Mg2+通过KDM6A介导H3K27me3去甲基化调控卫星细胞成肌分化在废用性肌萎缩中的作用与机制研究

Disused muscular atrophy is an important factor in delaying bone healing and remodeling, and there is still a lack of effective interventions for it. It is known that muscle satellite cells are the key cells to repair injured muscles whose myogenic differentiation genes expression is negatively regulated by lysine trimethylation located in the H3 histone 27 site (H3K27me3) in their promoters. In our previous studies, we found that Magnesium ions (Mg2+) could significantly antagonize muscular atrophy, but the specific mechanism was still unknown. Simultaneously, our preliminary experiments have initially confirmed that Mg2+ could promote the expression of myogenic differentiation genes, up-regulate histone demethylase KDM6A and reduce the level of H3K27me3, however, the specific theoretical framework needed to be further improved. Therefore, through some molecular biological means in this study, we will firstly clear the specific role of satellite cells in the progressive and recovery process of atrophic muscles and explore the effect of Mg2+ on its biological behavior. Further, the effect of H3K27me3 level in the myogenesis related gene promoter region mediated by KDM6A on the satellite cell myogenic differentiation will be detailedly investigated. Finally, the role of Mg2+ in the above pathways is clarified, and its specific molecular mechanism in satellite myoblast differentiation is also revealed. The expected results will provide theoretical and experimental basis for the prevention and treatment of disused muscular atrophy by Mg2+, which has important clinical significance.

废用性肌萎缩是延缓骨愈合及改建的重要因素，目前仍缺乏有效的干预措施。已知肌肉卫星细胞是修复损伤肌肉的关键细胞，其成肌分化基因的表达受启动子内H3组蛋白27位点赖氨酸三甲基化（H3K27me3）的负性调节。我们前期研究发现，镁离子（Mg2+）可显著拮抗肌肉萎缩，但具体机制不明；预实验初步证实Mg2+具有促进成肌分化基因表达、上调组蛋白去甲基化酶KDM6A及降低H3K27me3水平的作用，但具体理论构架有待进一步完善。为此，本研究拟通过分子生物学手段，首先阐明卫星细胞在废用性肌萎缩发生及恢复中的关键作用，初步明确Mg2+对卫星细胞生物学行为的影响；其次，探讨由KDM6A介导的H3K27me3去甲基化在卫星细胞成肌分化中的作用及机制;第三，明确Mg2+在上述途径中的作用，揭示其调控卫星成肌分化的具体分子机制。预期的研究结果将为Mg2+防治废用性肌萎缩的作用提供理论和实验依据，具有重要的临床意义。

目的：废用性肌萎缩是延缓骨愈合及改建的重要因素，目前缺乏有效的干预措施。肌肉卫星细胞是修复损伤肌肉的关键细胞，其成肌分化基因的表达受启动子内H3组蛋白27位点赖氨酸三甲基化（H3K27me3）的负性调节。前期研究发现，镁离子（Mg2+）可显著拮抗肌肉萎缩，但具体机制不明。本研究拟通过分子生物学手段，阐明卫星细胞在废用性肌萎缩发生及恢复中的关键作用，明确Mg2+通过KDM6A介导的H3K27me3去甲基化调控卫星细胞成肌分化中的作用及相关机制。.方法：体外分离小鼠肌肉卫星细胞，萎缩诱导并加入Mg2+后，检测其增殖能力及成肌分化相关基因及蛋白的变化；建立废用性肌萎缩小鼠模型，向萎缩后肢注射MgSO4溶液后观察Mg2+在废用性肌萎缩发生及恢复中的作用。选用小鼠的成肌细胞系C2C12，靶向上调、下调KDM6A的表达后，通过ChIP-seq确定受KDM6A/H3K27me3 机制途径调控的成肌分化相关基因，应用ChIP-qPCR 方法进行验证，而后对细胞成肌分化能力进行评估。经Mg2+处理的肌肉卫星细胞，检测细胞内KDM6A及前述成肌分化基因的表达和启动子区域内H3K27me3的含量变化，进一步结合KDM6A的靶向抑制（shRNA），明确KDM6A在Mg2+调节卫星细胞成肌分化中的关键作用。.结果：Mg2+在体外可明显提高萎缩状态的卫星细胞增殖活性，并且通过促进Myod、Myog和Myf5蛋白的转录与表达促进卫星细胞的成肌分化能力。Mg2+在废用性肌肉萎缩小鼠体内可拮抗肌肉萎缩并促进萎缩肌肉恢复，并且Mg2+处理组肌肉中Myod、Myog和Myf5蛋白的表达明显增加。小鼠C2C12细胞系中受KDM6A/H3K27me3机制途径调控的成肌分化相关基因为Myf5，靶向下调KDM6A的表达后，细胞成肌分化能力明显降低，而靶向上调KDM6A的表达可明显提高细胞的成肌分化能力。经Mg2+处理的卫星细胞KDM6A表达显著上调，从而增强了Myog的表达及启动子内H3K27me3的含量降低，促进了卫星细胞成肌分化作用。另外，以shRNA干扰KDM6A的表达后，Mg2+对卫星细胞的成肌分化促进作用显著降低。.结论：Mg2+可通过KDM6A介导H3K27me3去甲基化调控卫星细胞成肌分化能力来拮抗废用性肌肉萎缩并促进萎缩肌肉恢复，为其防治废用性肌萎缩的作用提供理论和实验依据，具有重要的临床意义。