P53 and STAT3 Dependent Expression of a Novel microRNA Restricts Hepatitis C Virus Infection Through Hyper-β-Oxidation of Fatty Acids by Impeding Cytosolic CAP350

Abstract

Background: The underlying complex molecular mechanism of Hepatitis C virus (HCV) mediated liver disease progression is still unresolved. Next generation sequencing of small RNAs provides genome-wide data about regulatory microRNAs. Here, we have identified undiscovered low abundant regulatory miRNAs and investigated the role of a novel human liver specific miRNAs in HCV mediated liver disease development. Methods: To discover unannotated miRNAs, unmapped sequences from small RNA transcriptome profiling of HCV infected and un-infected liver tissues were subjected to miRDeep2.pl pipeline. Targets predicted using MiRanda and Ingenuity pathway analysis (IPA). Northern-blot, Dicer knock down (K/D), Ago2-RNA immunoprecipitation (RIP), Stem-loop Real-time PCR (SLRTPCR), immunoblot (IB) analysis, luciferase assay, Chromatin immunoprecipitation (ChIP) etc. were used as required. Findings: Among seven predicted novel miRNAs, miR-c12 was found human liver specific miRNA. Dicer K/D and RIP assays showed that miR-c12 followed canonical miRNA biogenesis pathways. Its low expression in chronic HCV infected (CHC) tissues than normal liver indicated its protective role against HCV infection. Furthermore, upon HCV infection downregulation of p53 expression and hypermethylation of STAT3 binding site in the promoter of miR-c12 restricted its expression in CHC. IPA analysis revealed miR-c12 controlled mostly the fatty acid metabolism pathway. Reduced triglyceride accumulation and enhanced PPARα mediated fatty-acid catabolism upon miR-c12 retention in cells by targeting CAP350, a suppressor of PPARα, confirmed its role in lipid metabolism pathways. Interpretations: We discovered a previously unidentified low abundant human-liver specific miRNA having role in lipid homeostasis maintenance. Hence, miR-c12 together with anti-virals might be more effective therapeutic in restricting HCV infection. Funding Statement: Department of Biotechnology (DBT), Government of India [BT/PR11971/MED/29/890/2014]. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethics committee of the Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata, India and Indraprastha Apollo Hospital, New Delhi had approved the study. Blood and tissues were collected from patients who were willing to participate in the study with prior written consent.