Abstract
We aimed to investigate the effect of methotrexate (MTX) on microRNA modulation in rheumatoid arthritis fibroblast-like synovial cells (RA-FLS). RA-FLS were treated with MTX for 48 h. We then performed miRNA array analysis to investigate differentially expressed miRNAs. Transfection with miR-877-3p precursor and inhibitor were used to investigate the functional role of miR-877-3p in RA-FLS. Gene ontology analysis was used to investigate the cellular processes involving miR-877-3p. The production of cytokines/chemokines was screened by multiplex cytokine/chemokine bead assay and confirmed by ELISA and quantitative real-time PCR. The migratory and proliferative activities of RA-FLS were analyzed by wound healing assay and MKI-67 expression. MTX treatment altered the expression of 13 miRNAs (seven were upregulated and six were downregulated). Among them, quantitative real-time PCR confirmed that miR-877-3p was upregulated in response to MTX (1.79 ± 0.46-fold, p < 0.05). The possible target genes of miR-877-3p in RA-FLS revealed by the microarray analysis were correlated with biological processes. The overexpression of miR-877-3p decreased the production of GM-CSF and CCL3, and the overexpression of miR-877-3p inhibited migratory and proliferative activity. MTX altered the miR-877-3p expression on RA-FLS, and this alteration of miR-877-3p attenuated the abundant production of cytokines/chemokines and proliferative property of RA-FLS.
Highlights
Values are means ± standard deviation (SD). * p < 0.05 vs. scrambled controls. This is the first study to demonstrate that MTX alters the expression of microRNAs in rheumatoid arthritis fibroblast-like synovial cells (RA-fibroblast-like synovial cells (FLS))
Our findings demonstrated that (1) the expression of miR-877-3p was increased by the MTX treatment of RA-FLS, and (2) the upregulation of miR-877-3p led to
Our findings demonstrated that (1) the expression of miR-877-3p was increased by the MTX treatment of RA-FLS, and (2) the upregulation of miR-877-3p led to decreased productions of cytokines and chemokines and decreased cell migration
Summary
Rheumatoid arthritis (RA) is a systemic autoimmune disease with severe joint inflammation, characterized by progressive bone and cartilage destruction. FLS have an antiapoptotic property and an invasive phenotype that cause hyperplastic synovial tissue, leading to the destruction of cartilage and bone. The presence of FLS contributes to the local production of proinflammatory cytokines, chemokines, and proteolytic enzymes that degrade the extracellular matrix [1]. Several pharmacological mechanisms by which MTX attenuates the disease condition of RA have been reported. As a mechanism that is unique to RA pathogenesis, both the induction of apoptosis of RA-FLS and the inhibition of cytokine production from RA-FLS by MTX have been reported [7,8,9]. The precise mechanisms of how MTX inhibits cytokine production and exerts antiproliferative effects on RA-FLS remain unknown
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