Gastroenterology

Gastroenterology

Volume 133, Issue 4, October 2007, Pages 1166-1174
Gastroenterology

Clinical–liver, pancreas, and biliary tract
Reciprocal Effects of Micro-RNA-122 on Expression of Heme Oxygenase-1 and Hepatitis C Virus Genes in Human Hepatocytes

https://doi.org/10.1053/j.gastro.2007.08.002Get rights and content

Background & Aims: Heme oxygenase-1 (HO-1) is an antioxidant defense and key cytoprotective enzyme, which is repressed by Bach1. Micro-RNA-122 (miR-122) is specifically expressed and highly abundant in human liver and required for replication of hepatitis C virus (HCV) RNA. This study was to assess whether a specific miR-122 antagomir down-regulates HCV protein replication and up-regulates HO-1. Methods: We transfected antagomir of miR-122, 2’-O-methyl-mimic miR-122, or nonspecific control antagomir, into wild-type (WT) Huh-7 cells or Huh-7 stably replicating HCV subgenomic protein core through nonstructural protein 3 of HCV (NS3) (CNS3 replicon cells) or NS3-5B (9-13 replicon cells). Results: Antagomir of miR-122 reduced the abundance of HCV RNA by 64% in CNS3 and by 84% in 9-13 cells. Transfection with 2’-O-methlyl-mimic miR-122 increased HCV levels up to 2.5-fold. Antagomir of miR-122 also decreased Bach1 and increased HO-1 mRNA levels in CNS3, 9-13, and WT Huh-7 cells. Increasing HO-1 by silencing Bach1 with 50 nmol/L Bach1-short interfering RNA or by treatment with 5 μmol/L cobalt protoporphyrin or heme (known inducers of HO-1) decreased HCV RNA and protein by 50% in HCV replicon cells. Conclusions: Down-regulation of HCV replication using an antagomir targeted to miR-122 is effective, specific, and selective. Increasing HO-1, by silencing the Bach1 gene or by treatment with cobalt protoporphyrin or heme, decreases HCV replication. Thus, miR-122 plays an important role in the regulation of HCV replication and HO-1/Bach1 expression in hepatocytes. Down-regulation of miR-122 and up-regulation of HO-1 may be new strategies for anti-HCV intervention and cytoprotection.

Section snippets

Materials

The human hepatoma cell line, Huh-7, was purchased from the Japan Health Research Resources Bank (Osaka, Japan). Ferric (Fe+3)-protoporphyrin IX•Cl (hemin) and cobalt protoporphyrin (CoPP) were from Porphyrin Products (Logan, UT). Goat anti-human glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Mouse antihepatitis C core protein was purchased from Affinity BioReagents, Inc (Golden, CO). Mouse anti-HCV nonstructural

Down-Regulation of HCV RNA and Protein Expression by Antagomir of miR-122 in CNS3 and 9-13 Replicon Cells

miR-122 is specifically expressed and highly abundant in the human liver, in which it constitutes 70% of the total miRNA population.47, 50 There are 2 predicted binding sites for miR-122 in HCV viral 3′ and 5′ NCRs.53, 55 To assess down-regulation of antagomir of miR-122 on HCV expression, Huh-7 cells stably replicating HCV subgenomic (NS3-5B) replicon were used (Figure 1A). First, we compared the 2 cell lines stably replicating HCV core-NS3 or NS3-NS5B proteins with WT Huh-7 cells and cells

Discussion

The major findings of this study are that (1) an antagomir of miR-122 reduces the abundance of HCV mRNA and protein in CNS3 and 9-13 cells; (2) transfection with mimic-miR-122 increases HCV levels, whereas transfection with NSCA did not change the level of HCV, when compared with the cells that were not transfected; (3) antagomir of miR-122 decreased Bach1 and increased HO-1 mRNA levels in CNS3 and 9-13 replicon cells; (4) silencing Bach1 with Bach1-siRNA increases HO-1 mRNA levels and

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    The opinions expressed herein are those of the authors. They do not necessarily reflect the official views of the U.S.P.H.S. or The University of Connecticut Health Center.

    No conflicts of interest exist.

    Supported by NIH RO1-DK38825 and contracts NO-1 DK92326 and UO-1 DK065193.

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