title: Next generation sequencing of the Ago2 interacting transcriptome identified chemokine family members as novel targets of neuronal microRNAs in hepatic stellate cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Institute for Pathology, University Hospital of Cologne, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Noetel
authors.forename: Natalia
authors.initials: N
authors.lastname: Elfimova
authors.forename: Janine
authors.initials: J
authors.lastname: Altmüller
authors.forename: Christian
authors.initials: C
authors.lastname: Becker
authors.forename: Diana
authors.initials: D
authors.lastname: Becker
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lahr
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.forename: Hermann
authors.initials: H
authors.lastname: Wasmuth
authors.forename: Andreas
authors.initials: A
authors.lastname: Teufel
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Dienes
authors.forename: Margarete
authors.initials: M
authors.lastname: Odenthal
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism*; D004195:Disease Models, Animal; D055166:Hepatic Stellate Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D008103:Liver Cirrhosis / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000378:metabolism*; D058628:Myofibroblasts / Q000378:metabolism / Q000473:pathology; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D059467:Transcriptome / Q000235:genetics*; D015854:Up-Regulation
chemical_list: C466422:AGO2 protein, human; C582332:Ago2 protein, rat; D060565:Argonaute Proteins; D018925:Chemokines; C531888:MIRN125 microRNA, human; C581261:MIRN125 microRNA, rat; C545189:MIRN128 microRNA, human; C557010:MIRN128 microRNA, rat; C582333:MIRN9 microRNA, rat; C541874:MIRN92 microRNA, human; D035683:MicroRNAs