WTAP介导的PPARα m6A甲基化修饰在肝脏脂质沉积中的作用及机制研究

肝脏脂质沉积是非酒精性脂肪性肝病的始动因素。PPARα介导的脂肪酸氧化对于维持肝脏的脂代谢平衡至关重要，但其调控网络目前尚未完全阐明。我们前期发现，PPARαm6A修饰水平在脂肪肝患者及db/db、HFD小鼠肝脏中显著上调，同时伴随基因表达量明显降低。对m6A修饰蛋白的筛查发现RNA甲基化酶WTAP在脂肪肝中异常升高，与肝脏TG含量呈正相关。过表达WTAP可提高小鼠原代肝细胞PPARα m6A水平，降低其RNA稳定性以阻遏基因表达、诱导细胞脂质沉积。提示WTAP可通过PPARα RNA甲基化的调控影响肝脏脂稳态。为了解析该过程，本项目拟进一步从小鼠和细胞层面，通过WTAP过表达和敲除的代谢表型检测，结合m6A-seq、MeRIP、RIP、双荧光素酶等分子手段，明确WTAP介导的PPARα m6A修饰在肝脏脂代谢中的调控作用和具体机制，为脂肪肝、肥胖及相关的代谢综合征提供新的治疗靶点和依据。

Lipid deposition in hepatocytes is the initiating factor for the occurrence of non-alcoholic fatty liver disease. PPARα-mediated fatty acid oxidation is essential for maintaining the liver's lipid metabolism balance, but its regulatory network has not yet been fully elucidated. We found in the early stage that the PPARα m6A modification level was significantly up-regulated in the liver of NAFLD patients and db/db, HFD mice, accompanied by a significant decrease in PPARα gene expression. The screening of m6A modifying protein found that the RNA methylase WTAP was abnormally elevated in fatty liver, and its mRNA level was positively correlated with liver TG content. Overexpression of WTAP can increase the level of PPARα m6A in mouse primary hepatocytes, reduce its RNA stability and inhibit gene translation, and thus inducing hepatosteatosis. These results indicated that WTAP may affect lipid homeostasis in the liver via regulation of PPARα RNA methylation. In order to fully interpret this process, our project intends to further determine the role of WTAP role in metabolic phenotype through overexpression and knockout of WTAP in vivo in mouse model and in vitro in mouse primary hepatocytes. By utilizing m6A-seq, MeRIP, RIP, and dual luciferase reporter assays, we will further clarify the specific mechanisms of WTAP-mediated PPARα m6A modification in lipid metabolism in the liver. Our research may provide new therapeutic targets and experimental evidence for the curation of fatty liver, obesity and related metabolic syndromes.