METTL3 enhances dentinogenesis differentiation of dental pulp stem cells via increasing GDF6 and STC1 mRNA stability
Yue Pan, Ying Liu, Dixin Cui, Si-Han Yu, Yachuan Zhou, Xin Zhou, Wei Du, Liwei Zheng, Mian Wan
Abstract
Abstract Background The dentinogenesis differentiation of dental pulp stem cells (DPSCs) is controlled by the spatio-temporal expression of differentiation related genes. RNA N6-methyladenosine (m 6 A) methylation, one of the most abundant internal epigenetic modification in mRNA, influences various events in RNA processing, stem cell pluripotency and differentiation. Methyltransferase like 3 (METTL3), one of the essential regulators, involves in the process of dentin formation and root development, while mechanism of METTL3-mediated RNA m 6 A methylation in DPSC dentinogenesis differentiation is still unclear. Methods Immunofluorescence staining and MeRIP-seq were performed to establish m 6 A modification profile in dentinogenesis differentiation. Lentivirus were used to knockdown or overexpression of METTL3. The dentinogenesis differentiation was analyzed by alkaline phosphatase, alizarin red staining and real time RT-PCR. RNA stability assay was determined by actinomycin D. A direct pulp capping model was established with rat molars to reveal the role of METTL3 in tertiary dentin formation. Results Dynamic characteristics of RNA m 6 A methylation in dentinogenesis differentiation were demonstrated by MeRIP-seq. Methyltransferases (METTL3 and METTL14) and demethylases (FTO and ALKBH5) were gradually up-regulated during dentinogenesis process. Methyltransferase METTL3 was selected for further study. Knockdown of METTL3 impaired the DPSCs dentinogenesis differentiation, and overexpression of METTL3 promoted the differentiation. METTL3-mediated m 6 A regulated the mRNA stabiliy of GDF6 and STC1 . Furthermore, overexpression of METTL3 promoted tertiary dentin formation in direct pulp capping model. Conclusion The modification of m 6 A showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated m 6 A regulated in dentinogenesis differentiation through affecting the mRNA stability of GDF6 and STC1 . METTL3 overexpression promoted tertiary dentin formation in vitro, suggesting its promising application in vital pulp therapy (VPT).