The Pro-angiogenic Mechanisms of MiR-125a-5p in Inflammatory Arthritis
Sarah Wade, Mary Connolly, Douglas Veale, Ursula Fearon
Centre for Arthritis and Rheumatic Diseases, St. Vincent’s University Hospital, Dublin, Ireland.
MicroRNA belong to a class of small, evolutionarily conserved, noncoding RNAs that function as posttranscriptional repressors of gene expression. An accumulating body of evidence suggests that up to 50% of the human genome is regulated by miRNAs.
The aim of this study was to examine expression, regulation and function of miRNA-125a-5p in inflammatory arthritis.
Synovial tissue biopsies and primary synovial fibroblasts were obtained from patients with Psoriatic arthritis (PsA), Rheumatoid Arthritis (RA) and osteoarthritis (OA). MiR- 125a-5p levels were analyzed by real-time PCR and data was calculated by the deltaCt method using RNU6B as an endogenous control. To examine possible factors involved in regulating miR-125a-5p expression, primary synovial fibroblasts and microvascular endothelial cells (HMVEC) were cultured with candidate pro-inflammatory stimuli including; TLR ligands (PAM, PolyIC, LPS), pro-inflammatory cytokines (TNF-α, IL- 1β, IL-17) and growth factors (VEGF, Ang2). Overexpression/silencing of miR-125a-5p was analysed using a synthetic precursor of Pre or Anti-miR™-125, respectively. Cell invasion, tube formation and migration were examined using transwell invasion, angiogenic and wound repair assays, and proinflammatory mediators were quantified by ELISA.
Expression of miR-125a-5p was significantly higher in PsA and RA synovial biopsies and/or synovial fibroblasts compared to OA (p<0.05; p<0.05), with highest expresson observed in PsA (p<0.05). Angiogenic growth factor Ang2 induced miR-125a-5p in synovial fibroblasts and HMVEC (p<0.05), with no effect observed for TLR ligands or proinflammatory cytokines. Silencing of miR by transfection with anti-miR-125a-5p resulted in inhibition of cell invasion, angiogenic tube formation and IL-6 expression. This is page 47 consistent with in silico analysis where prediction algorithms identified members of the IL-6 signalling pathway (IL-6R, gp130) as potential targets of miR-125a-5p.
Our data provides evidence that miR-125a-5p is significantly increased in the inflamed joint, particularly in PsA. High miR-125a-5p expression in PsA and regulation by key angiogenic factor Ang2, is consistent with a possible role for miR-125a-5p in the regulation of angiogenic mechanisms. MiR- 125a-5p also mediated cell migration, angiogenesis and IL-6 expression, key processes involved in the pathogenesis of PsA and RA. In conclusion, miR-125a-5p may be an important regulator of pathogenic mechanisms in inflammatory arthritis and may represent a potential novel target for future therapeutic strategies.