The inhibition of miR-125b-5P prevented Treg differentiation through activating STAT3/HIF-1α pathway in PBMCs of ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the spine and joints. Its pathogenesis results from complex interactions among genetic, environmental, and immunological factors. Recent research has highlighted the role of regulatory T cells (Tregs) in maintaining immune homeostasis and suppressing excessive inflammation in AS. However, the mechanisms regulating Treg cell differentiation and function in AS remain poorly understood. This study aimed to elucidate the role of miR-125b-5p in regulating Treg cell differentiation and function in AS patients. Peripheral blood mononuclear cells (PBMCs) from AS patients and healthy controls (HC) were analyzed to investigate mechanisms underlying the Th17/Treg cell imbalance in AS. Flow cytometry, quantitative real-time PCR (qRT-PCR), Western blot, dual-luciferase reporter assay, enzyme-linked immunosorbent assay (ELISA), and co-immunoprecipitation (Co-IP) techniques were employed to assess the expression and regulatory functions of key molecules in Treg differentiation and function. The proportion of Treg cells was significantly decreased in PBMCs from AS patients, with decreased expression of FOXP3, IL-2, and IL-10. Meanwhile, we found that STAT3 was ur-regulaed in AS-PBMCs; STAT3 overexpression suppressed FOXP3 expression, whereas STAT3 knockdown restored it. Then, the online databases (ENCORT, miRWalk, TargetScan, miRTarBase, miRcode) predicted that STAT3 mRNA could be interacted by miR-125b-5p, which regulated its expression. The dual-luciferase reporter assay further demonstrated that miR-125b-5p can directly interact with 3’-UTR of STAT3 mRNA, which repressing its expression. Interestingly, MiR-125b-5p was significantly downregulated in AS PBMCs. Overexpression of miR-125b-5p increased Treg cell frequency and enhanced IL-10 and TGF-β1 secretion through inhibiting STAT3/HIF-1α axis. Additionally, we also found that STAT3 could interact with NOX2 proteins. NOX2 overexpression modulated the STAT3/HIF-1α pathway, affecting Treg cell differentiation. MiR-125b-5p regulates Treg cell differentiation and function by regulating STAT3 expression in AS patients. Moreover, NOX2 also affects Treg differentiation through the STAT3/HIF-1α pathway. These findings suggest new potential targets for immunomodulatory therapy in AS.