Abstract
OBJECTIVE: Enhanced expression of interleukin-6 (IL-6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel posttranscriptional regulator of IL-6 expression and is targeted by microRNA-9 (miR-9). We investigated the expression of MCPIP-1 in OA cartilage and explored whether targeting of MCPIP-1 by miR-9 contributes to enhanced IL-6 expression in OA. METHODS: Gene and protein expression in IL-1β-stimulated human OA chondrocytes/cartilage was determined by TaqMan assay and immunoblotting, respectively. Messenger RNA (mRNA) for MCPIP-1 and IL-6 expression at the single-cell level was analyzed using RNAscope. MCPIP-1 protein interaction with IL-6 mRNA was investigated using RNA immunoprecipitation. Transient transfections were used for the small interfering RNA (siRNA)-mediated knockdown and overexpression of MCPIP-1, its RNase-defective mutant miR-9, or antagomir. The role of signaling pathways was evaluated using small-molecule inhibitors. Binding of miR-9 with the "seed sequence" in the 3'-untranslated region of MCPIP-1 mRNA was investigated using a luciferase reporter assay. RESULTS: MCPIP-1 mRNA expression was low, but expression of miR-9 and IL-6 was high, in damaged OA cartilage. In IL-1β-stimulated OA chondrocytes, the expression of miR-9 and MCPIP-1 was mutually exclusive, and increased expression of miR-9 correlated with reduced MCPIP-1 expression and enhanced IL-6 expression. MCPIP-1 protein directly binds with IL-6 mRNA, and overexpression of wild-type MCPIP-1 destabilized the IL-6 mRNA. MCPIP-1 expression was altered by overexpression or inhibition of miR-9. Transfection with miR-9 mimics inhibited the reporter activity, and mutation of the "seed sequence" abolished the repression of reporter activity. CONCLUSION: These findings implicate miR-9-mediated suppression of MCPIP-1 in the pathogenesis of OA via up-regulation of IL-6 expression in IL-1β-stimulated human OA chondrocytes.