Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells

Hepatology. 2014 Mar;59(3):1118-29. doi: 10.1002/hep.26768. Epub 2014 Jan 27.


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.

Conclusion: 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 3' Untranslated Regions / genetics
  • Aniline Compounds / pharmacology
  • Animals
  • Benzylidene Compounds / pharmacology
  • Chemical and Drug Induced Liver Injury* / genetics
  • Chemical and Drug Induced Liver Injury* / metabolism
  • Chemical and Drug Induced Liver Injury* / pathology
  • Coculture Techniques
  • Connective Tissue Growth Factor / genetics*
  • Connective Tissue Growth Factor / metabolism
  • Epigenesis, Genetic / physiology
  • Exosomes / metabolism
  • Hep G2 Cells
  • Hepatic Stellate Cells / cytology
  • Hepatic Stellate Cells / physiology*
  • Humans
  • Liver Cirrhosis / genetics
  • Liver Cirrhosis / metabolism
  • Liver Cirrhosis / pathology
  • Mice
  • MicroRNAs / metabolism*
  • Primary Cell Culture
  • RNA, Messenger / genetics
  • Signal Transduction / drug effects
  • Signal Transduction / genetics


  • 3' Untranslated Regions
  • Aniline Compounds
  • Benzylidene Compounds
  • CCN2 protein, human
  • CCN2 protein, mouse
  • GW 4869
  • MIRN214 microRNA, human
  • MicroRNAs
  • Mirn214 microRNA, mouse
  • RNA, Messenger
  • Connective Tissue Growth Factor