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

Background: 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.

Objective: This study aimed to elucidate the role of miR-125b-5p in regulating Treg cell differentiation and function in AS patients.

Methods: 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.

Results: 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.

Conclusions: 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.

Supplementary Information: The online version contains supplementary material available at 10.1186/s13075-026-03762-5.