alcohol treatment,the cells treated with or without CQ(Chloroquine,an inhibitor of autophagosome-lysosome fusion).CQ treatment significantly up-regulated the level of LC3B-II protein(p < 0.05).Meanwhile,alcohol treatmen further enhanced the level of CQ-increased LC3B-II protein(p < 0.05).The numbers of GFP-LC3 dots were significantly up-regulated in cotreatment of CQ and alcohol,compare with alcohol or CQ treatment alone(p < 0.05).These data indicated that alcohol affects autophagy flux.(2)Alcohol induce autophagy through up-regulating PIASy expressionUnder the condition of overexpression of PIASy,overexpression of PIASy or alcohol treatment could both up-regulate the level of LC3B-II protein,promote the conversion of LC3B-I to LC3B-II,and down-regulate the level of p62 protein(p < 0.05).Furthermore,overexpression of PIASy significantly enhanced the alcohol-up-regulated expression of LC3B-II protein,the conversion of LC3B-I to LC3B-II,and alcohol-down-regulated the expression of p62(p<0.05).Silencing PIASy expression significantly decreased the level of LC3B-II protein,inhibited the conversion of LC3B-I to LC3B-II,and increased the p62 level(p<0.05).Furthermore,Silencing PIASy expression significantly inhibited the alcohol-upregulated the expression of LC3B-II protein,decreased the conversion of LC3 BI to LC3B-II,and increased the alcohol-down-regulated the expression of p62 protein(p<0.05).4.PIASy promotes HCV replication via inducing cellular autophagy.Under the condition of overexpression of PIASy,3-methyladenine(3-MA,an inhibitor of early stage of autophagy)treatment significantly down-regulated the levels of LC3B-II protein,inhibited the conversion of LC3B-I to LC3B-II,and increased the p62 protein level in overexpress PIASy group(p<0.05).Meanwhile,3-MA treatment significantly decreased the level of HCV core protein(p<0.05).Consistently,under the condition of silencing PIASy expression,3-MA treatment further enhanced the down-regulation of HCV core protein expression by silencing PIASy expression(p<0.05).5.PIASy induces autophagy through inducing the accumulation of SUMO1-conjugated proteins(1)The effect of PIASy on autophagy fluxUnder the condition of CQ treatment,overexpression of PIASy significantly enhanced CQ-induced LC3B-II protein level,the conversion of LC3B-I to LC3BII(p<0.05),and the numbers of GFP-LC3 dots(p<0.05).Silencing expression of PIASy,on the contrary,decreased the protein level of LC3B-II and the numbers of GFP-LC3 dots induced by CQ(p<0.05).(2)PIASy induces autophagy through inducing the accumulation of SUMO1-conjugated proteins.Under the condition of overexpression or slicensing PIASy expression,The levels of SUMO pathways were investigated by using the SUMO1 and SUMO2/3 antibodies.Overexpression of PIASy significantly promoted the SUMO modified proteins mediated by SUMO1(p<0.05),however,overexpression had little effect on the levels of SUMO modified protein mediated by SUMO2/3(p >0.05).Under the condition of silencing PIASy expression,the levels of protein modification mediated by SUMO1 was significantly decreased(p<0.05),and the levels of SUMO protein modification mediated by SUMO2/3 had no significantly affected(p>0.05).Overexpression of PIASy also upregulated the expression of autophagy related factors ATG5-12 and ATG7(p<0.05),and silencing PIASy expression down-regulated the expression of ATG5-12 and ATG7(p<0.05).Conclusion These results indicated that alcohol promotes HCV replication through alcohol-induced autophagy via upregulationg of PIASy expression.PIASy induces cellular autophagy through promoting SUMO protein modification mediated by SUMO 1.These findings provide a novel mechanism for the action of alcohol promoting HCV replication in the context of cellular autophagy,which may provide some clues for novel therapeutic strategies to improve the effects of antiviral therapy among alcohol-abused HCV-infected individuals. |
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