PRMT3突变靶向p53/caspase-3介导的晶状体细胞增殖异常致先天性白内障机制研究

Gene mutation is the leading cause of congenital cataract which can cause blindness. However, there are still limitations in eye surgery for infants. The research of mutant gene and pathogenic function is expected to make a breakthrough in non-surgical treatment of congenital cataract. To date, more than 50 candidate genes of congenital cataract have been identified, however, high-throughput sequencing only covers 60% of them and a large number of unknown genes and pathogenesis remains to be revealed. The applicant identified a novel pathogenic gene, protein-arginine methyltransferases 3 (PRMT3) in an autosomal dominant congenital cataract pedigree with cortical and posterior polar opacity, and found that the heterozygous mutation of PRMT3 might be the cause of cataract in this pedigree. This project aims to further elucidated the correlation between PRMT3 mutation targeting p53/caspase-3 mediated abnormal lens cell proliferation and congenital cataract development, and demonstrate a brand-new pathogenesis of PRMT3 mutation and congenital cataract. Moreover, the project intends to explore the phenotypic rescue effect as well as screen other potential targets and signaling pathways regulated by PRMT3 in the lens. The obtained results will help to preliminary clarify the nature of life phenomena of congenital cataract induced by PRMT3 mutation, further providing insights into non-surgical intervention in the occurrence, development and prognosis of the disease. It can provide innovative ideas and theoretical basis for the transformation into non-surgical treatment schemes including gene diagnosis, prevention and treatment of congenital cataract.

基因突变是致盲眼病先天性白内障的主要病因，而对先白婴幼儿行眼部手术治疗尚存局限性，突变基因及致病功能研究，可望在先白非手术治疗上有所突破。现已定位明确的先白候选基因超过50个，但高通量测序仅覆盖60%，尚有大量未知基因及致病机制亟待揭示。项目申请人前期在一个常染色体显性先天性后极性伴皮质混浊家系中，鉴定了新致病基因蛋白质精氨酸甲基转移酶3（PRMT3）的突变，发现该基因点突变可能是该家系白内障发生成因。本项目拟进一步揭示PRMT3突变靶向p53/caspase-3介导的晶状体异常细胞增殖和先白发生的相关性，论证PRMT3点突变与先白形成的全新发病机制。并探索PRMT3突变表型拯救效应、筛选晶状体内PRMT3调控的其他可能靶点。初步阐明PRMT3点突变致先白发病的生命现象本质，可望尽早在先白发生、发展过程中加以非手术干预。为后续转化为对先白患者基因诊断和防治等非手术治疗提供新思路和理论依据。

先天性白内障新致病基因的筛选及功能研究是探索疾病成因的重要环节，对揭示先白发病机制、丰富人类基因组研究资料、为先白非手术治疗提供相关学术依据均具有重要意义。本项目申请人及研究团队基于临床工作，在先白临床家系中首次筛选并鉴定了新致病基因蛋白质精氨酸甲基转移酶3（PRMT3）与先白发病的相关性。利用HLE-B3细胞系，揭示了PRMT3敲减可通过下调p53及下游凋亡因子 caspase 3表达，导致上皮细胞异常增殖和迁移。并利用p53缺失的人肺腺癌细胞系H1299，阐明了p53/caspase 3对细胞凋亡的调控是与PRMT3的甲基化修饰相关。进一步采用CRISPR-Cas9技术成功建立了Prmt3基因敲除斑马鱼系，在动物水平阐明了Prmt3null纯合子突变体中，p53/caspase 3介导的异常细胞增殖可导致晶状体发育过程中去核化障碍、晶状体分化关键蛋白ZL-1、AQP0呈不规则点状分布于上皮及延迟分化的内部晶状体纤维中。并通过晶状体后极部转换区（Transition zone, TZ）、赤道部生发区（Germinative zone, GZ）、内部分化区细胞核数目进行定量统计，揭示了Prmt3null纯合子突变体中晶状体发育中上皮间质转化（EMT）稳态失衡，晶状体上皮和纤维组织方式异常，赤道部晶状体上皮细胞过度迁移进入后囊转换区，导致后极部纤维实质积累。以上证据证实了p53/caspase 3介导的细胞增殖异常对先白发生发展的关键事件去核化、晶状体分化关键蛋白表达及EMT的调控。本项目进一步反向论证，在Prmt3null纯合子突变体证实了可以PRMT3或p53/caspase 3为靶点进行表型拯救。本项目较全面地揭示了由PRMT3主导的晶状体细胞增殖调控异常，致先白形成的全新发病机制，在先白发病机制上有崭新的突破。研究成果大大丰富了先天性白内障的疾病谱和基因组研究资料，将为先白的基因诊断与非手术防治奠定重要理论基础和学术依据。

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