miR-696与Connexin50基因间的调控对晶状体缝隙连接活性及晶状体发育的影响研究

Connexin 50 (Cx50) gene is the hot pathogenic gene which related to congenital cataracts and microlens. Based on our former work, we do effort to investigate the regulation between miR-696 and Cx50 gene, and the influence of this regulation on lens opacity and lens development. In our former study, we have identified miR-696 would be affected by Cx50 gene knocking down in mouse lens epithelial cells. In this project, by using the methods of microRNA mimics or inhibitor, and patch clamps, we will confirm the regulation between miR-696 and Cx50 gene and the influence of miR-696 on activity of gap junction formed by Cx50. Meanwhile, by using RNA-seq and luciferase assays, we will search the downstream gene of Cx50 from the angle of miR-696. This project will reveal the influence of miR-696 on lens opacity, and explain the occurrence of microlens caused by Cx50 gene mutations. The study will play a potential role in congenital cataract gene therapy.

Connexin 50 (Cx50) 基因是导致先天性白内障及小晶体的热点致病基因。本项目拟在前期筛查出的Cx50基因上游microRNA（miR-696）基础上，针对miR-696与Cx50基因间的调控关系，以及该调控对晶状体混浊及发育的影响进一步深入研究。课题将应用小鼠原代晶状体上皮细胞及Cx50基因敲除小鼠模型，利用microRNA模拟或抑制、膜片钳等技术明确miR-696与Cx50基因间的调控，探究miR-696对细胞缝隙连接活性的影响，同时利用RNA-seq、双荧光素酶报告基因法，以miR-696为切入点，筛选Cx50下游晶状体发育相关基因及miR-696对该基因的作用靶点。课题将探索miR-696对晶状体混浊的影响，以及Cx50基因突变或缺失引起小晶体的具体机制，旨在对先天性白内障专一有效的靶向治疗打下理论基础。

白内障是我国常见的致盲性眼病，在表观遗传学被人们所认识之前，先天性白内障多被认为单基因遗传疾病，而年龄相关性白内障及高度近视性白内障通常被认为是多基因遗传疾病。而当2011年Hughes AE在北爱尔兰家系中发现miR-184种子区域一突变导致先天性前极性白内障合并圆锥角膜病后，对白内障的研究开始出现新的方向，即向表观遗传学拓展，而以microRNA为靶点的基因治疗也开始成为白内障治疗的新的研究方向。本研究在小鼠原代晶状体上皮细胞及Cx43基因抑制的小鼠原代晶状体上皮细胞模型中抑制及模拟miR-696，利用western blot、免疫荧光及膜片钳技术研究miR-696对Cx50基因的调控，得出阴性结果，在细胞水平上未见miR-696对Cx50基因的正向调控。本项目进行拓展性研究，研究microRNA在高度近视性白内障及年龄相关性白内障中所起的作用，利用高通量测序miRNAseq技术，发现miR-140-5p在高近性白内障囊膜中高表达，miR-140-5p的改变可能参与高度近视性白内障的发生。而miR-222在年龄相关性白内障发展过程中含量递增，其靶基因PTEN在硬核晶体上皮细胞中蛋白表达下降。研究强烈提示在晶状体中，miR-222可能通过靶基因PTEN对晶状体衰老进行调控。MicroRNA-222及PTEN基因对晶状体衰老影响的研究将成为今后的研究方向。