METTL3-mediated m6A modification of circNOTCH2 attenuates osteogenic impairment of PDLSCs in inflammatory microenvironment via the miR-193b-3p/KCNJ2/NLRP3 axis

Background Persistent inflammation associated with periodontitis significantly impairs the osteogenic differentiation potential of periodontal ligament stem cells (PDLSCs). Therefore, maintaining the osteogenic capacity of PDLSCs under inflammatory conditions represents a key therapy target for reducing periodontal bone loss. This study aims to investigate the mechanism by which the m6A-modified circular RNA hsa_circ_0005737 (circNOTCH2) regulates osteogenic differentiation of PDLSCs in the inflammatory microenvironment. Methods CircRNAs were screened via bioinformatics analysis. Plasmid overexpression and siRNA knockdown were employed to modulate gene expression. The regulatory role of circNOTCH2 in inflammatory PDLSCs and periodontitis was assessed through transcriptomic sequencing, alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, and a murine model of periodontitis. The N6-methyladenosine (m6A) methylation modification of circNOTCH2 was validated by methylated RNA immunoprecipitation (MeRIP). The underlying molecular mechanisms of circNOTCH2 were investigated using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence (IF) staining, dual-luciferase reporter assays, and Western blot (WB) analysis. Results Under inflammatory conditions, the expression of circNOTCH2 is significantly upregulated. Transcriptomic sequencing analysis reveal that circNOTCH2 is primarily associated with osteogenic differentiation of stem cells and the NLRP3 inflammasome pathway. Interestingly, overexpression of circNOTCH2 promotes osteogenic differentiation in PDLSCs. Further investigation demonstrates that this pro-osteogenic effect is mediated through a compensatory upregulation of circNOTCH2. CircNOTCH2 acts as a molecular sponge for miR-193b-3p, leading to the upregulation of KCNJ2 expression. This subsequently inhibits NLRP3 inflammasome activation and counteracts the suppressive effects of inflammation on the osteogenic potential of PDLSCs. Furthermore, the study revealed that METTL3-mediated m6A modification enhances the stability of circNOTCH2. As an upstream regulatory molecule, METTL3 positively influences the regulatory role of circNOTCH2 on PDLSCs. Conclusions In summary, our findings demonstrate that m6A-modified circNOTCH2 is compensatorily up-regulated in inflammatory PDLSCs and alleviates inflammation-induced impairment of osteogenic differentiation via the miR-193b-3p/KCNJ2/NLRP3 axis. These findings suggest that circNOTCH2 may represent a significant molecular target for the treatment of periodontitis.