miR-708-5p Attenuates Osteoarthritis Progression via Multi-Target Modulation of the NOX4/NF-κB Axis and Cartilage Homeostasis

Objective To investigate the novel role of miR-708-5p in osteoarthritis (OA) and its potential as a therapeutic target through regulation of NOX4/NF-κB signaling. Methods Expression levels of miR-708-5p were analyzed in OA cartilage using GEO datasets and validated in interleukin (IL)-1β-treated primary human chondrocytes. Gain- and loss-of-function experiments were performed using miR-708-5p mimics and inhibitors to evaluate its effects on inflammation, extracellular matrix metabolism, apoptosis, and oxidative stress. Direct targeting of NOX4 by miR-708-5p was confirmed through bioinformatic prediction, luciferase reporter assays, and rescue experiments. Results miR-708-5p was significantly downregulated in OA cartilage and IL-1β-treated chondrocytes. Overexpression of miR-708-5p attenuated IL-1β-induced inflammatory responses by suppressing pro-inflammatory cytokines (IL-1β, IL-6, tumor necrosis factor [TNF]-α), inhibiting matrix-degrading enzymes (MMP3, ADAMTS-4), and enhancing anabolic factors (COL2A1, SOX9). miR-708-5p protected against chondrocyte apoptosis by regulating Bcl2/BAX and caspase-3 expression. It also increased chondrocyte proliferation in EdU assays and reduced reactive oxygen species (ROS) production. Mechanistically, miR-708-5p directly inhibited NOX4, reducing ROS generation and nuclear factor kappa B (NF-κB) activation. NOX4 overexpression reversed the protective effects of miR-708-5p, confirming the functional significance of this regulatory axis. Conclusion miR-708-5p is downregulated in OA and exerts chondroprotective effects. These findings suggest that restoring miR-708-5p expression may effectively suppress the NOX4/NF-κB axis and modulate chondrocyte inflammation, oxidative stress, apoptosis, and matrix degradation.