组蛋白和DNA甲基化相互作用调控内质网应激通路介导同型半胱氨酸致肝脏脂代谢紊乱的分子机制

How could homocysteine (Hcy) disrupt the balance of lipid transport in liver and cause atherosclerosis (As) while Hcy is not involved in lipid metabolism directly is not yet clear. The liver，the place of endogenous lipid metabolism ，is to be more prone to endoplasmic reticulum stress. Our previous study found Hcy could lead to liver cell dysfunction-induced lipid metabolism disorders, and DNA methylation is an important mechanism of Homocysteine-induced AS, and histone methylation is an important regulation way of encoding genetic information cooperated with DNA methylation. But the role of histone methylation and DNA methylation in Hcy-induced lipid metabolism disorder via ERS has not been reported. This project intends to target for ERS pathway gene, CHIP is used to detect the changes of H3K9me3 of ERS pathways related genes to determine its role of lipid metabolism disorder. Constructing carriers of histone methyltransferases(HMT) and demethylase(UTX)and intervent with antagonists to reverse H3K9 methylation. Interfere the balance of DNA methylation and histone methylation pattern bilaterally to explore the mechanism of interaction between DNA methylation and H3K9 methylation and the regulation of target genes, to study the effect of interaction for biological activities of liver cells and ERS pathways, to reveal epigenetic mechanisms of pathogenicity of Hcy and determine the key targets, to provide experimental basis for the therapy of As.

同型半胱氨酸(Hcy)并不直接参与脂质代谢，何以能显著扰乱肝脏脂质转运平衡引起血脂异常尚未清楚。肝脏作为内源性脂代谢的主要场所，易发生内质网应激(ERS)，且前期发现Hcy可引起肝细胞功能障碍致脂代谢紊乱，并DNA甲基化是Hcy致病重要机制，同时组蛋白甲基化是协同DNA甲基化编码遗传信息的重要调控方式，但组蛋白和DNA甲基化在Hcy经ERS致脂代谢紊乱的机制未见报道。本项目拟以ERS通路基因为靶基因，CHIP检测H3K9me3及其位点相关的ERS通路基因的变化，明确其在脂代谢紊乱中的作用；构建组蛋白甲基化酶(HMT)和去甲基化酶(UTX)载体及拮抗剂干预，逆转H3K9甲基化，通过双向干预组蛋白和DNA甲基化模式的平衡，探讨H3K9和DNA甲基化相互作用及对关键靶基因的调控机制，研究其对肝细胞生物学活性和ERS通路的影响，揭示Hcy致病的表观遗传学机制，确定关键靶点，为靶向治疗提供实验依据。

叶酸缺乏是已知的导致肝损伤的重要危险因素；然而，其潜在的致病机制仍不清楚。在本研究中，我们采用高同型半胱氨酸诱导肝脏损伤模型，使用载脂蛋白E敲除（ApoE-/-）小鼠喂养高蛋氨酸饮食，并发现高同型半胱氨酸可以通过下调囊性纤维化跨膜传导调节因子（CFTR）表达诱导内质网（ER）应激和肝细胞凋亡;该损伤过程可以通过补充膳食叶酸而减弱。CFTR表达的调节由CFTR启动子DNA甲基化和组蛋白H3（H3K27me3）上赖氨酸27的三甲基化介导。从机制上讲，叶酸可抑制同型半胱氨酸诱导的CFTR启动子DNA甲基化和H3K27me3，从而导致CFTR表达上调，ER应激和肝细胞凋亡减少。进一步的研究表明，叶酸抑制了DNA甲基转移酶1的表达和zeste同源物2的增强子，下调了细胞内S-腺苷甲硫氨酸（SAM）和S-腺苷同型半胱氨酸（SAH）的浓度，并上调了SAM/SAH比例，从而抑制了Hcy诱导的DNA高甲基化和CFTR启动子中的H3K27me3。总之，我们的结果发现通过调节CFTR表达为了解叶酸在同型半胱氨酸诱导的ER应激和肝细胞凋亡中的保护作用提供了新的思路。