Effect of miR-9 on Chondrocyte Apoptosis in Rats with Osteoarthritis by Regulating Notch1

Abstract

Objective: Notch1 is an important receptor in Notch signaling pathway. Abnormal Notch1 expression or function is associated with the pathogenesis of OA. There is evidence that the abnormal expression of miR-9 is related to the pathogenesis of OA. Bioinformatics analysis showed that there was a targeting relationship between miR-9 and Notch1’s 3′-UTR, suggesting that there might be a mutual regulation between them. In this study, we established an OA rat model to investigate whether miR-9 plays a role in regulating Notch1 expression, affecting cartilage matrix degradation and chondrocyte apoptosis in OA. Methods: OA model rats were divided into three groups: OA group, OA + agomir group, OA + miR-9 agomir group. The content of Hyp in articular fluid was measured by ELISA, the activity of caspase-3 was detected by kit, the mRNA expressions of miR-9, COL2A1, Notch1 and Hes5 was detected by qRT-PCR, and the protein expressions of Notch1 and Hes5 in articular cartilage was detected by Western blot. Chondrocytes were separated into control group, IL-1β +miR-NC group, IL-1β +miR-9 mimic group followed by analysis of mRNA expressions of miR-9, COL2A1, Notch1 and Hes5 were detected by qRT-PCR, protein expressions of Notch1 and Hes5 by Western blot, and cell apoptosis by flow cytometry. Results: Compared with Sham group, the levels of IL-1β , Hyp, Notch1, Hes5 and Caspase-3 in the synovial fluid of rats in OA group were increased significantly, while the mRNA expressions of miR-9, COL2A1 and Notch1 and Hes5 level in cartilage tissue were decreased significantly. Intra-articular injection of miR-9 agomir could significantly reduce the content of Hyp, the mRNA expressions of Notch1 and Hes5 and the activity of caspase-3 in cartilage tissue of OA rats, increase the mRNA expressions of miR-9 and COL2A1, and decrease the protein expressions of Notch1 and Hes5. After treated with IL-1β , the expressions of Notch1 and Hes5 were increased, the mRNA expressions of miR-9 and COL2A1 were decreased, and apoptosis of chondrocyte was increased in chondrocyte. After miR-9 mimic transfection, miR-9 and COL2A1 mRNA expressions were increased, Notch1 and Hes5 expressions were decreased, and apoptosis of chondrocyte was decreased under IL-1β treatment. Conclusion: Reduced miR-9 expression upregulates Notch1 expression and promotes the pathogenesis of OA. Increased miR-9 expression can inhibit Notch1 expression, reduce cartilage matrix degradation and inhibit chondrocyte apoptosis.