Background The hallmarks of rheumatoid arthritis (RA) is the expansive of fibroblast-like synovial cells (FLS) in affected joints, causing joint destruction. Inhibition of FLS proliferation is one of the therapeutic targets of RA. Moreover FLS have multilineage differentiation potential and can differentiate into osteoblasts. Therefore induction of osteogenic differentiation in proliferated FLS might become superior treatment. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been shown to regulate cell differentiation through regulation of gene expression post-transcriptionally.
Objectives To investigate the role of microRNAs during osteogenic differentiation of RA-FLS.
Methods RA-FLS was 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 and alizarin red staining. To investigate differentially expressed miRNAs during osteogenic differentiation, we performed miRNA array analysis. Expression of miRNA-218 (miR-218) during osteogenic differentiation of RA-FLS was analyzed by quantitative real-time PCR. 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. Finally, we analysed osteogenic differentiation after overexpression/knockdown of miR-218.
Results Differentiation of RA-FLS into osteoblast was evidenced by ALP staining, alizarin red staining and up-regulation of ALP and RUNX2 mRNA expression. The miRNA array analysis revealed that 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 and quantitative real-time PCR with additional samples confirmed that miR-218 was down-regulated during osteogenic differentiation of RA-FLS (20±5%, p<0.0001, n=5). To elucidate the functional consequence of the deregulation of miR-218 during osteogenic differentiation of RA-FLS, we searched for gene targets of miR-218 and examined osteogenic differentiation using gain-and-loss of function assays with miR-218. Microarray analysis revealed several genes correlated the Slit/ROBO pathway were decreased by overexpression of miR-218, consistently knockdown of miR-218 increased the expression of that genes. Interestingly, miR-218 promoted, not suppressed, osteogenic differentiation of RA-FLS.
Conclusions 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 genes related the Slit/ROBO pathway. Although miR-218 was down-regulated after differentiation into osteoblast, miR-218 promoted osteogenic differentiation. We need to examine changes in miR-218 expression with time course during osteogenic differentiation to clarify the role of miR-218 in osteogenic differentiation of RA-FLS.
Disclosure of Interest None declared