METTL3 enhances dentinogenesis differentiation of dental pulp stem cells via increasing GDF6 and STC1 mRNA stability

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 (m⁶A) 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 m⁶A methylation in DPSC dentinogenesis differentiation is still unclear.

Methods: Immunofluorescence staining and MeRIP-seq were performed to establish m⁶A 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 m⁶A 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 m⁶A 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 m⁶A showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated m⁶A 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).

Keywords: Dentinogenesis; Epigenesis; METTL3; Mesenchymal stem cells; RNA stability.

Fig. 1

The level of RNA m⁶A modification increased during dentinogenesis differentiation. A The expression of m⁶A gradually increased with the dentinogenesis differentiation in mice cervical loop. e Stem cells. f Transit-amplifying cells in labial cervical loop. g Transit-amplifying cells in lingual cervical loop. h)Odontoblasts. B The content of RNA m⁶A increased in DPSCs during the dentinogenesis differentiation. C Expression levels of RNA m.⁶A methylation-related genes, METTL3, METTL14, FTO, ALKBH5, were measured by real‐time RT‐PCR on 0, 7 and 14 days after mineralization induction (n = 3). D Western blotting analysis of METTL3 from DPSCs after mineralization induct for 0, 7 and 14 days. β‐ACTIN served as an internal control. *P < 0.05, ***P < 0.001

Fig. 2

The differential m⁶A-methylated peaks and differential m⁶A methylation distribution in DPSCs (n = 4). A The volcano diagram showed the differential m⁶A methylation peaks detected in DPSCs. B, C Pie diagrams revealed the annotation of hypermethylation and hypomethylation peaks. D GO Enrichment Top10. E KEGG pathway classification of differential peak related genes

Fig. 3

Joint Analysis of MeRIP-seq and RNA-seq (n = 4). A The volcano diagram showed the differential expression genes in DPSCs. B The combined analysis of differential m⁶A methylation and RNA expression

Fig. 4

Knockdown of METTL3 inhibited the dentinogenesis differentiation of DPSCs. A METTL3-kncokdown lentiviral were used to knockdown METTL3 (shMETTL3-a, shMETTL3-b) in DPSCs. The knockdown efficiency of METTL3 gene expression in DPSCs, detected by real‐time RT‐PCR (n = 3). B Alizarin red S staining on 14 d, and C)ALP staining on 7 d of DPSCs cultured in OM (n = 3), C The quantitative analysis and staining of ALP activity (n = 3). DThe knockdown efficiency of METTL3 gene expression detected by western blotting (n = 3). E Expression levels of dentinogenesis differentiation-related genes, OCN, RUNX2, OSX, DSPP, were measured by real‐time RT‐PCR on 0, 7 and 14 days after mineralization induction (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Overexpression of METTL3 promoted the dentinogenesis differentiation of DPSCs. A METTL3 overexpression lentiviral were used to infect DPSCs in order to over-express METTL3 (OE). The overexpression efficiency of METTL3 gene expression in DPSCs, detected by real‐time RT‐PCR (n = 3). B Alizarin red S staining on 14 d, and C ALP staining on 7 d of DPSCs cultured in OM (n = 3), C)The quantitative analysis and staining of ALP activity (n = 3). D The overexpression efficiency of METTL3 gene expression detected by western blotting (n = 3). E Expression levels of dentinogenesis differentiation-related genes, OCN, RUNX2, OSX, DSPP, were measured by real‐time RT‐PCR on 0, 7 and 14 days after mineralization induction (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

The mRNA stability of genes in DPSCs. A The mRNA stability of genes with hypermethylation and up-transcription, and B gene with hypermethylation and down-transcription in METTL3 knockdown DPSCs. C Genes with hypermethylation and up-transcription in METTL3 overexpression DPSCs (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

METTL3 promoted the formation of tertiary dentin. A Micro CT analysis of rat maxillae after pulp capping (n = 12). B The structure of mineralized tissue was detected by HE staining (n = 12). *P < 0.05, **P < 0.01