THU0483 Microrna-218 Regulates Osteogenic Differentiation of Fibroblast-Like Synovial Cells in Rheumatoid Arthritis
Authors: N. Iwamoto, T. Suzuki, A. Kawakami
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> Rheumatoid arthritis (RA) is characterized by the proliferation of fibroblast-like synovial cells (FLS) in affected joints. FLS have multilineage differentiation potential and can differentiate into osteoblasts[1]. Inducing osteogenic differentiation of FLS might be strong therapeutic option in RA. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been shown to regulate gene expression post-transcriptionally. <h3>Objectives</h3> In this study, we investigate the role of microRNAs during osteogenic differentiation of RA-FLS. <h3>Methods</h3> RA-FLSs were harvested from synovial tissue of patients with RA and differentiated in osteogenic medium containing dexamethasone, ascorbate, glycerophosphate, L-Glutamine, Pen/Strep and MCGS for up to 3 weeks. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) staining. To investigate differentially expressed miRNAs during osteogenic differentiation of RA-FLS, we performed miRNA array analysis. Expression of miRNA-218 (miR-218) during osteogenic differentiation of RA-FLS was analyzed by quantitative real-time polymerase chain reaction (PCR). Furthermore, to identify target of miR-218, RA-FLS was transfected with synthetic precursor miRNA (pre-miR)/inhibitors of miRNA (anti-miR) of miR-218 using Lipofectamine and then gene expression microarray was performed. <h3>Results</h3> RA-FLS could be differentiated into osteoblasts and the osteoblasts were evidenced by ALP staining. The miRNA array analysis revealed a specific expression profile differentially expressed miRNAs during osteogenic differentiation of RA-FLS. 36 miRNAs showed differentially expression. 12 miRNAs were up-regulated and 24 miRNAs were down-regulated as compared with that of untreated control. Among them we were interested in miR-218 because of that miRNA was most significantly down-regulated. To validate the microarray findings, quantitative real-time PCR with additional samples was performed. In agreement with the array data, miR-218 was significantly down-regulated during osteogenic differentiation of RA-FLS as compared with that of untreated controls (20±5%, p&lt;0.0001, n=5). To elucidate the functional consequence of the down-regulation of miR-218 during osteogenic differentiation of RA-FLS, we searched for gene targets of miR-218 by performing gene expression microarray using gain-and-loss of function assays with miR-218. Overexpression of miR-218 reduced the expression of phosphoglucomutase 5 (PGM5), Kruppel-like factor 15 (KLF15). Consistently, knockdown of miR-218 increased the expression of these genes. <h3>Conclusions</h3> This is the first demonstration to our knowledge that microRNAs regulate osteogenic differentiation of RA-FLS. Our result showed that miR-218 modulate osteogenic differentiation of RA-FLS through its target gene PGM5, KLF15. Further experiments are necessary to determine silencing of miR-218 could cause osteogenic on RA-FLS and this attractive hypothesis needs to be further tested in animal models. <h3>References</h3> De Bari, C., et al., Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001. 44(8): p. 1928-42. <h3>Disclosure of Interest</h3> : None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.3596