Abstract
Background: Dental pulp stem cells (DPSCs) exhibited self-renewal, pluripotency capacity and served as promising cells source in endodontic regeneration and tissue engineering. Meanwhile, the regenerative capacity of DPSCs is limited and reduced in long lifespan. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs. The methyltransferases complex and demethylases mediated m6A methylation and cooperated to impact various biological processes associated with stem cell fate determination. However, the biological effect of m6A methylation in DPSCs remained unclear. Methods: Cell surface markers and differentiation potential of primary DPSCs were identified and m6A immunoprecipitation with deep sequencing (m6A RIP-seq) was used to uncover characteristics of m6A modifications in DPSCs transcriptome. Expression level of m6A-related genes were evaluated in immature/mature pulp tissues and cells. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence and apoptosis were further analyzed by β-galactosidase, TUNEL staining and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq was used to functionally enrich overlapped genes and screen target of METTL3. Cell cycle distributions were assayed by flow cytometry and m6A RIP-qPCR was used to confirm METTL3 mediated m6A methylation in DPSCs. Results: Here, m6A peaks distribution, binding area and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relative high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. Furthermore, Conjoint analysis of m6A RIP and RNA-sequencing showed differentially expressed genes affected by METTL3 depletion was mainly enriched in cell cycle, mitosis and alteration of METTL3 expression resulted in cell cycle arrest which indicated METTL3 make essential effect in cell cycle control. To further investigate underlying mechanisms, we explored proteins interaction network of differentially expressed genes and Polo-like Kinase 1 (PLK1), a critical cycle modulator was identified as target of METTL3-mediated m6A methylation in DPSCs. Conclusions: These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and serve new insight in stem cells based tissue engineering.
Biodegradable and Biocompatible Graphene‐based Scaffolds for Functional Neural Tissue Engineering: A Strategy Approach Using Dental Pulp Stem Cells and Biomaterials
