1, 2 There is a continuing need to develop new antiviral therapies. The molecular mechanisms underlying HCV-associated liver injury remain imperfectly understood. However, studies have indicated that HCV directly induces oxidative stress in hepatocytes through multiple mechanisms that include chronic inflammation, increases in iron, and liver injury. Therefore oxidative stress has emerged as an important pathogenetic mechanism in chronic hepatitis C,3–5 and antioxidant and cytoprotective therapy has been proposed to treat hepatitis C. Heme oxygenase 1 (HMOX1) is a key cytoprotective enzyme, catalyzing heme degradation and generating ferrous

iron, carbon PLX4032 monoxide, and biliverdin, the latter two of which have antioxidant and anti-inflammatory activities in vivo.6–8 Bach1, a basic leucine zipper mammalian transcriptional repressor of HMOX1, negatively regulates HMOX1 gene expression. Bach1 forms antagonizing heterodimers with the Maf-related oncogene family. These heterodimers bind to Maf recognition elements and suppress expression of genes [e.g.,

X-396 ic50 HMOX1 and NAD(P)H:quinone oxidoreductase].9–12 microRNAs (miRNAs) are a class of small noncoding RNAs (≈22 nt) that regulate gene expression primarily through translational repression.13, 14 The exact mechanism by which target genes are down-regulated remains unclear. Sequence complementarity in the 6- to 8-bp seed regions at the end of miRNA–messenger RNA (mRNA) heteroduplexes seem to determine the specificity of miRNA–target RNA interactions.15 miR-196 was first recognized to have extensive and evolutionarily conserved complementarity to homeobox clusters,

groups of related transcription factor genes crucial for numerous developmental programs Dehydratase in animals, and to regulate homeobox gene expression.16, 17 A recent report indicated one perfect match between miR-196 and nonstructural (NS) 5A coding region of the HCV JFH1 genome (genotype 2a), and interferon-β treatment resulted in significant induction of miR-196 in the human hepatoma cell line Huh-7 and in primary murine hepatocytes.18 A search of the TargetScan 4.2 database revealed that at least two miR-196 seed match sites occur in the 3′-untranslated region (UTR) of Bach1 mRNA. This led us to propose that miR-196 is an miRNA that targets the HCV genome and the 3′-UTR of Bach1 mRNA, the latter leading to up-regulation of the HMOX1 gene. These dual effects are analogous to effects of miR-122, recently reported from our laboratory.19 In this study, we experimentally validated the computational prediction of miR-196 binding in the 3′-UTR of Bach 1 mRNA by luciferase reporter assay. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression, and inhibited HCV expression.