Reciprocal effects of micro-RNA-122 on expression of heme oxygenase-1 and hepatitis C virus genes in human hepatocytes

Abstract

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 mumol/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.

Figure 1. Organization of the HCV constructs CNS3 and 9–13

(A). Open reading frames (thick boxes) are flanked by the 5′- and 3′-UTRs (thin boxes). CNS3 is the Huh-7 cell line that expresses a zeo resistance gene (zeo), the IRES of the encephalomyocarditis virus (E–I), which directs translation of HCV sequences from core, envelop (E1 and E2), up to the aminoterminal domain of NS3. Replicon 9–13 is composed of HCV-IRES, the neomycin phosphotransferase (neo) gene, E–I, HCV sequences from NS3 through NS5B, and the 3′-NTR. Panels B and C are Western blot analysis of the HCV core, NS5A and GAPDH proteins from CNS3 and 9–13 replicon cell lines. 50 μg of protein were separated by 4–15% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-HCV core, anti-NS5A, and GAPDH specific antibodies. Representative results from one of three experiments are shown.

Figure 2. An antagomir of miR-122 reduces HCV RNA abundance in dose- and time-dependent manner in HCV replicon cells

Chemically synthesized miR-122 antagomir was introduced into CNS3 and 9–13 replicon cells by Lipofectamine 2000-mediated transfection, and total RNA was extracted 48 or 72 h later. qRT-PCR analysis of HCV core (A) and NS5A (B) mRNAs of cells treated with the indicated dose of miR-122 antagomir for 72 h as shown. Time-courses of replication of HCV core (C) and NS5A (D) treated with 50 nM of miR-122 antagomir are shown. Normalized values of miRNA in absence of miR-122 antagomir were set equal to 1. Data are presented as means ± SE from three samples. * differs from no treatment or zero time, p<0.05, ** differs from no treatment or zero time, p< 0.01.

Figure 3. An antagomir of miR-122 reduces HCV protein abundance in HCV replicon cells

Chemically synthesized miR-122 antagomir (50 nM) was introduced into 9–13 cells using Lipofectamine 2000-mediated transfection, and total protein was extracted 72 h later. Western blot analysis of HCV NS5A protein treated with the indicated doses of miR-122 antagomir is shown in panel A. 50 μg of protein were separated by 4–15% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-NS5A and GAPDH specific antibodies. Representative results from one of three experiments are shown. The relative NS5A protein amounts normalized to GAPDH, the invariant control, are shown in panel B. No miR-122 antagomir was set equal to 1.

Figure 4. An antagomir of miR-122 specifically down-regulates HCV mRNA in 9–13 cells

50 nM of non-specific control antagomir (NSCA), 2-O-methylated mimic miR-122 (mimic miR-122), and chemically synthesized miR-122 antagomir were introduced into 9–13 cells by Lipofectamine 2000-mediated transfection. Total RNA were extracted and the levels of HCV NS5A mRNAs were measured by qRT-PCR. No miRNA treatment was set equal to 1. Data are presented as means ± SE from three samples. * differs from none or other treatments, p<0.05.

Figure 5. An antagomir to miR-122 regulates Bach1 and HO-1 mRNAs in 9–13 cells

The miR-122 antagomir was transfected into 9–13 replicon cells by Lipofectamine 2000. Total RNA was extracted 72 hours later. HO-1 mRNA or Bach1 levels were quantified by qRT-PCR. A) Levels of Bach1 mRNA were down-regulated significantly in HCV replicon cells transfected with miR-122 antagomir. B) HO-1 mRNA increased >2-fold by miR-122 antagomir, compared with no miR-122 antagomir. Data are presented as means ± SE from three samples. * differs from non-treatments, p<0.05.

Figure 6. Silencing Bach1 gene up-regulates HO-1 and down-regulates HCV replication in 9–13 cells

9–13 cells were transfected with 50 nM Bach1-siRNA, antagomir of miR-122, or the combination of Bach1-siRNA and antagomir of miR-122 for 48 h, after which cells were harvested. Bach1 (A), HO-1 (B), and NS5A (C) mRNA levels were measured by quantitative RT-PCR, as described in the Methods. Data are presented as means ± SE from three samples.

Figure 7. Silencing Bach1 gene up-regulates HO-1 and down-regulates HCV replication in CNS3 cells

CNS3 cells were transfected with 50 nM Bach1-siRNA, antagomir of miR-122, or the combination of Bach1-siRNA and antagomir of miR-122 for 48 h, after which cells were harvested. HO-1 (A) and HCV Core (B) mRNA levels were measured by quantitative RT-PCR, as described in the Methods. Data are presented as means ± SE from three samples.

Figure 8. Up-regulation of HO-1 by CoPP down-regulates HCV replication in CNS3 cells

A) HO-1 mRNA was increased by treatment with 5 μM CoPP for 24 h. B) HCV core mRNA was down-regulated by CoPP in a dose-dependent fashion at 24h. C) HCV core protein was down-regulated by CoPP and heme. CNS3 cells were treated with 1–10 μM CoPP, or 2.5 μM heme for 24 h, after which cells were harvested. HO-1, HCV Core, and GAPDH mRNA levels were measured by quantitative RT-PCR. HO-1 and HCV Core were normalized by GAPDH. Data are presented as means ± SE from three samples. HCV Core and GAPDH proteins were measured by Western Blot. 50 μg of protein were separated by 4–15% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-HCV Core and GAPDH specific antibodies. Representative results from one of three experiments are shown. * differs from DMSO (0 μM) treatment, p<0.05; ** differs from DMSO treatment, p<0.001.