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OP0017 The tnf-alpha-induced MIR-18A activates rheumatoid arthritis synovial fibroblast through a positive feedback loop in NF-KAPPA B signalling
  1. M. Trenkmann1,2,
  2. M. Brock1,2,3,
  3. R.E. Gay1,2,
  4. R. Speich3,
  5. B.A. Michel1,2,
  6. S. Gay1,2,
  7. L.C. Huber1,2,3
  1. 1Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland
  2. 2Center of Experimental Rheumatology
  3. 3Clinic and Policlinic for Internal Medicine, University Hospital Zurich, Zurich, Switzerland

Abstract

Background The miR-17-92 cluster encodes six distinct microRNAs (i.e. miR-17, -18a, -19a, -19b, -20a and -92a) and has been found to be a crucial regulator in the pathogenesis of several diseases, e.g. autoimmune disorders. Rheumatoid arthritis (RA) is a chronic autoimmune disease of the joint in which the resident synovial fibroblast (SF) plays a major role in cartilage degradation and activation of immune cells.

Objectives To study expression and function of the miR-17-92 cluster in activated RASF.

Methods RASF were stimulated with 10ng/ml TNFa. Gene expression was analysed by SYBR Green quantitative real time PCR, Western blot and ELISA. RASF were transfected using Lipofectamin2000 (pre-miRs) or AMAXA Nucleofection (plasmids). Activity of the C13orf25 promoter and NF-kB was measured by reporter gene assay. The TNFAIP3 3’UTR was cloned into pmirGLO and reporter gene assays were carried out in pre-miR-18a-transfected Hek293 cells. The chemoattractive potential of RASF was determined using peripheral blood leukocytes (PBL, isolated by erythrocyte lysis) and conditioned medium (CM) from TNFa-stimulated RASF employed as chemoattractant in a transwell migration assay.

Results The miR-17-92 primary transcript (C13orf25) as well as the mature miRs-18a, -19a, -20a and -92a were significantly induced by TNFa in a time dependent manner with peak induction at 16h of stimulation (1.3- up to 1.7-fold, n=8, p<0.05). This induction was depending on a NF-kB-binding site in the C13orf25 promoter, as assessed by reporter gene assay. Transfection of pre-miRs-18a, -19a, -20a and -92a and stimulation of SF with TNFa revealed increased mRNA levels of MMP1 in miR-18a-transfected cells. Further experiments thus were focused on miR-18a. Pre-miR-18a transfection significantly increased the constitutive (MMP1, IL6, MCP1, RANTES) as well as the TNFa-induced expression (MMP1, IL6, IL8, MCP1, RANTES) of mediators of inflammation and joint destruction at mRNA and protein levels (by 1.9- up to 5.1-fold, n=8, p<0.05). In reporter gene assays, we studied the NF-kB signalling inhibitor TNFAIP3 (A20) as a potential target gene of miR-18a (predicted by www.targetscan.org). With the TNFAIP3 wt 3’UTR construct, miR-18a repressed luciferase activity which was rescued when using the construct in which the miR-18a seed match had been mutated (n=4, p<0.05), proving that TNFAIP3 is a direct target of miR-18a. In miR-18a-transfected RASF, TNFAIP3 protein expression was decreased by 30±19% (n=7, p<0.05). miR-18a enhanced the constitutive as well as TNFa-induced NF-kB activity in RASF by 1.83±1.25-fold and 1.48±0.43-fold (n=6, p<0.05). Measuring the chemoattractive potential of RASF in a transmigration assay, CM from miR-18a-transfected RASF induced more migration of PBL as compared to CM from scrambled RASF (n=8, p<0.05).

Conclusions In RASF, the miR-17-92 cluster is induced by TNFa via the NF-kB pathway and, by repressing TNFAIP3 miR-18a constitutes a positive regulator of NF-kB signalling. Thus, by enhancing the production of inflammatory cytokines and matrix degrading enzymes, miR-18a aggravates the activated phenotype of RASF.

Disclosure of Interest None Declared

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