Genome-wide transcriptomic analysis of hepatocyte-like cells upon ectopic miR-146a-5p expression

Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in cellular homeostasis within the liver and deregulation of the miRNome has been associated with liver disease including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross-talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here, we combined a hepatocyte-like based model system, high-throughput small RNA-sequencing, computational analysis and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least two-fold including miRNAs that were previously described to target genes associated with inflammation, fibrosis and cancer development. Further investigation demonstrated that miR-146a-5p was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes as well as in liver tissues from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p over-expression is related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p positively impacts on late steps of the viral replication cycle thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease. To explore the functional relevance of miR-146a-5p up-regulation, we performed a genome-wide transcriptomic analysis of hepatocyte-like cells upon ectopic miR-146a-5p expression. Hepatocyte-like cells (differentiated Huh7.5.1 cells) were reverse transfected with miR-146a-5p mimic (5 nM, Sigma-Aldrich; three samples) or non-targeting control (5 nM, GE Dharmacon; three samples) using RNAiMax reagent according to the manufacturer's instructions (Life Technologies). Seventy-two hours post-transfection, total RNA was extracted and subjected to genome-wide mRNA profiling using GeneChip Gene 1.0 ST array (Affymetrix TSA) at the IGBMC Microarray and Sequencing Platform (Illkirch, France).