长链非编码RNA MIAT调控Connexin50 表达影响晶状体缝隙连接通道的功能及其机制研究

Cx50 gene is the hot pathogenic gene which related to congenital cataracts and microphthalmia. We found in previous research that lncRNA MIAT ,which was specifically upregulated in the whole blood and aqueous humour of cataract patients,could affect the proliferation, apoptosis and migration of HLECs.In our former study, we have identified the correlation between MIAT and Cx50 by bioinformatics analysis and RNA pull down.Based on our work, we will do effort to investigate the regulation between MIAT and Cx50 gene, and the mechanism of MIAT mediating the expression of Connexin 50 to induce lens opacity.In this project,by using the methods of overexpression and silence of lncRNA，stem-loop RT-qPCR，we will confirm the dialogue between MIAT and Cx50 gene and the influence of MIAT on activity of gap junction formed by Cx50.Meanwhile, by using gene chips, Deletion-Mapping，we will screening the MIAT targeted genes, explore the mechanism of regulation between MIAT and Cx50,and then explain the occurrence of cataract and microphthalmia caused by Cx50 gene mutations.This project will identify the up-stream lncRNA of Cx50, and reveal the role of MIAT in Cx50 gene network, moreover may provide new insight towards congenital cataract gene therapy.

Connexin50（Cx50）基因是先天性白内障及小晶体的热点致病基因。文献报道lncRNA MIAT在白内障患者中特异性高表达，影响晶状体上皮细胞的分化、凋亡与迁移。课题组前期已证实MIAT与Cx50间存在较强调控相关性。为进一步明确MIAT与Cx50基因间的调控作用，阐明MIAT介导Cx50参与晶状体浑浊发生的机制，本项目拟在细胞水平，利用lncRNA过表达及沉默、茎环RT-qPCR等技术研究MIAT与Cx50基因间的调控关系及MIAT对晶状体缝隙连接通道的影响。同时，在动物水平利用基因芯片、Deletion-Mapping筛选MIAT靶基因及作用位点，探索MIAT对Cx50基因的调控机制，揭示Cx50基因突变引起白内障及小晶体的分子机制。本项目旨在探索Cx50基因上游lncRNA，并揭示MIAT在Cx50基因网络中的角色，为先天性白内障治疗确立新靶点提供科学依据。

白内障为全球首位的致盲性眼病，目前手术为唯一确定有效的治疗手段。有关晶状体表观遗传学的研究，推动了药物治疗白内障的进展。.缝隙连接蛋白基因是先天性白内障重要的致病基因。Connexin50（Cx50）基因突变或缺失可延缓晶状体纤维细胞成熟，下调晶状体上皮细胞增殖，导致小晶体及白内障发生。.本研究深入探索了Cx50上游非编码RNA的调控机制。在前期证实Cx50与MIAT间调控相关性的基础上，细胞水平通过MIAT过表达及沉默，蛋白印迹、染料弥散试验及膜片钳技术检测Cx50蛋白表达水平及Cx50缝隙连接活性，验证MIAT对Cx50基因的负向调控；组织水平鉴定MIAT下游靶基因，运用RNA-seq、siRNA、免疫荧光等方法明确MIAT介导miRNA-22-3p调控YAP，进而与Cx50发生相互作用，并探索了YAP核易位机制对Cx50缝隙连接的影响。本项目从多角度论证MIAT和Cx50基因间的调控作用，并揭示MIAT/miRNA-22-3p/YAP轴在晶状体浑浊发生过程中的角色。研究成果有望开拓基于非编码RNA的治疗策略，为白内障非手术治疗确立新的靶点提供科学依据。