Open AccessMicroRNA‐124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast‐like synoviocytes from patients with rheumatoid arthritis
Author(s) -
Nakamachi Yuji,
Kawano Seiji,
Takenokuchi Mariko,
Nishimura Kunihiro,
Sakai Yoshitada,
Chin Takaaki,
Saura Ryuichi,
Kurosaka Masahiro,
Kumagai Shunichi
Publication year - 2009
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.24475
Subject(s) - microrna , microbiology and biotechnology , transfection , monocyte , luciferase , chemistry , cytokine , reporter gene , cancer research , biology , gene expression , immunology , gene , biochemistry
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 G 1 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. Conclusion The results of this study suggest that miR‐124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.