ALKBH5介导NEIL2的m6A甲基化调控LECs氧化损伤在年龄相关性白内障发生中机制研究

氧化损伤是年龄相关性白内障（ARC）的重要发病机制。申请人以往研究表明表观遗传参与调控氧化损伤修复基因的功能和ARC的发生，而RNA甲基化（m6A）在ARC中的机制尚不清楚。前期预实验发现：ARC患者晶状体上皮细胞（LECs）中RNA去甲基酶ALKBH5高表达；碱基切除修复基因NEIL2的m6A修饰在ARC患者LECs中减少，提示可能是ALKBH5的潜在靶基因；NEIL2靶向结合并调控氧化损伤关键基因KEAP1。据此提出假说：ALKBH5通过m6A去甲基化下调NEIL2的表达，使得NEIL2与KEAP1相互作用下降，引起KEAP1表达升高导致LECs氧化损伤和ARC发病。本研究拟通过临床组织样本、细胞和小鼠模型，利用Crispr-Cas9、MeRIP-PCR、免疫共沉淀等方法，探讨ALKBH5介导NEIL2的m6A去甲基化靶向调控KEAP1在ARC发生中的机制，为ARC的防治提供新的思路。

Oxidative damage is a critical risk factor in the development of age-related cataract (ARC). Our previous projects have shown that epigenetics participate in the formation of ARC by regulating the functions of oxidative damage repair genes. However, the mechanism of RNA methylation, especially N6-methyladenine (m6A) modification in ARC formation is not clear.Our preliminary study found that m6A demethylase ALKBH5 was highly expressed in lens epithelial cells (LECs) of ARC patients, while the m6A methylation of NEIL2 was reduced, which suggesting that NEIL2 might be a potential target gene of ALKBH5.We also found that NEIL2 could interact with KEAP1 and regulate the expression of KEAP1, which known as an essential role in oxidative damage.On the basis of these, we speculate that m6A demethylase ALKBH5 might decrease the interaction between NEIL2 and KEAP1 by downregulating the expression of NEIL2, resulting in the increased expression of KEAP1, oxidative damage of LECs and ARC formation.For the purpose to explore the mechanism of ALKBH5-mediated m6A demethylation of NEIL2 for regulating KEAP1 in ARC formation, we used Crispr-cas9, MeRIP-PCR, co-immunoprecipitation and other methods from many aspects and levels such as tissue, cell, mouse model. In summary, we hope to provide new ideas for the prevention and treatment of ARC.