MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins - PubMed (original) (raw)

MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins

Weihong Hou et al. Hepatology. 2010 May.

Abstract

Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs ( approximately 22 nt) that are important regulators of gene expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were measured by way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The Dual Glo Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3'-untranslated region (UTR)-dependent luciferase activity but not in mutant Bach1 3'-UTR-dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3'-UTR-dependent luciferase activity.

Conclusion: miR-196 directly acts on the 3'-UTR of Bach1 messenger RNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Overexpression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection.

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Figures

Figure 1. In silico prediction of putative binding sites for miR-196 in the 3′-UTR of Bach1

(A) Schematic of seed region match between miR-196 and the first putative Bach1 3′-UTR site targeted. The first site is highly conserved in the human, mouse, rat, dog and pig. (B) Schematic of second seed region match between miR-196 and the putative Bach1 3′-UTR site targeted. The second site is poorly conserved in the human, mouse, rat, dog and pig.

Figure 2. miR-196 mimic down-regulates Bach1 protein levels and up-regulates expression of the HMOX1 gene in 9-13 cells

9-13 cells were transfected with 50 nM miR-196 mimic or miRNA mimic negative control (MMNC) by Lipofectamine 2000 as indicated. 24–48 h after transfection, the cells were harvested. Bach1 protein levels were assessed by Western blots with anti-Bach1 or GAPDH specific antibodies. The levels of Bach1 mRNA were quantified by qRT-PCR as described in Materials and Methods. The amounts of Bach1 protein and mRNA were normalized to GAPDH which did not vary with treatment. Values for cells with a mock transfection were set equal to 1. The levels of HMOX1 mRNA were quantified by qRT-PCR. The amounts of HMOX1 mRNA levels were normalized to GAPDH which did not vary with transfection. Values for cells with mimic negative control transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P<0.05. (A) miR-196 mimic down-regulated Bach1 protein levels. (B) The levels of Bach1 mRNA were not altered with miR-196 mimic transfection. (C) miR-196 mimic up-regulated HMOX1 mRNA levels. (D) miR-196 did not alter Cullin 3 mRNA levels.

Figure 3. miR-196 mimic represses the expression of a luciferase reporter containing Bach1 3′-UTR

(A) The Bach1 3′-UTR contains two seed match sites (highlighted) for miR-196. (B) Schematic representation of pGL3-Bach1, the firefly luciferase (f-luc) reporter construct utilized. (C) Inhibition of the f-luc activities of pGL3-Bach1 3′-UTR reporter by miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK (renilla) and with 50 nM miR-196 mimic, miR-16 mimic or miRNA mimic negative control (MMNC) by Lipofectamine 2000 as indicated. 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P<0.05.

Figure 4. miR-196 does not inhibit luciferase activity of reporter with mutant Bach1 3′-UTR

(A) Four nucleotides in two seed match sites of Bach1 3′-UTR were replaced. (B) miR-196 mimic decreased the f-luc activity of pGL3-Bach1-WT but not pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-196 mimic (10–50 nM) as indicated in Materials and Methods, 48 h after transfection, the luciferase reporter activities were measured using Dual Luciferase Assay System from Promega, firefly luciferase activities were normalized to renilla luciferase activities and total protein. (C) miR-155 mimic decreased the f-luc activities of both pGL3-Bach1-WT and pGL-Bach1-Mut reporter. 9-13 cells were co-transfected with 0.4 μg/mL of mutant pGL3-Bach1 or 0.4 μg/mL of pGL3-Bach1 and with 0.4 μg/mL of pRL-TK (renilla), and with miR-155 mimic (10–50 nM). 48 h after transfection, the luciferase reporter activities were assayed as described in Materials and Methods. Values for cells with a mock transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from negative control only, P<0.05.

Figure 5. Mutant miR-196 mimic does not alter the luciferase activity of Bach1 3′-UTR reporter but inhibits the luciferase activity of mutant Bach1 3′-UTR reporter

(A) Four nucleotides mutation were introduced to the seed match sites of miR-196. (B) Effect of mutant miR-196 on Bach1 3′-UTR reporter activity. 9-13 cells were co-transfected with 0.4 μg/mL of pGL3-Bach1, 0.4 μg/mL of pRL-TK and with 0, 10, 20 or 50 nM mimic negative control, miR-196 mimic or mutant miR-196 for 48 h, firefly and renilla luciferase activities were measured using Dual Luciferase Assay System from Promega. Firefly luciferase activities were normalized to renilla luciferase activities and total protein, as indicated in Materials and Methods. Values for cells without microRNA transfection were set equal to 1. (C) Restoration of seed match between mutant miR-196 and mutant Bach1 3′-UTR are shown. (D) Inhibition of mutant Bach1 3′-UTR reporter activity by mutant miR-196 mimic. 9-13 cells were co-transfected with 0.4 μg/mL of mutant reporter (pGL3-Bach1-Mut), 0.4 μg/mL of pRL-TK, and with 0, 10, 20 or 50 nM mutant miR-196 or wild type miR-196 mimic for 48 h, firefly and renilla luciferase activities were measured. Firefly luciferase activities were normalized to renilla luciferase activities and total protein. Values for cells without microRNA transfection were set equal to 1. Data are presented as means ± SE, n=3. * differs from mimic negative control, P<0.05.

Figure 6. miR-196 mimic represses HCV expression in HCV replicon cells

Cells were transfected for 24-48 h with 10–50 nM miR-196 mimic or Bach1-siRNA by Lipofectamine 2000, after which cells were harvested and total RNA and protein were extracted. The levels of HCV core and NS5A and host cell GAPDH mRNA and protein levels were measured by quantitative RT-PCR and Western blots as described in Materials and Methods. Data are presented as means ± SE, n=3. * differs from negative control only, P<0.05. (A) Down-regulation of HCV core and NS5A mRNA expression by miR-196 mimic in Con1 cells. (B) Down-regulation of HCV NS5A protein expression by miR-196 mimic in 9-13 cells. (C) Silencing Bach1 gene expression by Bach1-siRNA in Con1 cells. (D) Down-regulation of HCV core and NS5A mRNA expression by Bach1-siRNA in Con1 cells.

Figure 6. miR-196 mimic represses HCV expression in HCV replicon cells

Figure 7. Effect of miR-196 on HCV expression in Huh-7.5 cells transfected with Con1-WT or Con1-Mut RNA

Huh-7.5 cells were transfected with 2 μg/well of HCV Con1-WT or Con-Mut RNA by Lipofectamine 2000. After 24 h, cells were transfected with 50 nM of miR-196 mimic, or miRNA mimic negative control (MMNC) as control. After 48 h, cells were harvested and total proteins were extracted. HCV core, NS5A and GAPDH protein levels were measured by Western blots. Data are presented as means ± SE, n=3. * differs from control P<0.05. (A) In silico prediction of putative binding sites for miR-196 in HCV genotype1 Con1 genome and the four nucleotides mutation introduced to the binding sites of miR-196. (B) Effects of miR-196 on HCV core and NS5A protein levels in Con1-WT-transfected Huh-7.5 cells. (C) Effects of miR-196 on HCV core and NS5A protein levels in Con1-Mut-transfected Huh-7.5 cells. (D) Comparison of the effects in Con1-WT- vs Con1-Mut- transfected Huh-7.5 cells.

Figure 8. miR-196 mimic inhibits HCV expression in the HCV J6/JFH1-based cell culture system

Huh-7.5 cells were transfected with 2 μg/well of HCV J6/JFH1 RNA by Lipofectamine 2000. After 48 h, cells were transfected with miR-196 mimic, or miRNA mimic negative control (MMNC). For HCV infection, naïve Huh-7.5 were cultured with 1 mL of cell culture supernatants harvested from J6/JFH1-transfected cells, as described in Materials and Methods. After 48 h of exposure to the supernatants, cells were transfected with miR-196 mimic, or miRNA mimic negative control (MMNC) for 48 h. Cells were harvested and total RNA and proteins were extracted. HCV RNA was quantified by qRT-PCR, and HCV NS3 and GAPDH protein levels were measured by Western blots. Data are presented as means ± SE, n=3. * differs from control P<0.05. (A) Down-regulation of HCV J6/JFH1 RNA levels by miR-196 mimic in Huh-7.5 cells transfected with J6/JFH1 RNA. (B) Down-regulation of HCV J6/JFH1 RNA and (C) protein levels by miR-196 mimic in Huh-7.5 cells infected with J6/JFH1 hepatitis C virus secreted into the culture supernatant.

References

1. Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology. 2002;36:S237–244. - PubMed
1. Russo MW, Fried MW. Side effects of therapy for chronic hepatitis C. Gastroenterology. 2003;124:1711–1719. - PubMed
1. Choi J, Ou JH. Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus. Am J Physiol Gastrointest Liver Physiol. 2006;290:G847–851. - PubMed
1. Okuda M, Li K, Beard MR, Showalter LA, Scholle F, Lemon SM, Weinman SA. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology. 2002;122:366–375. - PubMed
1. Ghaziani T, Shan Y, Lambrecht RW, Donohue SE, Pietschmann T, Bartenschlager R, Bonkovsky HL. HCV proteins increase expression of heme oxygenase-1 (HO-1) and decrease expression of Bach1 in human hepatoma cells. J Hepatol. 2006;45:5–12. - PubMed

MicroRNA-196 represses Bach1 protein and hepatitis C virus gene expression in human hepatoma cells expressing hepatitis C viral proteins - PubMed (original) (raw)