Abstract
BackgroundDental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of m6A methylation in DPSCs.Methodsm6A immunoprecipitation with deep sequencing (m6A RIP-seq) demonstrated the features of m6A modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by β-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and m6A RIP-qPCR was used to confirm METTL3-mediated m6A methylation.ResultsHere, m6A peak distribution, binding area, and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of m6A RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated m6A methylation in DPSCs.ConclusionsThese 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 served new insight into stem cell-based tissue engineering.
Highlights
Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity
After odontogenic medium induction for 3, 7, and 14 days, the early differentiation stage of DPSCs was evidenced by strong alkaline phosphatase (ALP) straining and upregulated osteo/odontogenesis-related markers such as RUNX2, ALP, and COL1 (Fig. 1b, c) while late differentiation stage identified by significant calcium mineralization in alizarin red staining (ARS) and mRNA expression of BSP, DSPP, and OCN (Fig. 1d, e)
Consistent with this study, we discovered the expression of Polo-like kinase 1 (PLK1) in DPSCs and elevated PLK1 expression following methyltransferase like 3 (METTL3) inhibition resulted in the S phase arrested which suggested a regulatory role of PLK1 in cell cycle control of DPSCs
Summary
Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. We aim to explore the biological effect of m6A methylation in DPSCs. Adult stem cells residing in various tissues play an essential role in maintaining tissue homeostasis and ensuring regenerative needs. Adult stem cells residing in various tissues play an essential role in maintaining tissue homeostasis and ensuring regenerative needs These cells may remain quiescent in a specialized microenvironment for a quite long time and can still differentiate into specific cell types once there are physical damages or activating signals [1]. The regenerative and self-renewal capacity of stem cells highlight the therapeutic approaches as regenerative medicine in local injury repair and in tissue engineering. DPSCs can continuously grow up to thirty passages and still maintain pluripotent to differentiate into odontogenic, chondrogenic, adipogenic, and neurogenic cells [3]
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