microRNA-218 modulates gene expression in fibroblast-like synovial cells in rheumatoid arthritis

Objective: Fibroblast-like synovial cells (FLS) have multilineage differentiation potential including osteoblasts. We aimed to investigate the role of microRNAs during the osteogenic differentiation of rheumatoid arthritis (RA)-FLS. Methods: MicroRNA(miRNA) array analysis was performed to investigate the differentially expressed miRNAs during the osteogenic differentiation. Expression of miR-218-5p (miR-218) during the osteogenic differentiation was determined by quantitative real-time PCR. Transfection with miR-218 precursor and inhibitor were used to confirm the targets of miR-218 and to analyse the ability of miR-218 to induce osteogenic differentiation. Results: The miRNA array revealed that 12 miRNAs were up-regulated and 24 miRNAs were down-regulated after osteogenic differentiation. We observed that miR-218 rose in the early phase of osteogenic differentiation and then decreased. Micro array analysis revealed the mir-218 modulate the expression of ROBO1 in RA-FLS. The induction of miR-218 in RA-FLS decreased ROBO1 expression, and promoted osteogenic differentiation. Number of sample:4 sample Method: For a transient transfection approach with the aim to inhibit or enhance the miR-218 function, RA-FLSs were transfected with synthetic precursor miRNA (pre-miR), with inhibitors of miR-218 (anti-miR), or with scrambled controls (Pre-miR/Anti-miR Negative Control #1; Ambion/Applied Biosystems, Foster City, CA) at a final concentration of 100 nM with the use of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After transfection we extracted RNA using mirVana miRNA Isolation kit(Ambion/Applied Biosystems). We investigated the gene expression after transfection of pre/anti-miR-218 using microarray.