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.

Metrohealth Medical Center/CWRU, Cleveland, OH
Metrohealth Medical Center/CWRU

Background:

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.

Methods:

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.

Results:

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.

Conclusions:

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
DOI: 10.1002/art.28401

Abstract Supplement

Meeting Menu

2010 ACR/ARHP