title: MicroRNA-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis.
abstract: OBJECTIVE
  To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression.
  
  
  METHODS
  Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription-polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay.
  
  
  RESULTS
  We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G1 phase. We identified a putative consensus site for miR-124a binding in the 3'-untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3'-untranslated regions of CDK-2 and MCP-1 mRNA.
  
  
  CONCLUSIONS
  The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.
authors.affiliation: Department of Clinical Laboratory, Kobe University Hospital, and Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nakamachi
authors.forename: Seiji
authors.initials: S
authors.lastname: Kawano
authors.forename: Mariko
authors.initials: M
authors.lastname: Takenokuchi
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Nishimura
authors.forename: Yoshitada
authors.initials: Y
authors.lastname: Sakai
authors.forename: Takaaki
authors.initials: T
authors.lastname: Chin
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Saura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
authors.forename: Shunichi
authors.initials: S
authors.lastname: Kumagai
mesh_terms: D017209:Apoptosis / Q000502:physiology; D001172:Arthritis, Rheumatoid / Q000209:etiology / Q000503:physiopathology*; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000032:analysis / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000032:analysis; D018844:Cyclin-Dependent Kinases / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000502:physiology*; D010003:Osteoarthritis / Q000503:physiopathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000166:cytology / Q000503:physiopathology*; D014162:Transfection
chemical_list: D018932:Chemokine CCL2; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases