Background & aims: After myofibroblastic transdifferentiation, hepatic stellate cells (HSC), mainly involved in liver fibrosis by extracellular matrix production, exhibit an altered growth factor profile including increased expression of neuronal mediators. Here, we analyzed putative targets of neuronal microRNAs miR-9, miR-125b, and miR-128 by deep sequencing of the transcript population, interacting with the miRNA/Argonaute 2 (Ago2) complex in myofibroblastic HSC.
Methods: MicroRNA expression was quantified by real-time PCR in primary HSC, isolated from the rat or human liver. Myofibroblastic HSC were transfected either with mimics or inhibitors of miR-9, miR-125b, and miR-128. RNA from immunoprecipitated Ago2-miRNA/transcript complexes was purified and used for next generation sequencing. Additionally, gene expression was investigated in quiescent and activated primary HSC, treated with the miR-128 mimic or inhibitor, by microarray analysis.
Results: During myofibroblastic transdifferentiation of HSC, miR-9, miR-125b, and miR-128 expression was markedly increased. Transcriptome analysis of Ago2 bound mRNA by deep sequencing identified a broad spectrum of transcripts that interact with neuronal miRNAs in myofibroblastic HSC. In particular, in HSC overexpressing miR-128, many members of the chemokine family were bound to the Ago2 repression complex. Furthermore, a comprehensive profiling of gene expression demonstrates the high impact of neuronal miRNAs on the chemokine network.
Conclusions: Ago2 immunoprecipitation followed by deep sequencing is a useful tool to identify novel miRNA targets. Upregulation of neuronal miR-9, miR-125b, and miR-128 during myofibroblastic transition and the identified interaction with a wide range of chemokines and chemokine receptors suggest a prominent role of neuronal miRNAs in the inflammatory response of HSC during fibrosis.
Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.