Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
MicroRNA-199a-Mediated Regulation of Cyclooxygenase-2 Expression in Human OA Chondrocytes.
Akhtar1, Nahid, Rasheed2, Zafar, Haqqi1, Tariq M.
Recent evidence implicate the deregulated expression of microRNAs (miRNAs) in the pathogenesis of osteoarthritis (OA). Interleukin-1b (IL-1b)-induced expression of cyclooxygenase-2 (COX-2) correlates with the production of prostaglandin E2 and cartilage degradation in OA. Here we determined the posttranscriptional regulation of COX-2 expression by miRNAs in IL-1b-stimulated human OA and normal chondrocytes.
Human chondrocytes were derived by enzymatic digestion of OA cartilage (OA chondrocytes) and cartilage from trauma patients with no history of OA (normal chondrocytes). Chondrocytes were stimulated with IL-1b (5ng/ml) in vitro. Total RNA was prepared using TRIZOL reagent and miRNAs were purified using the mirVANA system. Single stranded cDNA was synthesized using stem loop-specific primers and the expression of miRNAs of interest was quantified using TaqMan miRNA Expression Assay and their target mRNA was identified using bioinformatics. Transfection of OA chondrocytes with a 3'UTR reporter construct and pre-miRNAs was employed to verify the miRNA:mRNA interaction. OA chondrocytes transfected with pre-miRNAs and anti-miRNAs were analyzed for the expression of COX-2 mRNA and protein by qRT-PCR using specific primers and Western immunoblotting, respectively.
IL-1b- stimulation of OA chondrocytes resulted in the downregulation of two miRNAs- miR-101_3 and miR-199a*-that potentially target COX-2 mRNA. Kinetic analysis showed that in OA chondrocytes, expression of miR-101_3 was down-regulated at 6 h (2.1-fold ± 1.0; n=7) but no significant change was observed at 24 h (n=11) post stimulation. Normal chondrocytes showed down-regulation of miR-101_3 (1.7-fold ± 0.53; n=3) at 24 h but no change at 6 h post-stimulation with IL-1b. Expression level of miR-199a* in OA chondrocytes stimulated with IL-1b at 24 h (3.3-fold ± 1.5; n=11, p<0.05) and at 6 h (0.94-fold ± 0.86; n=7) inversely correlated with the COX-2 protein expression level. Similar results were obtained with normal chondrocytes stimulated with IL-1b. Significantly lower expression of miR-199a* was observed in freshly isolated OA cartilage compared to normal cartilage (n=5; p<0.001). Over-expression of miR-199a* in OA chondrocytes inhibited the IL-1b-induced expression of COX-2 protein significantly compared to control OA chondrocytes (p<0.05). Transfection of OA chondrocytes with miR-199a* inhibitor enhanced the IL-1b-induced expression of COX-2 protein. Co-transfection of OA chondrocytes with a luciferase reporter construct containing the 3'UTR of human COX-2 mRNA and pre-miR-199a* suppressed the luciferase activity significantly (p<0.05). No inhibition of luciferase activity was observed in OA chondrocytes transfected with negative control miRNA. Inhibition of NF-kB and p-38MAPK activation with specific inhibitors altered the expression of miR199a* in human OA chondrocytes suggesting the regulation of miR-199a* by activation of these pathways.
Our data implicate miR-199a* in the posttranscriptional regulation of COX-2 expression in OA chodrocytes and identify miR-199a* as a novel therapeutic target for the treatment/prevention of OA.
To cite this abstract, please use the following information:
Akhtar, Nahid, Rasheed, Zafar, Haqqi, Tariq M.; MicroRNA-199a-Mediated Regulation of Cyclooxygenase-2 Expression in Human OA Chondrocytes. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :633