OP0246 MIR-214-3P PROTECTS AGAINST OSTEOARTHRITIS BY DIRECTLY TARGETING NF-ĸB PATHWAY

Abstract

Background:Osteoarthritis (OA) is a degenerative disease associated with changes in the articular cartilage and bone, severely affecting patients’ mobility and quality of life. Multiple factors including mechanical stress, metabolic alteration and inflammatory mediators are involved in the complex pathogenesis of OA[1]. Interventions targeting these pathogenic factors may contribute to the treatment of OA. MiRNAs are single strand non-coding small RNAs, which are regulated in chondrogenesis and OA[2,3]. Recent studies demonstrated that miRNAs are involved in the regulation of NF-κB signaling pathway by different mechanisms[4]. These interactions suggest that NF-κB related miRNAs may be used as potential biomarkers and drug therapeutic targets in clinical treatment of OA. However, the relationship between miR-214-3p and NF-κB pathway remains poorly understood in OA.Objectives:This study aimed to test the expression and biological function of miR-214-3p in OA, and explore its mechanism in osteoarthritic chondrocytes.Methods:Articular primary chondrocytes were isolated from human cartilage samples, which were acquired from patients with end-stage knee OA at the time of total knee replacement surgery (n = 27), according to protocols approved by the Ethic Committee of Zhujiang Hospital. Real time PCR (RT-PCR) and in situ hybridization (ISH) were used to detect the expression of miR-214-3p in OA and non-OA cartilage tissues. Interference of miR-214-3p was conducted using inhibitor, while overexpression of miR-214-3p was performed with mimics. Metabolism of extracellular matrix was detected by RT-PCR, western blotting and immunofluorescence in vitro. Flow cytometry were conducted to determine cell apoptosis. A luciferase reporter assay, was used to evaluate the interaction between miR-214-3p and its downstream target. Human chondrocytes were cotransfected with miR-214-3p and the IKBKB-overexpressing plasmid to confirm the interaction between miR-214-3p and NF-ĸB pathway. For in vivo studies, experimental OA was induced in 12-week-old male C57BL/6J mice by destabilization of the medial meniscus (DMM) surgery with miR-214-3p agomir intra-articular (IA) injection (once weekly for 12 days) or by IA injection (once weekly for 12 days) of miR-214-3p antiagomir. Mice were sacrificed 10 weeks after the first IA injection, and subjected to histological analyses.Results:MiR-214-3p was significantly reduced in human OA cartilage. The decreased expression of miR-214-3p in the OA cartilage tissues was directly associated with excessive apoptosis and imbalance between anabolic and catabolic factors of ECM. Mechanistically, we determined that miR-214-3p directly targeted IKBKB/IKK-b and thereby suppressed the activation of NF-ĸB pathway. IKBKB overexpression attenuated the inhibitory effect of miR-214-3p on NF-ĸB pathway. Furthermore, inhibition of miR-214-3p in mice joints triggered spontaneous cartilage loss and OA development, while IA injection of miRNA-214-3p agomir alleviated OA in the DMM mouse model.Conclusion:Our results reveal an important role of miR-214-3p in OA progression. MiR-214-3p was down-regulated while IKBKB was upregulated in OA. MiR-214-3p inhibits the NF-kB signaling pathway and suppresses the progression of OA through targeting IKBKB. Thus, miR-214-3p maybe a therapeutic target for OA.