Background The hallmarks of rheumatoid arthritis (RA) is the expansive of fibroblast-like synovial cells (FLS) in affected joints, causing joint destruction. FLSs are resistant to programmed cell death resulting in aggressive, invasive phenotype like a cancer invades and the hyperplastic synovial tissue destroys cartilage and bone. Therefore inhibition of FLS proliferation and is one of the therapeutic targets of RA. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been shown to regulate cell differentiation through regulation of gene expression post-transcriptionally. Recently, several studies reported anti-cancer drugs modulate miRNA expression and that have been considered as one of important mechanism of cellular action to drug1,2.
Objectives To investigate the changes in microRNA expression profiles in response to MTX.
Methods RA-FLS was treated with MTX with 1nM 48 hours, that could inhibit IL-6 production from RA-FLS. To investigate differentially expressed miRNAs in response to MTX, we performed miRNA array analysis. Expression of miR-887–3p in response to MTX of RA-FLS was analyzed by quantitative real-time PCR. To investigate the functional role of miR-887–3p, RA-FLS was transfected with synthetic precursor miRNA (pre-miR)/inhibitors of miRNA (anti-miR) of miR-887–3p using Lipofectamine and then gene expression microarray was performed. The cytokine/ chemokine production was screened by Multiplex cytokine/chemokine bead assays and confirmed by ELISA. Finally, we analysed migratory activities of RA-FLS by scratch assay.
Results After 48 h of treatment with MTX, 7 miRNAs were up-regulated and 6 miRNAs were down-regulated as compared with that of untreated control. Among them quantitative real-time PCR with additional samples confirmed that miR-887–3p was up-regulated in response to MTX treatment of RA-FLS (1.79±0.46 -fold, p<0.05, n=7). To elucidate the functional consequence of the deregulation of miR-887–3p of RA-FLS, we performed gain-and-loss of function assays with miR-887–3p. Microarray analysis with gene ontology analysis revealed several genes correlated with cell signalling was modulated by miR-887–3p. Multiplex bead assay showed that overexpression of miR-887–3p decreased cytokine/chemokine production of RA-FLS such as TNF-a, GM-CSF, CCL4. Among these candidates, the secretion of GM-CSF was consistently and strongly decreased from RA-FLS transfected with pre-miR-887–3p. Furthermore overexpression of miR-887–3p reduced migratory activity of RA-FLS in scratch assay.
Conclusions Our result showed that MTX altered micro RNA expression profiles in RA-FLS. MiR-887–3p might be downstream effector of MTX in suppression of its cytokine production and invasive phenotype. This knowledge may also be useful for the development of novel therapeutic strategies for RA based on other treatments able to boost the cellular reservoir of miR-877–3p.
Potenza N, Mosca N, Zappavigna S et al. MicroRNA-125a-5p Is a Downstream Effector of Sorafenib in its Antiproliferative Activity Toward Human Hepatocellular Carcinoma Cells. J Cell Physiol. 2016 Dec 16.
Shah MY, Pan X, Fix LN et al. 5-Fluorouracil drug alters the microRNA expression profiles in MCF-7 breast cancer cells. J Cell Physiol. 2011; 226(7):1868–1878.
Disclosure of Interest None declared