As the most prevalent, reversible internal modification in nuclear RNAs, m6A modifications involved in various biological processes including stem cell fate determination, embryonic development and cell cycle(20–22). The methyltransferases complex and demethylases allowed m6A marks to play a regulatory role in post-translational modifications of multiple levels, cooperated to influence different life stages of stem cells(10). Dental pulp stem cells derived from cranial neural crest cells, are promising stem cells for tissue regeneration. Though both immature and mature dental pulp stem cells retain pluripotency and self-renewal, the immature teeth showed significant better regenerative potential(8, 9). We found high gene expression level of methyltransferases-METTL3 was detected in immature DPSCs. Our data identified the binding area, motif and distribution of m6A peaks in DPSCs by m6A RIP-sEq. METTL3 inhibition in immature DPSCs induced cell senescence and apoptosis. Conjoint analysis of RNA-seq and m6A RIP-seq Alteration of METTL3 in DPSCs led to cell cycle arrest which tightly relates to stem cell self-renewal and fate decision. Our data indicated that METTL3 may support sufficient regenerative potential in DPSCs by maintaining cell cycle progression.
Over 80% transcripts were methylated during development which occur to effect the self-renewal and pluripotency of stem cells(23). m6A modification can influence stability, altered splicing, exporting, translation of mRNA in a fine, cell-specific manner. Methyltransferases complex is responsible to distinguish and target the redundant consensus RRm6ACH ([G/A/U][G > A]m6AC[U > A > C]) motif which enriched in 3’ and 5’ UTR of RNA transcripts especially long internal exons(24). In our study, m6A RIP-seq of DPSCs also revealed that the majority m6A marks distributed in 3’UTR (45.25%), then 5’UTR (22.99%) and methylated levels was higher in exons than introns. The clustered m6A modifications in 3’UTR around the stop codon influenced stability, localization, and translation of RNA while in 5’UTR effected translation of mRNA in a cap-independent manner(14, 25). METTL3 is the only catalytically active subunit in complex that can catalyze methylation of adenosines to form m6A modification(26). In the previous study METTL3 can independently read, regulate m6A modify near 3’UTR of mRNA and promote translation of this specific mRNA(27). Here we identified METTL3 was the essential m6A-related gene differentially expressed in different stages of DPSCs which might result in regenerative capacity alteration.
As a powerful stem cell fate determiner, m6A methylation modified the transcripts in a cell-dependent manner which lead to divergent effects, somehow even conflicting. METTL3 knockdown and m6A depletion limited self-renewal and promoted cell differentiation in epiblast stem cells while improved pluripotency, blocked regeneration in embryonic stem cells(13, 21, 28). The depletion of METTL3-mediated m6A methylation in stem cells of different status would disrupt the balance and boost cells into totally contrary fates(23). m6A modification affect large-scale of genes governing specific biological processes and contributed to maintaining balanced status of stem cells in multiple pathways(13, 15, 16). In our study, METTL3 inhibition in immature DPSCs promoted senescence and cell apoptosis which could impair stem cells self-renewal and fate decision. Enrichment analysis of overlapped genes in m6A RIP-seq and shMETTL3 RNA-seq of DPSCs indicated METTL3 was most likely to make effect in cell apoptosis by interplaying with cycle-mediated factors.
The functional stem cells apoptosis cooperated with proper cell cycle progress which is essential for cellular homeostasis. Dynamic activation of cell cycle was tightly related to self-renewal of bone marrow stem cells and maturation of hematopoietic stem cells (29). The transcriptome and epigenetic landscape alterations in cell cycle correspond with different status of development, maturation and differentiation(30). Prime stem cells usually stayed in quiescence as cells resource, expansion of progenitor cells need high cycling rate or short cycle time, while some differentiated cells withdraw from cell cycle which could still reenter cell cycle with certain signals(30, 31). m6A modifications were reported to participate in stem cells self-renew and pluripotency by regulating cell cycle processes. In cortical neurogenesis, the reduction of m6A modification resulted in longer cell cycle which related to insufficient differentiation and delayed generation(32). The methyltransferases complex consisted by METTL3, METTL14 and WTAP, inhibition of anyone could contribute to cell cycle arrest and disrupt the adipogenesis of adipocytes(33). METTL3 was also reported to be a tumor suppressor by modulating G1-phase cell cycle arrest via p21 expression(34). We found METTL3 reduction in DPSCs led to the cell cycle arrest and this was consisted with previous studies.
Cell cycle progress is controlled by robust cell cycle-related genes and checkpoints which were essential for pluripotency, self-renewal and homeostasis of stem cells. The mitotic cell cycle can be briefly divided into four phases, DNA duplication and synthesis -S phase, Mitosis cell division -M phase and gaps between these two stages. As critical mitotic regulator, PLK1 make effect in centrosome maturation, cytokinesis execution, bipolar spindle formation and chromosome segregation. PLK1 expression was tightly associated with cell cycle stages which initiated at relative low level and accumulated during S phase, then reach a peak in G2/M phase(35, 36). PLK1 was widely studied as a target in cancer therapy while its expression was at a relative low level in normal cells and there were no prior studies made in DPSCs till now. Our date suggested elevated PLK1 expression after METTL3 inhibition contributed to S phase arrested which suggested an essential role of PLK1 in cell cycle progression of DPSCs. PLK1 mediated G2 And S-Phase Expressed 1 (GTSE1) phosphorylation and p53 inactivation which led to G2 checkpoint recovery, DNA damage checkpoints termination and chromosome instability(37). PLK1 overexpression in mammary epithelial and murine cells contributed to mitosis defection and aberration in chromosome segregation, cytokinesis which supported unscheduled PLK1 expression disrupted mitosis and defected genomic stability(38, 39).