METTL3介导SOX9的m6A RNA甲基化调控牙髓干细胞成牙本质向分化的机制研究

The odontogenic differentiation of dental pulp stem cells is a key program in the study of restorative dentin formation and regeneration. N6 methyladenine (m6A) methyltransferase METTL3-mediated m6A RNA methylation plays a key role in injury response and stem cell fate determination, but it has not been reported in the differentiation of dental pulp stem cells. In the preliminary experiment, we found METTL3 expression was up-regulated in the pulp tissue of the restorative dentin formation area, and METTL3-mediated m6A modification was induced during odontogenic differentiation of pulp stem cells. We further explored the target genes of METTL3 by RNA-seq, the expression of SOX9 was significantly changed. So, how does METTL3 regulate the differentiation of dental pulp stem cells? Is it related to the m6A modification of SOX9? In this study, we intend to construct dental pulp stem cells transfected with METTL3 inhibition, over-expression and enzyme activity mutation lentivirus vector to investigate the regulatory mechanism of METTL3 on odontogenic differentiation at gene and protein levels. We use m6A co-immunoprecipitation to investigate that SOX9 was modified by METTL3 and verify it in vivo model. This study is helpful to reveal the mechanism of RNA epigenetics during the differentiation of dental pulp stem cells, and provide new ideas and potential targets for the preservation of living pulp.

牙髓干细胞分化为成牙本质样细胞，形成修复性牙本质，是牙髓损伤修复的核心所在。N6甲基腺嘌呤(m6A)甲基转移酶METTL3介导m6A RNA甲基化在损伤应答、干细胞命运决定中发挥关键作用，但在牙髓干细胞分化中尚无报道。申请人预实验发现，在修复性牙本质形成区的牙髓组织中METTL3表达上调，且在牙髓干细胞成牙本质向分化过程中METTL3介导m6A修饰水平升高。RNA-seq进一步筛选METTL3的靶基因，SOX9表达变化显著并具有相关性。那么，METTL3如何调控牙髓干细胞分化，是否与SOX9的m6A修饰有关？本课题拟构建METTL3抑制、过表达和酶活性突变的牙髓干细胞，探讨METTL3对成牙本质向分化进程的调控作用，以m6A免疫共沉淀分析METTL3靶向修饰SOX9的机制，并结合体内模型进行验证。本研究有助于揭示牙髓干细胞分化过程中的RNA表观遗传学机制，为活髓保存提供新的思路和潜在靶点。

牙髓干细胞分化为成牙本质样细胞，形成修复性牙本质，是牙髓损伤修复的核心所在。如何调动牙髓干细胞固有的修复潜能，促进其成牙本质向分化是临床亟待解决的问题。N6甲基腺嘌呤(m6A)作为最常见的mRNA甲基化修饰方式，广泛调控基因的转录后水平表达，参与组织修复再生。m6A甲基转移酶METTL3在损伤应答、干细胞分化及自我更新中发挥重要作用。本课题主要研究RNA甲基化m6A修饰在牙髓干细胞分化中的生物学功能及作用。从牙髓干细胞成牙本质向分化中的m6A修饰图谱及关键因子METTL3的表达模式入手，探索METTL3对牙髓干细胞分化、自我更新和矿化基质形成的影响及分子机制。结果发现，以m6A免疫共沉淀测序揭示牙髓干细胞成牙本质向分化过程中的m6A-RNA表达图谱，其中3’非编码区（3’UTR）的m6A修饰峰增加。METTL3在牙髓干细胞分化过程中表达显著上调，是介导m6A修饰的关键酶。体外慢病毒转染敲低METTL3会导致牙髓干细胞周期紊乱，抑制其成牙本质向分化，促进成脂向分化；而过表达METTL3有助于牙髓干细胞成牙本质向分化。构建裸鼠皮下异位矿化模型，体内验证METTL3促进牙髓干细胞分化及矿化基质形成。联合m6A免疫共沉淀以及转录组测序结果筛选靶基因，NOG在牙髓干细胞分化过程中3’UTR的m6A修饰增加，基因表达下调。NOG的m6A修饰受METTL3调控，进而影响其基因表达及下游Smad信号通路的激活。METTL3抑制NOG基因和蛋白的稳定性，从而调控牙髓干细胞的分化。本项目的实施有助于阐明METTL3介导m6A修饰在牙髓干细胞分化中的作用机制，进一步完善牙髓生物学的表观遗传学体系，并为牙髓损伤修复的治疗方式提供新思路。