The major novel findings of this work are that (1) miR-196 has functional binding sites in the 3′-UTR of Bach1 mRNA and (2) down-regulation of Bach1 by either miR-196 or Bach1-siRNA represses HCV expression. Functionality of miR-196 in regulation of Bach1 is demonstrated by several lines of evidence, including initial in silico
analysis (), by expected effects of miR-196 mimics (), and by the expected effects of miR-196 mimics on luciferase reporter constructs containing the wild type and mutant Bach1 3′-UTR (, and , and Suppl. Figure 1
). In addition, we demonstrate that down-regulation of Bach1 by either miR-196 or Bach1-siRNA leads to the up-regulation of the HMOX1 gene () and to down-regulation of HCV expression in replicon cells (the genotype 1b Con1 strain) () and the HCV J6/JFH1-generated cell culture system (). These results suggest that over-expression or up-regulation of miR-196 represents a potential new additional therapeutic strategy for chronic HCV infection, as well as other conditions in which up-regulation of HMOX1 may be desirable.
Based upon a large number of experimental and clinical studies carried out during the past several years, it is now generally accepted that HCV infection produces an increase in oxidative stress in infected hepatocytes. One important mediator of such increased oxidative stress is the HCV core protein (27
). In parallel with these observations are a series of observations in numerous systems, including experimental systems with expression of HCV, showing that HMOX1 helps to protect numerous cells and tissues against the potentially damaging effects of excess oxidative stress. These actions are based upon the ability of HMOX1 to decrease “free” or loosely-bound heme, which can act as a potent prooxidant, and to increase production of carbon monoxide, biliverdin, and bilirubin, which have potent antioxidant and anti-inflammatory and antifibrogenic effects (6
). HMOX1 has also emerged as an important anti-apoptotic enzyme (31
). Over-expression or induction of HMOX1 suppresses HCV replication and increases resistance of hepatocytes to oxidant injury (19
Regulation of expression of the HMOX1 gene is complex. However, we and others have shown that among the important sites for regulation are a series of expanded AP-1 sites, also called antioxidant responsive elements (31
), Maf protein responsive elements [MARE], and metalloporphyrin-responsive elements [MPRE] in the 5′ untranslated region of HMOX genes, across many species (32
). Bach1 plays a key role in tonic repression of expression of the HMOX1 gene. It does so by forming heterodimers with small Maf proteins and blocking transcriptional activation of the gene. Bach1 contains several consensus binding sites (all containing CP motifs), when they bind heme, lead to a change in conformation of the protein with marked reduction in affinity for Maf proteins and subsequent derepression and increase in activity of HMOX1 gene expression (9
In view of the above, it is not surprising that HMOX1 activity might be increased in HCV infection, and, indeed, we and others have shown this to be the case (5
). Nevertheless, in some other experimental systems and also in some clinical studies, a decrease in expression of HMOX1 has been observed in the setting of chronic hepatitis C (37
). These findings suggest the possibility that patients with genetic or other factors that lead to lower levels of HMOX1 gene expression may be at increased risk for development of chronic hepatitis C infection after acute HCV exposure and/or with greater risks of development of more rapidly progressive liver disease due to HCV infection. In this regard, there are at least two known genetic factors that influence levels of expression of HMOX1, namely, the length of GT repeats in the 5′-untranslated region and the presence of a single nucleotide polymorphism at position -413 (A/T, rs2071746) in the HMOX1 promoter region (39
The discovery of miRNAs in C. elegans
ushered in a new and exciting area of study. The numbers of miRNAs continues to grow, and additional mRNAs and candidate genes regulated by them continue to be identified. With respect to the liver, miR-122 was identified as the most abundant miRNA expressed in hepatocytes (accounting for ~70% of total miRNA’s) and shown to have major effects on several enzymes of cholesterol metabolism (41
). Unexpectedly, miR-122 was also shown to be required for HCV expression (19
), at least in cell culture systems. More recent work has shown that the effects of miR-122 depend upon the context and location of its cognate seed sequence binding sites. The sites in the 5′ region are mostly associated with up-regulation of expression, whereas those in the 3′ untranslated region are mostly associated with repression of expression (44
). Our current work adds miR-196 as a down regulator of HCV expression ( & ) and an attractive candidate as new therapeutic agents for chronic HCV infection.
Our study has limitations. Effects of miR-196 on, Bach1, HMOX1, and HCV thus far have been shown only in cell culture models, and the suppression of HCV expression has been moderate, not extremely high. The field of HCV research has been stymied by the lack of simple and robust animal models. Among non-human species, only chimpanzees have thus far been capable of being infected with HCV, and the disease in them is generally relatively mild. They are also extraordinarily difficult and expensive to maintain. Recently, murine models have been developed, based upon immune deficient animals into which human hepatocytes can be implanted without rejection, and these hepatocytes then infected with the hepatitis C virus (45
). Another recent model has been able to establish this in non-immunodeficient mice in which the host animal hepatocytes undergo necrosis and apoptosis and can be rescued with human hepatocytes (47
). Thus, over-expression of a combination of miR-196 and other selected miRNAs in order to decrease the viral output further in cell cultures and murine models are currently under study in our laboratory.
In summary, we demonstrate functional miR-196 binding sites in the 3′-UTR of Bach1, which lead to down-regulation of Bach1 gene expression, up-regulation of HMOX1 gene expression, and down-regulation of HCV expression. These findings add to the growing panoply of miRNAs that influence expression of genes and proteins of the hepatitis C virus and of HMOX1, a key cytoprotective enzyme. They suggest potential new additional therapies for chronic HCV infection and, perhaps, for other diseases characterized by increased oxidative stress.