Alcohol Facilitates HCV RNA Replication Via Up‐Regulation of miR‐122 Expression and Inhibition of Cyclin G1 in Human Hepatoma Cells

Abstract

Background 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. Methods 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. Results 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-κB and could be prevented by NF-κB inhibition. Conclusions 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.

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease

“…Our present observation in livers with ALD appears to be contrary to previous in vitro findings where we have shown an increase in MIR122 during acute exposure to EtOH in hepatoma cell lines 43,50 . However, exposure in vitro represents an acute exposure model rather than chronic alcohol consumption.…”

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde

“…There is no direct evidence that ethanol metabolites, and particularly AGS, suppress HCV replication. In contrast, AGS exposure upregulated miR-122, which usually enhances HCV replication (20,45,48) and has been shown to be elevated in alcoholic liver disease patients (49). In addition, here, AGS treatment increased miR-34a, known to be a proapoptotic (10,11).…”

MicroRNAs in liver disease

“…In this context, alcohol consumption is known to enhance HCV replication, and a role for miR-122 in this process has been demonstrated; 71 alcohol treatment in HCV-infected hepatoma cells increases levels of miR-122, which in turn modulates cyclin G1 expression. 71 In addition, miR-122 upregulation by alcohol was associated with increased levels of the P body protein, GW182 and heat shock protein 90, which contributed to enhance HCV replication.…”

“…In this context, alcohol consumption is known to enhance HCV replication, and a role for miR-122 in this process has been demonstrated; 71 alcohol treatment in HCV-infected hepatoma cells increases levels of miR-122, which in turn modulates cyclin G1 expression. 71 In addition, miR-122 upregulation by alcohol was associated with increased levels of the P body protein, GW182 and heat shock protein 90, which contributed to enhance HCV replication. 72 An additional consideration is that regulation of HCV replication by miR-122 has been investigated in isolated hepatocytes or hepatoma cells whereas changes in total liver levels of miR-122 expression might reflect miR-122 regulation in other cell types.…”