WTAP-mediated m6A modification upregulates the circCDK8 to promote ferroptosis in periodontal ligament stem cells.

Abstract

Impaired osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is a key factor in the failure of alveolar bone repair. Ferroptosis, a form of regulated cell death, has been implicated in osteogenic dysfunction. Wilms' tumor 1-associated protein (WTAP) regulates RNA fate through m6A modification; however, whether WTAP modulates ferroptosis in PDLSCs remains unknown. This study aims to investigate the role of WTAP in LPS-induced ferroptosis in PDLSCs. LPS-treated hPDLSCs were used as cell models. Osteogenic differentiation and inflammation of hPDLSCs, ferroptosis biomarkers, and the expression circular RNA CDK8 (circCDK8), microRNA let-7a-5p (let-7a-5p), transferrin receptor (TFRC), and Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were measured. m6A enrichment on circCDK8 and its binding to Insulin-like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3) were analyzed. Interactions among circCDK8, let-7a-5p, and TFRC, as well as Y-box Binding Protein 1 (YBX1) recruitment to circCDK8 and ACSL4, were confirmed. Following LPS induction, the expression of WTAP, circCDK8, TFRC, and ACSL4 was upregulated in hPDLSCs, while let-7a-5p expression was downregulated. WTAP knockdown enhanced osteogenic differentiation, reduced inflammation, and decreased ferroptosis in hPDLSCs. Mechanistically, WTAP-mediated m6A modification stabilized circCDK8 through IGF2BP3 binding. CircCDK8 then promoted ferroptosis via two parallel routes: competitively sponging let-7a-5p to upregulate TFRC, and recruiting YBX1 to stimulate ACSL4 expression. Overexpression of circCDK8 or knockdown of let-7a-5p inhibited osteogenic differentiation and promoted ferroptosis. These findings reveal that WTAP drives ferroptosis in PDLSCs through m6A-dependent stabilization of circCDK8, which activates the let-7a-5p/TFRC and YBX1/ACSL4 axes, offering mechanistic insights for alveolar bone repair.