二至丸通过Mettl3/14介导m6A动态修饰促骨髓间充质干细胞成骨分化抗骨质疏松的表观机制

RNA腺嘌呤甲基化（m6A）修饰是骨髓间充质干细胞（BMSC）分化的关键调控因素，m6A修饰异常引起成骨基因表达异常且骨丢失。补肾名方二至丸临床用于治疗骨质疏松，调控BMSC成骨分化是重要作用环节，但对其调控的深层次机制未明。预实验发现二至丸拆方促BMSC甲基转移酶Mettl3/14表达，改变m6A修饰特征，提出Mettl3/14介导m6A动态修饰是二至丸促成骨抗骨质疏松的重要机制。本项目在前期基础上，系统研究二至丸调控BMSC成骨分化过程m6A修饰的动态特征、Mettl3/14的表达变化规律及功能，鉴定关键靶标基因及Hippo等相关信号网络调控作用，并以计算模拟及实验评价Mettl3/14与二至丸成分的靶点配体关系，论证Mettl3/14的药靶作用，联合体内实验以验证上述假说，探寻二至丸抗骨质疏松的新靶点，也为肾主骨生髓与BMSC相关理论的生物学阐释提供思路，提升二至丸的应用与开发价值。

RNA adenine methylation (m6A) is a key regulatory factor in the differentiation of bone marrow mesenchymal stem cells (BMSC). The abnormal modification of m6A results in the abnormal expression of osteogenic genes and bone loss. The kidney-tonifying prescription of Erzhi Pill has obvious anti-osteoporotic effect in clinic and can regulate the osteogenesis of BMSC, but the deep mechanism of its regulation is unclear. We found that the components of Erzhi Pill could up-regulate the expression of mettl3/14 in BMSC and changed the modification of m6A. It is suggested that mettl3/14-mediated dynamic modification of m6A is an important mechanism of Erzhi Pill in treating osteoporosis through promoting osteogenesis. In this project, on the basis of the previous research, we systematically study the dynamic characteristics of m6A modification and the expression level of mettl3/14 as well as its functional role in the process of osteogenesis from BMSC regulated by Erzhi Pill. The key target genes are identified. The network related to osteogenic differentiation, including hippo pathway, is analyzed. Further, the target-ligand interaction mode of mettl3/14 and components from Erzhi Pill is evaluated by molecular docking analysis. The target role of mettl3/14 for Erzhi Pill is validated by experimental interference. The hypothesis above is verified by combination of in vivo experiments. It is helpful for exploring the new drug target for treating osteoporosis by Erzhi Pill, providing ideas for the biological interpretation of theory of kidney governing bone and tonifying marrow related to BMSC, and enhancing the application and development value of Erzhi Pill.