环状RNAcirc-MRE11A通过ATM/p53/p21通路介导晶状体上皮细胞衰老在年龄相关性白内障发病中的作用机制

Age-related cataract (ARC) formation is tightly related with cellular senescence of lens epithelial cells (LECs), but the mechanism of intermediated pathways remains to be determined. Recently, studies found the extraordinary function of circular RNAs (circRNAs) in gene regulation and their role in cellular senescence. Our preliminary data showed an upregulation of circ-MRE11A and oxidative damage repair gene ATM that is a gene of upstream of the p53/p21 signaling pathway in LECs of ARC. Bioinformatics analysis indicates circ-MRE11A could regulate ATM expression by hsa-miR-31-5p. Combined with two studies sponsored by The Natural Science Foundation of China, we hypothesize circ-MRE11A is involved in the regulation of ATM epigenetically and post-transcriptionally in the process of LEC senescence, which results the formation of cellular senescence. We will use ARC LECs, cell lines, Zebrafish and ARC mouse model to unveil the relationship between circ-MRE11A and ATM and the molecular mechanisms of circ-MRE11A in cellular senescence and ARC pathogenesis by FISH, RIP, RNA pull down and FR-EMSA. The results would provide a new insight to the disease mechanism and add a novel strategy for ARC precision medicine by targeting circRNAs.

年龄相关性白内障（ARC）的形成与晶状体上皮细胞（LEC）衰老密切相关，但机制未阐明。近期发现环状RNA（circRNA）有很强的基因调控功能并参与细胞衰老。我们研究发现circ-MRE11A和p53/p21信号通路上游ATM基因在ARC患者LEC中表达上调。生物信息学预测circ-MRE11A通过miR-31-5p调控ATM，结合我们前两个国自然课题推测其可能参与ATM转录前（表观遗传）和/或转录后（微小RNA）调控致细胞衰老。申请人拟以人白内障LEC、细胞株、基因敲除斑马鱼、ARC小鼠为三种研究对象。首先在人LEC中明确circ-MRE11A与ATM的关系，再在细胞株中通过FISH、RIP、RNA沉淀和FR-EMSA探明circ-MRE11A调控ATM的机制。最后动物模型眼内注射circ-MRE11A干预质粒，验证延缓ARC的可能。为设计延缓LEC衰老的ARC精准防治提供新的理论基础。

年龄相关性白内障(Age-related cataract ,ARC)是全球范围内主要的致盲性眼病。尽管其病因及发病机制仍不明确，但晶状体上皮（lens epithelial cells ,LECs）的氧化损伤引起的细胞衰老是其主要病理基础。本项目采用临床病例对照研究，通过高通量测序检测在ARC组(3例)及对照组(3例) LECs中表达差异的环状RNA（circRNA），我们发现circ-MRE11A在ARC患者LECs中表达增加，然后在30例ARC患者（皮质型、核型、后囊下型各10例）和10例对照组患者中进行了验证。采用荧光原位杂交（FISH）技术检测circ-MRE11A在细胞中的定位，同时Sanger测序和Northern Blot验证circ-MRE11A的序列及环状结构。并且发现在紫外线（UVB）照射下人类晶状体上皮细胞株（SRA01/04）中circ-MRE11A上调，随后敲除circ-MRE11A增强了细胞活力和细胞周期，而circ-MRE11A的过度表达则表现出相反的趋势。通过RNA免疫沉淀技术我们发现circ-MRE11A可以与UBXN1蛋白结合，使得氧化损伤修复基因ATM的过度激活，并启动ATM/p53/p21衰老信号通路，导致LECs细胞周期停滞和衰老。最后将过表达circ-MRE11A的重组腺相关病毒载体注射到年龄相关样小鼠玻璃体腔中。注射4周时，腺相关病毒载体编码的GFP蛋白可在小鼠晶状体中表达。注射后8周观察到注射组的LECs衰老和晶状体混浊。本项目的研究发现circ-MRE11A可能与ARC的发生发展相关，其主要机制是通过结合UBXN1过渡活化ATM而诱导LEC的衰老，最终导致ARC的发生。我们在本项目研究基础上，继续深入探讨circRNA的m6A甲基化修饰这一前沿热点，发现circRNA甲基化修饰参与其表达调控，是circRNA的上游调控机制。总之，该项目的研究发现circ-MRE11A调节氧化损伤修复基因的表达，进而影响其DNA的修复功能，可作为ARC防治的新靶点。已发表SCI论文8篇，总影响因子32.229，中文核心期刊文章1篇，参加国家级会议2次。