was significantly downregulated in spinal tuberculosis (STB) tissues and peripheral blood.
expression was found to be upregulated in STB tissues.
The knockdown of miR-221-3p led to increased production of pro-inflammatory cytokines.
Overexpression of miR-221-3p resulted in reduced inflammation.
ALKBH5 directly demethylates pri-miR-221-3p, impairing its maturation.
Silencing ALKBH5 restored miR-221-3p levels and decreased cytokine secretion.
Simplified
BACKGROUND: The interplay between N6-methyladenosine (m6A) modification and microRNAs (miRNAs) in the pathogenesis of spinal tuberculosis (STB) remains incompletely understood. This study aimed to investigate the regulatory mechanism involving the m6A demethylase and specific miRNAs in STB.
METHODS: miRNA sequencing was conducted to identify differentially expressed miRNAs in STB tissues. The expression levels of and ALKBH5 were validated via qRT-PCR in tissue samples and peripheral blood from 50 STB patients and healthy controls. The diagnostic potential of these biomarkers was assessed using ROC curve analysis. The expression of ALKBH5 and miR-221-3p was assessed by qRT-PCR, immunohistochemistry (IHC), and immunofluorescence. ALKBH5-mediated m6A modification of pri-miR-221-3p was analyzed by methylated RNA immunoprecipitation (MeRIP) Functional effects on inflammatory responses were evaluated by measuring TNF-α, IL-1β, and IL-6 levels following functional experiments, combined with ELISA assays.
RESULTS: miR-221-3p was significantly downregulated in STB tissues, peripheral blood, and BCG-infected macrophages, indicating its potential as a diagnostic marker. In contrast, ALKBH5 expression was upregulated. Functionally, knockdown of miR-221-3p increased the production of pro-inflammatory cytokines and exacerbated inflammation, whereas its overexpression had the opposite effect. Mechanistically, ALKBH5 was found to directly demethylate pri-miR-221-3p, as confirmed by MeRIP, thereby impairing its maturation. Silencing ALKBH5 restored miR-221-3p expression and reduced cytokine secretion.
CONCLUSION: miR-221-3p may serve as a potential diagnostic biomarker for STB. ALKBH5 upregulation promotes STB progression by inhibiting miR-221-3p maturation via m6A demethylation, which enhances the inflammatory response. Targeting the ALKBH5/miR-221-3p axis may offer a novel therapeutic approach for STB.
Key numbers
0.793
AUC for
Area under the ROC curve for in STB diagnosis.
50 of 100
50 patients
Total number of patients included in the study.
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Declarations. Ethics approval and consent to participate: This study was conducted in accordance with the principles of Helsinki Declaration. The study approval was obtained from the Ethics Committee of General Hospital of Ningxia Medical University (KYLL-2022-0133). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
