Background/Purpose: Synovial fibroblasts (SF) from the joints of RA patients display an activated and invasive phenotype.  They produce a number of pro-inflammatory cytokines and proteolytic enzymes and in consequence play a crucial role in perpetuating inflammation and mediating local damage in RA.  Several microRNA are known to be involved in the aggressive phenotype of RASF, therefore, we set out to identify microRNA which were differentially expressed in these cells at a very early stage of disease.

Methods: SF were obtained from patients with undifferentiated arthritis (symptom duration <3 months).  Patients were classified as resolving arthritis or very early RA depending on whether they fulfilled the ACR criteria for classification within the subsequent 18 months.  microRNA expression was measured by TaqMan low density array (TLDA).  For functional experiments SF were isolated from RA patients undergoing joint replacement surgery.  SF were stimulated with IL-1β (1 ng/ml), TNFα (10 ng/ml), TGFβ (10 ng/ml), BLP (300 ng/ml), poly I:C (10 µg/ml) or LPS (100 ng/ml) for 24h.  For long term stimulations cells were cultured with TGFβ (10 ng/ml) for 72h, or TNFα (10 ng/ml) for 7 days.  Endogenous miR-204 was down regulated using Lipofectamine transfection with specific miRNA inhibitors. Total RNA was isolated, and miR-204 expression quantified by Taqman qPCR.  The concentrations of IL-6, IL-8, MMP1 and MMP3 were measured in cell culture supernatants by ELISA.  Adhesion and proliferation of cells were all quantified using the xCELLigence system.

Results: TLDA analysis identified miR-204 as an interesting candidate which was down regulated in SF from very early RA patients.  PCR in additional samples confirmed that miR-204 was significantly lower in SF derived from very early RA patients compared to those from patients with resolving arthritis (p<0.05) or healthy controls (p<0.001).  miR-204 expression was also reduced in SF following stimulation with poly I:C (x-fold: 0.63 ± 0.04, p<0.05), and after prolonged exposure to TGFβ (x-fold: 0.58 ± 0.06, p < 0.001) or TNFα (x-fold: 0.64 ± 0.11, p<0.05).  Compared to transfection with a control inhibitor, inhibition of miR-204 in SF resulted in a significant increase in the production of IL-6 (1332 ± 173.6 vs 2087 ± 274.5 pg/ml), IL-8 (78.26 ± 22.76 vs 212.4 ± 38.33 pg/ml), MMP1 (127.8 ± 21.47 vs 360.6 ± 92.94 pg/ml) and MMP3 (129.4 ± 62.29 vs 518.4 ± 78.6 pg/ml).  We were also able to show that miR-204 inhibition results in decreased attachment and proliferation of SF.

Conclusion: The reduced miR-204 expression observed in SF from very early RA patients suggests that this microRNA is not altered as a consequence of chronic inflammation, but instead represents an early change in the development of RA.  This novel finding, together with the increase in basal cytokine and MMP production observed following miR-204 inhibition, suggest an important functional role of this microRNA in regulating SF activation.  We therefore hypothesize that miR-204 targets a master regulator of inflammatory pathways in SF and its down regulation in early RA contributes to the activated and aggressive phenotype observed in RASF.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reduced-expression-of-mir-204-in-early-ra-promotes-inflammatory-pathways-in-synovial-fibroblasts/