title: NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis.
abstract: BACKGROUND
  MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.
  
  
  METHODS
  Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.
  
  
  RESULTS
  In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.
  
  
  CONCLUSIONS
  This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
authors.forename: Ginette
authors.initials: G
authors.lastname: Tardif
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Pelletier
authors.forename: Hassan
authors.initials: H
authors.lastname: Fahmi
authors.forename: David
authors.initials: D
authors.lastname: Hum
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Mohit
authors.initials: M
authors.lastname: Kapoor
authors.forename: Johanne
authors.initials: J
authors.lastname: Martel-Pelletier
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D035683:MicroRNAs / Q000096:biosynthesis*; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D051900:Smad3 Protein / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis
chemical_list: D035683:MicroRNAs; C544302:Mirn140 microRNA, human; D050778:NFATC Transcription Factors; C494010:NFATC4 protein, human; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases