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Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells.

ABSTRACT: Clinical studies demonstrate synergistic liver damage by alcohol and hepatitis C virus (HCV); however, the mechanisms by which alcohol promotes HCV infection remain obscure. The liver-specific microRNA-122 (miR-122) regulates HCV replication and expression of host genes, including Cyclin G1. Here, we hypothesized that alcohol regulates miR-122 expression and thereby modulates HCV RNA replication.The J6/JFH/Huh-7.5 model of HCV infection was used in this study. Real-time quantitative polymerase chain reaction, Western blotting, electrophoretic mobility shift assay, and confocal microscopy were used for experimental analysis.We found that acute alcohol exposure (25 mM) significantly increased intracellular HCV RNA as well as miR-122 levels in Huh-7.5 and Huh-7.5/CYP2E1 expressing cells in the presence and absence of J6/JFH-HCV infection. Expression of the miR-122 target, Cyclin G1, was inhibited by alcohol both in J6/JFH-infected and uninfected Huh-7.5 cells. The use of a miR-122 inhibitor increased Cyclin G1 expression and prevented the alcohol-induced increase in HCV RNA and protein levels, suggesting a mechanistic role for alcohol-induced miR122 in HCV replication. We discovered that siRNA-mediated silencing of Cyclin G1 significantly increased intracellular HCV RNA levels compared with controls, suggesting a mechanistic role for Cyclin G1 in HCV replication. Alcohol-induced increase in miR-122 was associated with increased nuclear translocation and DNA binding of the nuclear regulatory factor-?B and could be prevented by NF-?B inhibition.Our novel data indicate a miR-122-mediated mechanism for alcohol increasing HCV RNA replication. We show for the first time that Cyclin G1, a miR-122 target gene, has regulatory effects on HCV replication and that alcohol increases HCV replication by regulating miR-122 and Cyclin G1.

SUBMITTER: Hou W

PROVIDER: S-EPMC4171687 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells.

Hou Wei W Bukong Terence N TN Kodys Karen K Szabo Gyongyi G

Alcoholism, clinical and experimental research 20121105 4

<h4>Background</h4>Clinical studies demonstrate synergistic liver damage by alcohol and hepatitis C virus (HCV); however, the mechanisms by which alcohol promotes HCV infection remain obscure. The liver-specific microRNA-122 (miR-122) regulates HCV replication and expression of host genes, including Cyclin G1. Here, we hypothesized that alcohol regulates miR-122 expression and thereby modulates HCV RNA replication.<h4>Methods</h4>The J6/JFH/Huh-7.5 model of HCV infection was used in this study. ...[more]

PMID: 23126531

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Project description:Both alcohol and hepatitis C virus (HCV) infection could induce cellular autophagy in liver cells, which is considered to be essential for productive HCV replication. However, whether alcohol-induced autophagy is involved in the pathogenesis of HCV infection is still poorly understood. Alcohol treatment could induce autophagy in Huh7 cells (a hepatoma cell line that supports HCV JFH-1 replication), evidenced by the increase of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the decrease of p62 level in alcohol-treated cells compared with control cells. Alcohol treatment also significantly increased PIASy (a member of the PIAS family) expression, which can act as a SUMO (small ubiquitin-like modifier protein) E3 ligase to regulate a broader range of cellular processes including autophagy. Overexpression or the silencing expression of PIASy in alcohol-treated Huh7 cells could increase or decrease autophagic activation caused by alcohol treatment, respectively, and thus affect HCV replication correspondingly. In the absence of alcohol, overexpression or silencing expression of PIASy increase or decrease the level of cellular autophagy, judged by the changes of LC3B-II and p62 levels in the presence or absence of chloroquine (CQ), a lysosome inhibitor. More importantly, in the presence of 3-methyladenine (3-MA), an inhibitor in the early stage of autophagy, the effects of overexpression or silencing expression of PIASy on HCV replication were largely blocked. Furthermore, PIASy could selectively drive the accumulation of SUMO1-conjugated proteins, along with upregulation of the expression of several important autophagy factors, including ATG7 and ATG5-ATG12. In conclusion, alcohol promotes HCV replication through activation of autophagy in Huh7 cells, which partly attributes to its induction of PIASy expression. PIASy-enhanced accumulation of SUMO1-conjugated proteins may contribute to its inducing effect of autophagy. Our findings provide a novel mechanism for the action of alcohol-promoting HCV replication in the context of cellular autophagy.

Project description:miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.

Dataset Information

Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells.

Publications

Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells.

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