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

Abstract

ObjectiveTo 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.MethodsExpression 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.ResultsmiR-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.ConclusionmiR-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.

Keywords: NOX4/NF-κB signaling; cartilage homeostasis; chondrocyte inflammation; miR-708-5p; osteoarthritis.

Figure 1.

miR-708-5p expression is downregulated in osteoarthritic cartilage and IL-1β-stimulated chondrocytes. (A, B) Expression levels of miR-708-5p in normal and osteoarthritis (OA) cartilage tissues from GEO datasets (A) GSE105027 and (B) GSE143514. (C, D) Time-dependent suppression of (C) miR-708-5p and its host gene (D) Odz4 expression in human chondrocytes following stimulation with IL-1β (10 ng/ml) for indicated periods (0, 1, 6, 12, or 24 hours). Expression levels were determined by real-time PCR and normalized to GAPDH. Data are presented as mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001 compared to control group.

Figure 2.

miR-708-5p inhibits IL-1β-induced inflammatory responses in human chondrocytes. (A) RT-qPCR analysis of miR-708-5p expression in chondrocytes transfected with miR-708-5p mimics or negative control (NC). (B-D) Effects of miR-708-5p overexpression on inflammatory mediators. RT-qPCR analysis of (B) IL-1β and (C) IL-6 mRNA expressions. (D) Western blot analysis and quantification of IL-6 and TNF-α protein levels in chondrocytes transfected with miR-708-5p mimics or NC and treated with IL-1β (10 ng/ml) for 24h. β-Actin served as loading control. (E) RT-qPCR analysis of miR-708-5p expression in chondrocytes transfected with miR-708-5p inhibitor or inhibitor negative control (NC). (F-H) Effects of miR-708-5p inhibition on inflammatory mediators. RT-qPCR analysis of (F) IL-1β and (G) IL-6 mRNA expressions. (H) Western blot analysis and quantification of IL-6 and TNF-α protein levels in chondrocytes transfected with miR-708-5p inhibitor or NC and stimulated with IL-1β (10 ng/ml) for 24h. (I-L) ELISA analysis of IL-6 and TNF-α production in culture supernatants. Concentration of (I, K) IL-6 and (J, L) TNF-α in supernatants from chondrocytes transfected with (I, J) miR-708-5p mimics or (K, L) miR-708-5p inhibitor and stimulated with IL-1β (10 ng/ml) for 24h. Data are presented as mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared to IL-1β + NC group.

Figure 3.

miR-708-5p regulates extracellular matrix homeostasis in IL-1β-stimulated chondrocytes. (A-C) RT-qPCR analysis of mRNA expression for (A) MMP3, (B) ADAMTS4, and (C) COL2A1 in chondrocytes transfected with miR-708-5p mimics and stimulated with IL-1β (10 ng/ml) for 24h. (D) Western blot analysis and densitometric quantification of catabolic (MMP3, ADAMTS-4) and anabolic (SOX9, COL2A1) proteins in chondrocytes transfected with miR-708-5p mimics or negative control (NC) and stimulated with IL-1β (10 ng/ml) for 24h. β-Actin served as loading control. (E-G) RT-qPCR analysis of mRNA expression for (E) MMP3, (F) ADAMTS-4, and (G) COL2A1 in chondrocytes transfected with miR-708-5p inhibitor and stimulated with IL-1β (10 ng/ml) for 24h. (H) Western blot analysis and quantification of MMP3, ADAMTS-4, SOX9, and COL2A1 proteins in chondrocytes transfected with miR-708-5p inhibitor or inhibitor NC and stimulated with IL-1β (10 ng/ml) for 24h. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared to IL-1β + NC group.

Figure 4.

miR-708-5p modulates IL-1β-induced chondrocyte apoptosis. (A-B) RT-qPCR analysis of mRNA expression for (A) Bcl2 and (B) caspase-3 in chondrocytes transfected with miR-708-5p mimics or negative control (NC) and treated with IL-1β (10 ng/ml) for 24h. (C) Western blot analysis and densitometric quantification of apoptotic markers Bcl-2-associated X protein (BAX) and cleaved caspase-3 in chondrocytes transfected with miR-708-5p mimics or NC and stimulated with IL-1β (10 ng/ml) for 24h. β-Actin served as loading control. (D-E) RT-qPCR analysis of mRNA expression for (D) Bcl2 and (E) caspase-3 in chondrocytes transfected with miR-708-5p inhibitor or NC and treated with IL-1β (10 ng/ml) for 24h. (F) Western blot analysis and quantification of BAX and cleaved caspase-3 proteins in chondrocytes transfected with miR-708-5p inhibitor or NC and stimulated with IL-1β (10 ng/ml) for 24h. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared to IL-1β + NC group.

Figure 5.

miR-708-5p regulates chondrocyte proliferation under IL-1β stimulation. (A-B) Human chondrocytes were transfected with (A) miR-708-5p mimics or (B) miR-708-5p inhibitor or respective negative controls (NC) for 24h, then treated with IL-1β (10 ng/ml) for 24h. Cell proliferation was assessed by EdU incorporation assay (green) with DAPI nuclear counterstaining (blue). Quantification is shown as the percentage of EdU-positive cells (right panels). ***P < 0.001 compared to untreated control; ## P < 0.01, ### P < 0.001 compared to IL-1β + respective NC group.

Figure 6.

NOX4 is a direct target of miR-708-5p. (A) Predicted secondary structure of miR-708-5p and sequence alignment showing the binding site between miR-708-5p and NOX4 3'UTR. The wild-type (WT) sequence and mutated (MUT) binding site are shown with mutated nucleotides highlighted in red. (B-C) Effects of miR-708-5p on NOX4 expression: (B) Relative NOX4 mRNA expression and (C) representative Western blot with quantification of NOX4 protein expression in chondrocytes transfected with miR-708-5p mimic or negative control (NC). (D-E) Effects of miR-708-5p inhibition on NOX4 expression: (D) Relative NOX4 mRNA expression and (E) representative Western blot with quantification of NOX4 protein expression in chondrocytes transfected with miR-708-5p inhibitor or negative control (NC). β-Actin served as loading control for all Western blots. (F-G) Luciferase reporter assay results showing relative luciferase activity in chondrocytes co-transfected with miR-708-5p mimic or NC along with (F) wild-type NOX4 3'UTR or (G) mutated NOX4 3'UTR reporter constructs. **P < 0.01, ***P < 0.001 compared to respective NC.

Figure 7.

miR-708-5p modulates IL-1β-induced oxidative stress and NOX4/NF-κB signaling in chondrocytes. (A-D) Effects of miR-708-5p on ROS production and NOX4 expression. Human chondrocytes were transfected with miR-708-5p (A-B) mimics or (C-D) inhibitors and treated with IL-1β (10 ng/ml) for 24h. (A, C) ROS production visualized with DCFH-DA staining (green fluorescence, bottom panels) and corresponding phase contrast images (top panels). (B, D) RT-qPCR analysis of NOX4 mRNA expression under the same treatment conditions. (E-F) Western blot analysis and quantification of NOX4 protein expression in IL-1β-stimulated chondrocytes after transfection with miR-708-5p (E) mimics or (F) inhibitor, with β-Actin as loading control. (G-H) Western blot analysis and quantification of NF-κB pathway activation showing phosphorylated NF-κB (p-NF-κB) and total NF-κB in IL-1β-stimulated chondrocytes transfected with miR-708-5p (G) mimics or (H) inhibitor relative to appropriate controls. * P < 0.05, ** P < 0.01 compared to untreated control; # P < 0.05, ## P < 0.01 compared to IL-1β + respective negative control (NC) group.

Figure 8.

The miR-708-5p/NOX4/NF-κB axis modulates IL-1β-induced inflammatory and catabolic responses in chondrocytes. (A) Western blot analysis and densitometric quantification of MMP3, MMP13, iNOS, NOX4, and caspase3 protein expression in human chondrocytes. Cells were co-transfected with miR-708-5p mimics and/or NOX4 overexpression plasmid (pcNOX4) as indicated, then stimulated with IL-1β (10 ng/ml) for 24h. β-Actin served as loading control. (B) Cell viability assessed by MTT assay in chondrocytes under the same treatment conditions as in (A). Results expressed as OD570 nm. (C-D) ELISA analysis of (C) IL-6 and (D) TNF-α production in culture supernatants of chondrocytes treated as in (A). ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ### P < 0.001 compared to IL-1β treated group; & P < 0.05, && P < 0.01, *** P < 0.001 compared to IL-1β + miR708-5p group.