Background: Hepatitis C virus (HCV) infection and the resulting liver diseases are a major health problem both in the United States and worldwide. The natural history of HCV in the liver is characterized by a slow progression to cirrhosis, end-stage liver diseases, and in some cases hepatocellular carcinoma. miRNAs have been implicated in many human diseases and viral infections are known to modulate miRNA expression. This study is aimed to identify the role of HCV induced changes in miRNAs; in particular miRNA 449a, in mediating CCL2 induced inflammatory responses.

Methods: Computational sequence analyses and reporter assays using the CCL2 promoter region were carried out to analyze transcription factors. mRNA and miRNA expression analyses were done using q-PCR. Gene knockdowns in primary human hepatocytes were carried out using siRNA transfections. Localization in the cells wase determined by immunostaining. miRNA target validations were done using q-PCR and western blot.

Results: Reporter analysis using -2000 bp of the CCL2 promoter region and localization demonstrated that CCL2 expression is regulated by the cytokine IL6. Further, the IL6 dependent CCL2 expression was mediated by miRNA-449a in human hepatocytes. Computational analyses followed by biochemical assays identified IL6 Receptor (IL6R) is a direct target of miRNA-449a. siRNA mediated knockdown of IL6R and expression of miRNA-449a significantly reduced IL6 mediated CCL2 expression (>2 fold). Further, computational analysis followed by reporter studies demonstrated that the transcription factor STAT3, a component of the IL6 signaling pathway is required for IL6 mediated CCL2 expression.

Conclusions: Results demonstrate that miR-449a plays an important role in modulating expression of the inflammatory biomarker CCL2 through targeting the components of the IL6 signaling pathway following HCV infection. Therefore, definition of HCV induced changes in microRNAs and transcription factors will be important in defining the mechanisms for the accelerated development of allograft fibrosis following liver transplantation of HCV recipients.

« Back to 2013 American Transplant Congress