Abstract
BACKGROUND: Human Periodontal Ligament Stem Cells (PDLSCs) represent a subset of mesenchymal stem cells originating from the periodontal ligament, a connective tissue responsible for anchoring teeth to alveolar bone. This study explored the functional role of Methyltransferase-Like-1 (METTL1)-mediated N7-methylguanosine (m7G) modification in regulating the osteogenic differentiation capacity of PDLSCs. METHODS: PDLSCs were isolated from periodontal ligament tissues. Osteogenic differentiation was assessed through Alkaline Phosphatase (ALP) activity assays and Alizarin Red S (ARS) staining. Expression levels of Runt-related Transcription Factor-2 (RUNX2), Osteocalcin (OCN), and Osteopontin (OPN) were quantified using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Western blot analysis was employed to detect protein expression of METTL1, NOP2/Sun RNA Methyltransferase-2 (NSUN2), and WD Repeat Domain-4 (WDR4). Total m7G content in PDLSCs was measured via m7G dot blot assays. The interaction between METTL1 and RUNX2 was validated using luciferase reporter assays. RESULTS: The present findings demonstrated that METTL1 exhibited elevated expression levels in PDLSCs, with further upregulation during osteogenic induction. METTL1 knockdown significantly impaired osteogenic differentiation, characterized by decreased ALP activity, reduced ARS staining intensity, and downregulated osteogenic marker gene expression. Mechanistically, the authors identified that METTL1 enhanced m7G modification of key osteogenic genes, including RUNX2, OCN, and OPN, thereby improving their mRNA stability and translational efficiency. Notably, forced overexpression of RUNX2 partially reversed the osteogenic differentiation defects induced by METTL1 suppression. CONCLUSION: METTL1 promotes osteogenic differentiation in PDLSCs through m7G modification-mediated stabilization of RUNX2 expression. This discovery unveils a novel epigenetic regulatory mechanism involving m7G modification in periodontal tissue regeneration, offering potential therapeutic targets for bone defect repair applications.