MicroRNA‐9 Promotion of Interleukin‐6 Expression by Inhibiting Monocyte Chemoattractant Protein–Induced Protein 1 Expression in Interleukin‐1β–Stimulated Human Chondrocytes

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.