title: Derepression of MicroRNA-138 Contributes to Loss of the Human Articular Chondrocyte Phenotype.
abstract: OBJECTIVE
  To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs).
  
  
  METHODS
  The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays.
  
  
  RESULTS
  Expression levels of miR-138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR-138 is able to repress the expression of COL2A1 by directly targeting Sp-1 and hypoxia-inducible factor 2α (HIF-2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR-138 in the RNA-induced silencing complex and confirmed binding of Sp-1 to the COL2A1 promoter region in HACs.
  
  
  CONCLUSIONS
  We propose that an evolutionary pressure helps to suppress expression levels of miR-138 in human cartilage, thus enabling expression of appropriate tissue-specific matrix genes. Inhibition of miR-138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Christine I
authors.initials: CI
authors.lastname: Seidl
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Aida
authors.initials: A
authors.lastname: Martinez-Sanchez
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Murphy
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D024043:Collagen Type II / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007719:Knee Joint; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D060565:Argonaute Proteins; D051792:Basic Helix-Loop-Helix Proteins; D024043:Collagen Type II; D035683:MicroRNAs; D016329:Sp1 Transcription Factor; D000099162:Endothelial PAS Domain-Containing Protein 1; C466422:AGO2 protein, human; C499177:COL2A1 protein, human; C546110:MIRN138 microRNA, human; C108209:SP1 protein, human