State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Pediatric Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China.
Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University, Shenzhen, Guangdong, China.
BMC Oral Health. 2023 Apr 11;23(1):209. doi: 10.1186/s12903-023-02836-z.
The dentinogenesis differentiation of dental pulp stem cells (DPSCs) is controlled by the spatio-temporal expression of differentiation related genes. RNA N6-methyladenosine (mA) 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 mA methylation in DPSC dentinogenesis differentiation is still unclear.
Immunofluorescence staining and MeRIP-seq were performed to establish mA 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.
Dynamic characteristics of RNA mA 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 mA regulated the mRNA stabiliy of GDF6 and STC1. Furthermore, overexpression of METTL3 promoted tertiary dentin formation in direct pulp capping model.
The modification of mA showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated mA 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).
牙髓干细胞（DPSCs）的 dentinogenesis 分化受分化相关基因的时空表达控制。RNA N6-甲基腺苷（mA）甲基化是 mRNA 中最丰富的内部表观遗传修饰之一，影响 RNA 处理、干细胞多能性和分化的各种事件。甲基转移酶样 3（METTL3）是必需的调节因子之一，参与牙本质形成和根发育过程，而 METTL3 介导的 DPSC dentinogenesis 分化中的 RNA mA 甲基化机制尚不清楚。
通过免疫荧光染色和 MeRIP-seq 建立 dentinogenesis 分化中的 mA 修饰谱。慢病毒用于敲低或过表达 METTL3。通过碱性磷酸酶、茜素红染色和实时 RT-PCR 分析 dentinogenesis 分化。通过放线菌素 D 测定 RNA 稳定性。通过大鼠磨牙建立直接牙髓盖髓模型，以揭示 METTL3 在第三期牙本质形成中的作用。
通过 MeRIP-seq 证明了 dentinogenesis 分化中 RNA mA 甲基化的动态特征。甲基转移酶（METTL3 和 METTL14）和去甲基酶（FTO 和 ALKBH5）在牙本质形成过程中逐渐上调。甲基转移酶 METTL3 被选为进一步研究。METTL3 敲低会损害 DPSCs 的 dentinogenesis 分化，而过表达 METTL3 则促进分化。METTL3 介导的 mA 调节 GDF6 和 STC1 的 mRNA 稳定性。此外，过表达 METTL3 促进直接牙髓盖髓模型中的第三期牙本质形成。
在 DPSCs dentinogenesis 分化过程中，mA 的修饰表现出动态特征。METTL3 介导的 mA 通过影响 GDF6 和 STC1 的 mRNA 稳定性来调节 dentinogenesis 分化。METTL3 过表达促进体外第三期牙本质形成，提示其在活髓治疗（VPT）中有应用前景。
