Study On The Mechanism(s) Of Mycophenolic Acid Inhibiting HCV Replication By Regulation Of Autophagy

Objective Previous studies have shown that mycophenolic acid(MPA.An activated metabolic component of a clinical immunosuppressant mycophenolate mofetil)could inhibit the replication of hepatitis C virus(HCV),but the mechanism(s)remains largely unknown.This study aims to reveal the novel mechanism(s)of mycophenolic acid inhibiting HCV replication,on the aspect of autophagy.We expect to provide a new anti-HCV strategy based on suppression of cellular autophagy.Methods In vitro cultured hepatoma cell line Huh7 cells were used in this study.First,this study investigated the effect of MPA on cellular autophagy in Huh7 cells.We used MPA to treat Huh7 cells with or without autophagosomal inhibitor BafA1 pretreatment.The expression levels of LC3 mRNA were detected by RT-PCR.The protein levels of LC3B-I and LC3B-II(autophagic marker)were determined by Western blot.The effect of MPA on autophagic flux in Huh7 cells was determined by the changes of LC3 expression.Second,we investigated the effect of MPA on HCV-induced or tunicamycin(autophagy inducer)-induced cellular atuophagy.With or without BafA1(autophagosomal inhibitor)pretreatment,MPA was used to treat Huh7 cells with /without HCVinfection or with /without tunicamycin treatment.The expression of LC3 mRNA was measured by RT-PCR,and the levels of LC3B-I,LC3B-II protein were determined by Western blot.Simultaneously,autophagic vacuoles were also visualized by transmission electron microscopy(TEM).Thus,we examined the inhibitory effect of MPA on HCV-induced or tunicamycin-induced cellular autophagy.On that basis,we used Human Autophagy PCR array to screen and identify the autopahgy-related genes that are regulated by MPA treatment,then used gene overexpression and siRNA silencing to determine the role of key factors responsible for the actions of MPA to block autophagy,thereby to inhibit HCV replication.Results1.MPA treatment could suppress cellular autophagy in Huh7 cells.In the absence or presence of BafA1,at LC3 mRNA level,no significant effect(p>0.05)was observed in MPA-treated cells compared to control cells.However,the level of LC3B-II as well as conversion of LC3B-I to LC3B-II in MPA-treated cells significantly decreased in MPA-treated cells compared to those in untreated control cells.Therefore,the results imply that MPA treatment could suppress cellular autophagy,evidenced by decreased level of LC3B-II.2.HCV infection or tunicamycin treatment could induce cellular autophagy in Huh7 cells.Although at LC3 mRNA level,no significant effect(p>0.05)was observed in tunicamycin-treated cells or HCV-infected cells compared to control cells.However,the level of LC3B-II as well as conversion of LC3B-I to LC3B-II in HCV-infected cells or tunicamycin-treated cells significantly increased compared to those in control cells.These data indicate that HCV infection or tunicamycin treatment could activate cellular autophagy in Huh7 cells.3.MPA treatment inhibited HCV-induced or tunicamycin-induced cellular autophagy in Huh7 cells.In the absence or presence of Baf A1,at LC3 mRNA level,no significant effect of MPA treatment(p>0.05)was observed in HCV-infected cells or tunicamycin-treated cells compared to control cells.However,MPA treatment could significantly decrease HCV-or tunicamycininduced LC3B-II level and conversion of LC3B-I to LC3B-II.These results indicate that MPA treatment could suppress HCV-or tunicamycin-induced autophagy in Huh7 cells.4.MPA treatment inhibited the expression of autophagy-related genes ATG3,ATG5,ATG7.The Human Autophagy RT-PCR array screening results show that,in HCV-uninfected and HCV-infected Huh7 cells,MPA treatment could down-regulate the expression of ATG3,ATG5,ATG7 at mRNA level compared to MPA-untreated cells,respectively(more than 2-fold change).5.Overexpression and silencing expression of ATG3,ATG5,ATG7 confirmed the role of ATG3,ATG5 and ATG7 in MPA-modulated inhibition of HCV replication.Either at HCV RNA level or at HCV core protein level,overexpression of ATG3,ATG5 or ATG7 could restore HCV replication partially,evidenced by the increased levels of HCV RNA or core protein expression in pATG-transfected cells compared to MPA-treated cells with blank plasmid transfection.The results indicate that overexpression of ATG3,ATG5 or ATG7 could restore partially HCV replication inhibited by MPA treatment.In contrary,in MPA-treated cells with silencing expression of ATG3,ATG5 and ATG7 could further decrease HCV replication,either at HCV RNA level or at HCV core protein level,compared to the MPA-treated cells transfected with scrambled siRNA.Thus,silencing expression of ATG3,ATG5 and ATG7 could enhance inhibitory effect of MPA on HCV replication.Conclusions Our study indicates that MPA could inhibit cellular autophagy as well as HCV-induced or tunicamycin-induced autophagy.MPA treatment inhibits cellular autophagy through down-regulating the expression of ATG3,ATG5 and ATG7 in Huh7 cells,thereby inhibits HCV replication in Huh7 cells.This study provides a new molecular mechanism for the inhibitory action of MPA on HCV replication.Furthermore,these results provide a scientific basis for the clinical treatment and prognosis of patient with liver transplanation due to HCV infection.