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 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.MicroRNA (miRNA) are a well-established class of small (ϳ22 nucleotides) endogenous noncoding RNAs that influence the stability and translation of messenger RNA (mRNA) (1). Using various computational and experimental approaches, hundreds of miRNA have been identified in numerous animal species. The miRNA genes are transcribed by RNA polymerase II as primary miRNA (pri-miRNA) (2,3). The RNase III enzyme Drosha then processes the nuclear pri-miRNA, yielding a ϳ70-nucleotide molecule known as precursor miRNA (pre-miRNA) (4), which is exported from the nucleus. Maturation of the pre-miRNA into miRNA is then mediated by the cytoplasmic enzyme Dicer (5), after which the mature miRNA is loaded into the RNA-induced silencing complex (RISC)

Nfib hemizygous mice are protected from hyperoxic lung injury and death

“…The role of miRNAs in posttranscriptional silencing of MCP‐1 is well studied (Nakamachi et al. 2009), and may contribute to better survival in hemizygous mice. Overexpression of some of the redox genes, including those involved in expression of AOE activity is typical following exposure to hyperoxia.…”

The Role of miRNA in Rheumatoid Arthritis

“…However, their analyses were focused on miR-146a but not on miR-363 and miR-498. We compared synovial fibroblast derived from patients with RA (RASF) with those from patients with OA (OASF) for their expression of a panel of 156 miRNAs with quantitative stem-loop RT-RCR (Nakamachi et al, 2009). We found that the miR-124a level significantly decreased in RASF.…”

“…In culture, RA fibroblast like synoviocytes (FLS) proliferate and secrete a variety of cytokines/ chemokines/angiogenic factors, including fibroblast growth factor, granulocytemacrophage colony stimulating factor, interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory proteins 1 , and they present various adhesion molecules on their surfaces. Since the first report by Stanczyk et al in 2008, several reports have described the altered miRNAs in the joint and/or peripheral blood leucocytes of patients with RA, including miR-155, miR-146 and others (Table 1) ( Stanczyk, et al, 2008;Nakasa, et al, 2008;Pauley, et al, 2008;Murata, et al, 2010;Li, et al, 2010;Fulci, et al, 2010;Nakamachi, et al, 2009. Niimoto, et al, 2010.…”

Inflammatory memories: Is epigenetics the missing link to persistent stromal cell activation in rheumatoid arthritis?

“…Expression of several microRNAs has been described to be altered in RASFs and to influence proliferation rates and production of pro-inflammatory cytokines and MMPs. Levels of miR-155 and miR-146a are constitutively elevated in cultured RASFs, as well as in RA synovial tissues, synovial fluid and PBMCs, whereas the expression of miR124a is decreased in RASFs compared to OASFs [44][45][46][47][48]. Expression of miR-203 is constitutively upregulated in RASFs, and overexpression of miR-203 promotes production of IL-6 and MMPs in these cells [49].…”