Mycophenolic Acid Regulates Autophagy Key Factor Expression And Its Effect On Hcv Replication

Objective This study was to investigate the effects of mycophenolic acid(MPA)on HCV replication through mediating the autophagy pathway in Huh7 cells and to elucidate the molecular mechanisms of MPA's inhibitory effect on HCV replication..Methods The effects of MPA on HCV replication and autophagy(LC3B-? as a marker of autophagy)were observed in Huh7 cells in vitro.Real-time quantitative PCR(RT-PCR)was applide to screen autophagy-related genes,including Beclinl,ATG3,ATG5,ATG7,Bad,Bax,Bcl-xl and GABARAP,whose expression might be mediated by MPA treatment in Huh7 cells with or without HCV JFH-1 infection,Further validation of the PCR results were confirmed by Western blot assay.Results1.MPA inhibited HCV replication in Huh7 cells.The inhibitory effects of MPA on HCV replication were exhibited as dose-dependent and time-dependent fashions(P<0.05).The HCV RNA level in MPA treatment group was significantly lower than that in control group(P<0.05).Consistent with the results of RNA level,MPA treatment decreased the expression of HCV Core protein(0.64 ±0.06 vs 0.96 ±0.07,P<0.01,compared with control)in HCV JFH-1 infected Huh7 cells.2.MPA inhibited cellular autophagy in Huh7 cells(LC3B-II as a marker of autophagy).Compared with control group,MPA treatment reduced the mRNA level of LC3B in uninfected Huh7 cells(0.67 ±0.15 vs 1.01 ±0.14)as well as in HCV JFH-1-infected Huh7 cells(1.01 ±0.14 vs 0.72 ± 0.08),and the difference was statistically significant(P<0.01).MPA also reduced the level of LC3B-? protein in uninfected or HCV-infected Huh7 cells(1.03±0.07 vs 1.477±0.04);(0.41 ±0.06 vs 0.93+0.11),the difference was also statistically significant(P<0.01).Simultaneously,the level of total LC3B(LC3B-I+LC3B-II)protein in MPA treatments,either in uninfected or in HCV-infected Huh7 cells was lower than that in control group,respectively(P<0.01).3.MPA inhibited the expression of autophagy-related genes.(1)The screening results in uninfected Huh7 cells:compared with control group,MPA treatment down-regulated the expression of Beclinl,ATG3,ATG5,ATG7,Bad,Bax,Bcl-xl and GABARAP at mRNA level(P<0.01).(2)The screening results in HCV-infected Huh7 cells:compared with control group,MPA treatment down-regulated the expression of Beclin1,ATG3,ATG5,ATG7,Bad and Bax at mRNA level(P<0.01)except the Bcl-xl and GABARAP.(3)The expression of ATG3,ATG5 and ATG7 protein in MPA treatment was lower than that in control group,either in uninfected or in HCV-infected Huh7 cells(1.05±0.13 vs 1.39±0.07;0.56±0.06 vs 0.88±10.03;0.78±0.08 vs 0.96±0.06);(0.71 ± 0.09 vs 1.12±0.12;0.66±0.08 vs 1.08±0.05;0.76±0.05 vs 1.13±0.11),and the differences were statistically significant(P<0.01).4.HCV infection increased the expression of LC3,Beclinl,ATG3,ATG5,ATG7,Bad,Bax,Bcl-xl and GABARAP at mRNA level,by comparing their expression in HCV JFH-1 infected Huh7 cells and uninfected cells(P<0.01).Conclusions MPA treatment could inhibit HCV replication and block cellular autopahgy.MPA treatment down-regulated the expression of several important autophagy-related genes,including Beclin1,ATG3,ATG5,ATG7,through which inhibiting cellular autophagy and further inhibiting HCV replication.These results suggest that MPA has a antiviral ability against HCV replication by down-regulating the expressions of ATGs and blocking cellular autophagy.