miR-29a-3p targets the PI3K/Akt signaling pathway to suppress inflammation and MMP-9-dependent matrix degradation in spinal tuberculosis

OBJECTIVE: Spinal tuberculosis (STB), a prevalent form of extrapulmonary tuberculosis, remains incompletely understood at the mechanistic level. This study investigates the role of miR-29a-3p in modulating STB-associated inflammation and extracellular matrix degradation via targeted regulation of the PI3K/Akt signaling pathway, thereby identifying novel molecular targets for improved diagnosis and therapeutic intervention. METHODS: Twenty patients with histopathologically confirmed STB and twenty age- and sex-matched controls with Intervertebral disc degeneration were prospectively enrolled. Genome-wide miRNA profiling was performed on spinal tissue specimens and BCG-infected THP-1-derived macrophages using small RNA sequencing, followed by qRT-PCR validation. Macrophage polarization and mycobacterial infection were modeled by PMA-induced differentiation of THP-1 cells and subsequent challenge with Bacillus Calmette-Guérin (BCG). Functional gain- and loss-of-function assays were conducted via transfection of miR-29a-3p mimics or inhibitors. Cytokine secretion (IL-6, IL-1β, TNF-α) and MMP-9 protein expression were quantified by ELISA, Western blotting, immunofluorescence microscopy, and qRT-PCR. Target prediction and dual-luciferase reporter assays validated direct binding of miR-29a-3p to the 3'-UTR of PI3K. Rescue experiments employed co-treatment with the selective PI3K inhibitor LY294002 to determine pathway-specific dependency. RESULTS: miR-29a-3p was significantly downregulated in both STB patient-derived spinal tissue specimens and BCG-infected THP-1-derived macrophages, concomitant with marked upregulation of MMP-9. Overexpression of miR-29a-3p markedly suppressed the secretion of pro-inflammatory cytokines-including IL-6, IL-1β,and TNF-α-as well as MMP-9 protein expression in BCG-infected macrophages; conversely, miR-29a-3p knockdown enhanced the production of these mediators. Mechanistically, miR-29a-3p directly bound to the 3'-UTR of PI3K-thereby inhibiting phosphorylation-dependent activation of the PI3K/Akt signaling axis. Crucially, pharmacological inhibition of PI3K with LY294002 reversed the pro-inflammatory response and matrix degradation effects induced by miR-29a-3p inhibition, confirming the functional dependence of this pathway. CONCLUSION: miR-29a-3p negatively regulates STB-associated inflammatory responses and MMP-9-mediated matrix degradation by targeting the PI3K/Akt signaling pathway, establishing a novel miR-29a-3p-PI3K/Akt-inflammatory cytokines/MMP-9 regulatory axis. This molecule represents a promising candidate biomarker and therapeutic target for the clinical diagnosis and targeted intervention of STB.