Background 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. 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.
Objectives In this study, we investigate the role of microRNAs during osteogenic differentiation of RA-FLS.
Methods 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.
Results 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<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.
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 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.
De Bari, C., et al., Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001. 44(8): p. 1928-42.
Disclosure of Interest : None declared