抗氧化应激相关基因DNA甲基化在高度近视并发性白内障发病中的作用和机制研究

High-myopic cataract (HMC), accounting for 30% of cataract surgery in China recently, is a type of complicated cataract with great risk of blindness. Its proportion is increasing every year. However, the study of HMC-associated pathogenesis has been quite scarce. Our previous study found that compared to patients with age-related cataract (ARC), high myopia was associated with a significantly greater risk of dark nucleus, which multiplied the difficulties during cataract surgery. And such dark nucleus might be caused by hypermethylation of anti-oxidative gene (CRYAA) promoter and decreasing of lens structural protein expression in the high-myopic eyes. However, it might be only part of the story. In order to further clarify the changes of other anti-oxidative related genes in HMC eyes, we then carried out preliminary screening with high-throughput DNA methylation sequencing and microarray analysis in the HMC lenses, and 4 anti-oxidative related genes (Nrf2/SOD1/TFAP2C/GSTP1) with significantly elevated DNA methylation status comparing to ARC cases were identified. In the current study, we plan to carry out pyrosequencing in a large sample to verify the DNA methylation status and expression of the 4 selected genes in HMC cases. We will also analyze the binding of the selected genes with upstream transcription related factors and the changes of their downstream pathways after DNA hypermethylation. Finally, we will validate the intervention effects of DNA methylation inhibitor on the 4 selected genes in HMC. Based on these studies, we manage to clarify the role and mechanisms of DNA methylation of anti-oxidative related genes in the pathogenesis of HMC, and to provide the basis for its early non-surgical intervention in the future.

高度近视并发性白内障（HMC）是高致盲风险的白内障类型，已占我国白内障手术量的30%，且比例逐年上升，但其发病机制的研究却一直匮乏。我们前期研究发现HMC极易并发手术风险极高的黑核，而DNA甲基化增加导致抗氧化应激基因之一的CRYAA与晶状体结构蛋白表达下降加速了HMC发病；为进一步明确HMC中其他抗氧化应激相关基因的改变，我们进行了高通量DNA甲基化组测序及表达谱芯片分析，初步筛选了HMC较年龄相关性白内障晶状体中DNA甲基化明显升高的4组抗氧化应激相关基因：Nrf2/SOD1/TFAP2C/GSTP1。本研究拟用焦磷酸测序大样本量验证HMC中这些基因的DNA甲基化状态及表达，观察其甲基化升高后与上游转录相关因子的结合及下游相关通路的改变，并评估DNA甲基化抑制剂对其的干预效应。旨在较全面地阐明抗氧化应激相关基因DNA甲基化对HMC的作用和机制，为早期非手术干预提供依据。

高度近视并发性白内障（HMC）是高致盲风险的复杂白内障，目前占据我国白内障手术量的30%，且该比例持续逐年上升，但其发病机制一直未能揭晓，相关机制研究较为匮乏。课题组前期研究发现HMC容易并发手术风险极高的棕黑核白内障，而在此过程中存在抗氧化应激基因和晶状体重要结构蛋白、分子伴侣CRYAA基因的启动子区域CpG岛的高度DNA甲基化和表达下调现象，可能参与了HMC的发生；为了进一步明确HMC中其他抗氧化应激相关基因的改变，我们开展了本课题。通过前期的高通量DNA甲基化测序、表达谱芯片分析和文献阅读，我们初步筛选出GSTP1/NRF2/OGG1/TXN/TXNRD1/TXNRD2等6个基因作为备选，通过Sequenom MassArray检测比较其启动子区域的CpG位点在HMC和对照组年龄相关性白内障（ARC）的晶状体前囊膜中的甲基化水平，发现GSTP1和TXNRD2的启动子CpG位点在HMC中较ARC处于高甲基化水平，并且两基因在HMC中的表达水平也显著下调。荧光素酶报告基因实验证实了GSTP1和TXNRD2中的DNA甲基化差异位点在转录激活中具有重要意义。通过过表达和敲减GSTP1/TXNRD2和一系列抗氧化应激水平检测，我们证实了GSTP1和TXNRD2在晶状体上皮细胞中具有抗氧化能力。此外，在HMC中甲基化转移酶1（DNMT1）也较ARC表达水平更高，而经甲基转移酶抑制剂处理后的晶状体上皮细胞中GSTP1和TXNRD2的表达有所上调。最终，为了模拟高度近视眼中的高氧化环境，我们使用H2O2处理晶状体上皮细胞，观察到GSTP1和TXNRD2的启动子区域CpG位点的甲基化水平出现了时间依赖性的增长和基因表达的最终下调。因此通过本课题探索，我们提出了高度近视并发性白内障发病的DNA甲基化机制假说：在高度近视眼中，晶状体较正常眼处于更高的氧化环境，形成了由更高的氧化应激刺激与氧化应激导致抗氧化应激基因高甲基化介导的低表达构成的恶性循环，故而高度近视眼中白内障发生较早且进展更快。