Importin α3蛋白在晶状体上皮细胞衰老中的作用及其表观遗传学调控机制的研究

Cataract is the leading cause of blindness worldwide and age-related cataract (ARC) is the most prevalent type. As ARC is a complex disorder caused by multiple risk factors, it is wise to investigate these factors and study mechanisms of the disease in order to design strategies for treatment and prevention. In the former work, we have successfully conducted a genome-wide association study (GWAS) analysis of ARC and identified KPNA4 as the new susceptibility gene for ARC. Here, we do effort to investigate the function of KPNA4 and its epigenetic regulation. With the model of senescent human lens epithelial cells in vitro, we will confirm the role of importin α3 in lens aging by using the methods of qRT-PCR, Western-Blot, overexpressive plasmid vector and RNA interference. In addition, gene chips, luciferase assays and miRNA Mimics will be used for detection of specific miRNA targeting 3'UTR of KPNA4. This may explain why the functional polymorphism at 3'UTR of the KPNA4 gene confers risk for ARC. If this project can be performed smoothly, it would reveal the mechanism of cell senescence and apoptosis induced by importin α3 and further improve our understanding of the molecular mechanism underlying lens opacity. Moreover, the specific miRNA and its binding site identified in this project would play a potential role in ARC prevention and gene therapy. As government invested a huge expediture on cataract surgery, our society and economy will benefit from the reducing incidence of this disease.

本项目拟在前期年龄相关性白内障人群中利用GWAS筛查和定位相关易感基因的基础上，对新发现的易感基因KPNA4的功能和表观遗传学调控机制进行研究。应用建立的人晶状体上皮细胞氧化损伤模型，利用qRT-PCR、Western Blot、过表达质粒载体、干扰RNA等技术首次探讨importin α3蛋白在晶状体上皮细胞衰老凋亡通路中的作用；同时采用基因芯片、双荧光报告载体、miRNA模拟体等方法，寻找调控importin α3蛋白表达的特异miRNA，为KPNA4基因3'端易感多态性位点提供表观遗传学解释。若该项目能顺利实施，将阐明importin α3蛋白启动细胞衰老和凋亡的机制，揭示晶状体混浊发生发展的分子机制，同时可以筛选出具有靶向作用的miRNA及其特异性作用靶点，为今后研制有效、专一的靶向防治打下基础，可望有效抑制和延缓全球首要致盲疾病年龄相关性白内障的发病，具有积极的社会和经济效益。

项目拟在前期年龄相关性白内障人群中利用GWAS筛查和定位相关易感基因的基础上，对新发现的易感基因KPNA4的功能和表观遗传学调控机制进行研究。本项目应用建立的人晶状体上皮细胞氧化损伤模型，顺利完成了对importinα3蛋白在晶状体上皮细胞衰老及凋亡中的功能及机制研究。研究发现importinα3蛋白是细胞内的转运蛋白，与晶状体上皮细胞衰老及凋亡的调控机制有相关作用。在importinα3蛋白与晶状体上皮细胞凋亡中的功能及机制研究中，运用质粒转染技术，过表达KPNA4质粒转染293T细胞后，设立正常组和过表达KPNA4基因组，通过Western-Blot、细胞免疫荧光等生物学技术检测该基因过表达引起的分子水平的影响及其与凋亡蛋白间的相互作用。并运用CRISPR/Cas9 技术敲除斑马鱼 KPNA4，即在 KPNA4 CDS 序列中设计 gRNA，通过 Cas9 敲除 KPNA4 CDS 片段，导致基因缺失突变，通过 mRNA 注射修复技术验证得到 KPNA4 特异性敲除的稳定斑马鱼系，为今后更深入的importinα3蛋白作用机制研究提供了有力的动物模型基础，并为今后研制有效、专一的靶向防治打下基础，可望有效抑制和延缓全球首要致盲疾病年龄相关性白内障的发病，具有积极的社会和经济效益。