Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Epigenetically Regulated MicroRNA-126 Influences Migratory Potential and Apoptosis of Synovial Fibroblasts in Rheumatoid Arthritis.
Frank1, Mojca, Filkova1, Maria, Stanczyk1, Joanna, Connolly1, Mary, Kolling2, Christoph, Michel1, Beat A., Rozman3, Blaz
Increased migration and decreased apoptosis are main characteristics of the aggressive behaviour of rheumatoid arthritis (RA) synovial fibroblasts (SF). Increased migration has been implicated in spreading RA from affected to unaffected joints, resulting in polyarticular disease. Dysregulation of several microRNAs (miRs) has been found to play a role in cell migration, apoptosis and inflammation.
To analyse the expression of most relevant miRs in RA, their regulation by proinflammatory and epigenetic mechanisms in SF and their functional role in shaping the aggressive behaviour of RASF.
TaqMan Single Assays were used to analyse the expression of 260 miRs in human RASF and osteoarthritis (OA) SF (n=3 each). MiR-126 was further quantified in RASF (n=10), OASF (n=9), RA (n=10), OA (n=6) synovial tissue and RA (n=20) or healthy control sera (n=17) by Taqman Real Time PCR. Let-7a was used as an endogenous control. The regulation of miR-126 expression was investigated in RASF (n=5) treated with TNFa, IL-1b, IFN-b, Poly(I:C) or LPS and in RASF (n=6) exposed to hypoxia (24h, 1% O2). Additionally, RASF and OASF were treated with 5'azacytidine (5'AZA, 0.1, 0.5 and 1mM, 5 days) or Trichostatin A (TSA, 1mM, 24h). The functional role of miR-126 (migration/scratch and Annexin-V apoptosis assays) was studied in SF following Lipofectamine transfection (48h) with pre-miR-126 (100nM), mature miR-126 (25, 50nM), anti-miR-126 (25, 50nM) or scrambled controls.
MiR-126 was significantly upregulated in RASF (dCt±SE=7.93±2.43) vs. OASF (dCt=9.91±0.81, p=0.022) and in sera from RA patients (dCt=-1.79±1.01) compared to healthy controls (dCt=-1.26±0.61, p=0.031), while no significant difference was found between RA and OA synovial tissue. Proinflammatory cytokines, TLR ligands or hypoxia (1% O2) had no effect on the expression of miR-126 in RASF. DNA demethylation of RASF (x-fold: 1.78±0.53, n=2) and OASF (1.58±0.21, n=4) with 5-AZA and treatment of OASF (1.50±0.34, n=6, p=0.031) and RASF (1.70±0.34, n=4) with TSA upregulated miR-126. Transfection with pre-miR-126 decreased the migration of SF in the scratch assay by 86±0.05% in all of OA and by 48±16% in 4 out of 6 RA patients. Anti-miR-126 had no significant effect on migration. Apoptosis significantly increased in SF (n=7) transfected with mature miR-126 (2.67±0.43; p=0.008) and anti-miR-126 (1.71± 0.25, p=0.031).
MiR-126 is epigenetically upregulated in synovial fibroblasts by acetylation and demethylation and significantly alters the migration and apoptotic ability of SF. Therefore it may serve as a potential new therapeutic target in RA possibly influencing the spread of RA to unaffected joints.
To cite this abstract, please use the following information:
Frank, Mojca, Filkova, Maria, Stanczyk, Joanna, Connolly, Mary, Kolling, Christoph, Michel, Beat A., et al; Epigenetically Regulated MicroRNA-126 Influences Migratory Potential and Apoptosis of Synovial Fibroblasts in Rheumatoid Arthritis. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :42