Abstract
OBJECTIVE: Cyclooxygenase-2 (COX-2) expression is associated with the pathogenesis of chronic inflammation and pain in osteoarthritis (OA). A study was undertaken to determine whether interleukin-1β (IL-1β)-mediated induction of COX-2 can be regulated by microRNAs (miRNAs) in OA. METHODS: Human chondrocytes were stimulated with IL-1β in vitro. Total RNA was prepared using Trizol reagent. Gene expression was quantified using TaqMan Assays and miRNA targets were identified using bioinformatics. Transfection with reporter construct and premiRNA and antimiRNA was employed to verify suppression of target mRNA. Expression of COX-2 proteins was determined by immunoblotting. The role of activated p38-MAPKs was evaluated using specific inhibitor. RESULTS: The 3'UTR of COX-2 mRNA contained the 'seed-matched' sequences for miR-199a* and miR-101_3. Increased expression of COX-2 correlated with the downregulation of miR-199a* and miR-101_3 in IL-1β-stimulated normal and OA chondrocytes. miR-199a* directly suppressed the luciferase activity of a COX-2 3'UTR reporter construct and inhibited the IL-1β-induced expression of COX-2 protein in OA chondrocytes. Modulation of miR-199a* expression also caused significant inhibition of IL-1β-induced upregulation of mPGES1 and prostaglandin E(2) production in OA chondrocytes. Activation of p38-MAPK downregulated the expression of miR-199a* and induced COX-2 expression. Treatment with antimiR-101_3 increased COX-2 expression in IL-1β-stimulated chondrocytes, but overexpression of miR-101_3 had no significant effect on COX-2 protein expression. CONCLUSIONS: miR-199a* is a direct regulator of COX-2 expression in OA chondrocytes. IL-1β-induced activation of p38-MAPK correlates inversely with miR199a* expression levels. miR-199a* may be an important regulator of human cartilage homeostasis and a new target for OA therapy.