miR-584-5p alleviates osteoarthritis by targeting HIF1A to regulate chondrocyte function and extracellular matrix metabolism

Osteoarthritis (OA) is a chronic disease characterized by the degeneration of articular cartilage and secondary hyperosteogeny, typically located in weight-bearing joints. This study was undertaken to assess the expression profile, the diagnostic value, and the molecular mechanism of miR-584-5p in OA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of miR-584-5p and hypoxia-inducible factor-1 A (HIF1A) in serum samples from OA patients and healthy controls. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic efficacy of miR-584-5p for OA. Pearson correlation analysis was applied to explore the correlations between miR-584-5p expression and clinical scores (Lysholm, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Visual Analogue Scale (VAS) as well as HIF1A expression. In vitro, human chondrogenic HC-A cells were induced with interleukin-1β (IL-1β) to establish an OA cell model. Cell counting kit-8 (CCK-8) assay and flow cytometry were used to assess chondrocyte proliferation and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) kits were employed to quantify the secretion levels of inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β. Dual-luciferase reporter assay was conducted to verify the direct targeting relationship between miR-584-5p and HIF1A. miR-584-5p was significantly downregulated in OA patients, and its expression level decreased progressively with the increase of Kellgren-Lawrence (K-L) grade. ROC curve analysis confirmed that miR-584-5p had a high diagnostic value for OA with an area under the curve (AUC) of 0.897, a sensitivity of 80.2% and a specificity of 86.2%. Pearson correlation analysis showed that miR-584-5p expression was positively correlated with Lysholm scores and negatively correlated with WOMAC and VAS scores in OA patients. In the IL-1β-induced HC-A cell model, miR-584-5p mimic significantly promoted chondrocyte proliferation, inhibited cell apoptosis, and reduced the secretion of IL-6, TNF-α and IL-1β. Additionally, miR-584-5p mimic downregulated the mRNA expression of extracellular matrix (ECM)-degrading enzymes including matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), while upregulated the expression of ECM synthesis-related molecules including aggrecan (ACAN) and collagen type II alpha 1 chain (COL2A1). Notably, all these protective effects of miR-584-5p mimic were abrogated by transfection with HIF1A overexpression plasmid (oe-HIF1A). miR-584-5p is a potential novel diagnostic biomarker for OA with high sensitivity and specificity, and its expression level can reflect the clinical severity of OA. Mechanistically, miR-584-5p alleviates IL-1β-induced chondrocyte injury, inflammatory response and ECM metabolic imbalance in OA by directly targeting and inhibiting HIF1A.