分泌型HSP90通过m6A RNA去甲基化酶FTO促进后发性白内障的机制研究

Posterior capsular opacification (PCO) remains a major complication of intraocular lens exchange surgery. The microenvironment of capsular bag regulates abnormal proliferation, migration and fibrosis of lens epithelial cells (ECs), which is the main pathological feature of PCO. Epithelial-mesenchymal transition (EMT) causes cell migration and fibrosis. We found the lens ECs of PCO extracellular secreted HSP90 (eHSP90). However, the mechanism of eHSP90 in PCO progression is unknown. The conditioned medium of lens capsular bag that removed eHSP90 by neutralizing antibody inhibited the migration and fibrosis of lens ECs, and upregulated the expression of m6A RNA demethylase FTO. Furtherly, the overexpression of FTO in lens ECs downregulated Snail, the important transcriptional factor of EMT. We hypothesis the microenvironmental eHSP90 regulates EMT through FTO, and then promotes PCO in lens ECs. In this study, by using in vivo rabbit IOL-PCO model and in-vitro-cultured rat lens capsular bag, and m6A-IP-qPCR, etc., we will: 1. investigate the functional domain and membrane-associated protein of eHSP90 that promoting EMT; 2. study the molecular mechanism that eHSP90 promoting EMT through FTO; 3. explore the molecular pathway that membrane-associated protein of eHSP90 downregulating FTO. The aim of the grant is to clarify the mechanism of the eHSP90 that promotes PCO and provide new PCO-therapy possibility of targeting eHSP90.

后发性白内障（PCO）是白内障人工晶体置换术常见并发症，其主要特征是囊袋微环境调控晶状体上皮细胞异常增生、迁移和纤维化。EMT是迁移和纤维化重要原因。我们发现PCO上皮细胞向细胞外分泌HSP90（eHSP90），但其在PCO中的作用不清。我们通过中和抗体清除eHSP90可抑制晶状体上皮细胞迁移和纤维化，并促进m6A RNA去甲基化酶FTO表达；过表达FTO下调EMT关键因子Snail。我们推测eHSP90参与囊袋微环境组成，通过FTO调控细胞EMT，促进PCO发生。本研究以兔IOL-PCO和大鼠晶状体囊袋为模型，通过m6A-IP-qPCR等方法，①明确eHSP90调控EMT的功能结构域和膜相关蛋白；②研究eHSP90通过FTO调控EMT的分子机制；③探讨eHSP90通过膜相关蛋白下调FTO的信号通路，以期揭示微环境eHSP90调控PCO病理发生机制，为寻找治疗PCO新靶点提供理论依据。

后发性白内障（PCO）是白内障人工晶体置换术常见并发症，其主要特征是囊袋微环境调控晶状体上皮细胞异常增生、迁移和纤维化。我们前期发现PCO上皮细胞向细胞外分泌HSP90（eHSP90），但其在PCO中的作用不清。本项目以人晶状体上皮细胞和大鼠晶状体囊袋为模型，研究发现：①晶状体上皮细胞通过非经典的自噬相关途径分泌HSP90。②重组蛋白GST-HSP90α诱导晶状体上皮细胞发生终末分化，即分化为晶状体纤维细胞。GST-HSP90α蛋白诱导大鼠囊袋晶状体上皮细胞伸长；上调晶状体纤维分化的关键转录因子PROX1及其下游晶状体纤维相关蛋白高表达，如β-和γ-晶状体蛋白。③eHSP90通过与受体LRP1结合激活LRP1-AKT介导的YAP降解来上调PROX1表达。④eHSP90促进晶状体囊袋残留上皮细胞分化的结构域为M结构域。因此，晶状体囊袋残留上皮细胞上调并分泌HSP90，eHSP90诱导晶状体上皮细胞向晶状体纤维细胞终末分化。重组HSP90蛋白是晶状体再生过程中潜在的新型分化调节因子。本研究有助于晶状体再生和治疗后发性白内障提供靶点和理论依据。