MicroRNA-124a Is a Key Regulator of Proliferation and Monocyte Chemoattractant Protein 1 Secretion in Fibroblast-like Synoviocytes From Patients With Rheumatoid Arthritis
MicroRNA-124a Is a Key Regulator of Proliferation and Monocyte Chemoattractant Protein 1 Secretion in Fibroblast-like Synoviocytes From Patients With Rheumatoid Arthritis
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DOI：
10.1002/art.24475
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发表时间：
2009-05-01
影响因子：
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通讯作者：
Kumagai, Shunichi
中科院分区：
文献类型：
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作者：
Nakamachi, Yuji;Kawano, Seiji;Kumagai, Shunichi
Objective.为了阐明microRNA（miRNA）在类风湿关节炎（RA）发病机制中的作用，我们分析了RA患者滑膜细胞的miRNA表达。从RA患者手术标本中获得的滑膜细胞与从骨关节炎（OA）患者中获得的滑膜细胞进行比较，用定量茎环逆转录-聚合酶链反应检测其156种miRNA的表达。鉴定RA滑膜细胞中与OA滑膜细胞相比表达减少或增加的miRNA，并通过计算机分析预测其靶基因。我们使用了一种体外系统，通过将前体转染到滑膜细胞中来增强特定miRNA的表达，然后进行增殖、细胞周期和凋亡测定，以及用于细胞因子产生的酶联免疫吸附测定。通过Western blot分析和荧光素酶报告基因分析检测转染对预期靶蛋白和信使RNA（mRNA）的影响。我们发现，与OA滑膜细胞相比，RA滑膜细胞中的miR-124 a水平显著降低。将前体miR-124 a转染到RA滑膜细胞中显著抑制其增殖并将细胞周期阻滞在G（1）期。我们在细胞周期蛋白依赖性激酶2（CDK-2）和单核细胞趋化蛋白1（MCP-1）mRNA的3 '非翻译区鉴定了miR-124 a结合的推定共有位点。在RA滑膜细胞中诱导miR-124 a显著抑制CDK-2和MCP-1蛋白的产生。荧光素酶报告基因检测结果表明，miR-124 a特异性抑制CDK-2和MCP-1 mRNA 3 '端非翻译区的报告基因活性。本研究结果提示miR-124 a是RA滑膜细胞转录后调控机制中的关键miRNA。
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.