title: Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells.
abstract: Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC, is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150 nm in diameter, negatively charged (-26 mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR-214-transfected activated HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild-type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled by way of exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels.
  
  
  CONCLUSIONS
  Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs.
authors.affiliation: Research Institute at Nationwide Children's Hospital, Columbus, OH.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Alyssa
authors.initials: A
authors.lastname: Charrier
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Ruju
authors.initials: R
authors.lastname: Chen
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
authors.forename: Kitty
authors.initials: K
authors.lastname: Agarwal
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: L James
authors.initials: LJ
authors.lastname: Lee
authors.forename: Michael E
authors.initials: ME
authors.lastname: Paulaitis
authors.forename: David R
authors.initials: DR
authors.lastname: Brigstock
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001597:Benzylidene Compounds / Q000494:pharmacology; D056486:Chemical and Drug Induced Liver Injury / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018920:Coculture Techniques; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000502:physiology; D055354:Exosomes / Q000378:metabolism; D056945:Hep G2 Cells; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D000814:Aniline Compounds; D001597:Benzylidene Compounds; C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C468773:GW 4869; C547397:MIRN214 microRNA, human; D035683:MicroRNAs; C534781:Mirn214 microRNA, mouse; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor