LncRNA对年龄相关性白内障氧化损伤基因表观遗传和microRNA的调控及干预研究

Our previous studies, sponsored by The Natural Science Foundation of China, reported that the ARC formation was correlated with the DNA-microRNA binding site sequence variation and epigenetics changes of DNA oxidative damage related genes (ODGs). However, we do not study on the translational regulation and intervention of ODGs. We hypothesis that lncRNAs could regulate the ODGs expression before transcription and post transcription in lens of ARC patients. The lncRNAs can cause the diseases which have a closed relationship with other age related diseases. In current research, we use ARC patients, mouse and zebrafish as the object of study. At first, we detect the expression of lncRNAs in lens samples of ARC. Then the different expression lncRNAs (fold changed >1.5) will be confirmed by qRT-PCR. Next, we’ll transfect the lncRNA mimics, repressors and control plasmid to lens epithelial cells (LECs) to investigate the change of expression of ODGs and the cellular phenotypes. The FISH is used to detect the intracellular location of lncRNAs. We also detect the DNA methylation status and histone modification of ODGs. Using dual-luciferase reporter gene, we’ll detect the regulated roles of lncRNAs and microRNAs for ODGs expression. Finally, we’ll use the intraocular lncRNA injection to zebrafish ODGs knockout model to observe the molecular levels and pathological changes in the zebrafish lens. The changes will help us to confirm the relationship between the lncRNAs and phenotypes of zebrafish lens and to elucidate the pathogenesis of ARC. This study will give us a new insight to prevent the different types ARC by the targeted lncRNA which play a vital role for the precision medicine of ARC patients.

我们之前的年龄相关性白内障（ARC）国自然课题研究发现氧化损伤基因（ODGs）microRNA 结合位点等DNA突变、表观遗传与ARC的发生有关，但未探讨其调控及干预。今假设与全身病密切相关的lncRNA也可在ARC患者ODGs转录前、转录后调控表达，引起ARC。我们拟以ARC患者、小鼠、斑马鱼为对象，先在患者晶状体中筛选出表达差异明显的lncRNA；然后转染lncRNA模拟物、阻遏物和对照质粒至晶状体上皮细胞，观察该干预对ODGs的调控及表型影响。同时通过FISH实验定位lncRNA调控，检测ODGs甲基化、组蛋白修饰等表观遗传改变，应用双荧光素酶实验确定lncRNA、microRNA对ODGs表达的调控。再通过敲除ODGs的斑马鱼眼内注射lncRNA，观察其晶状体的分子、病理改变，明确lncRNA对白内障表型的干预。以阐明ARC的发病机制，为设计以lncRNA为靶点的精准治疗奠定基础。

年龄相关性白内障（ARC）是全球首位的致盲性眼病，其病因及发病机制仍不明确。本项目采用病例对照研究，通过高通量测序检测在年龄相关性白内障组（6例）及对照组（6例）差异表达的lncRNA，利用Cytoscape预测相应的靶基因，然后在60例年龄相关性白内障患者和20例对照组患者中进行差异lncRNA及靶基因的验证；同时构建lncRNA过表达慢病毒转染入细胞系，检测细胞学变化以了解lncRNA的功能。采用FISH技术检测lncRNA在细胞中的位置，利用miRdb和targetscan预测可能相关的miRNA，并通过双荧光素酶报告基因验证miRNA，lncRNA，靶基因间的调控机制；再通过CRISPR/Cas9基因编辑技术敲除AB品系斑马鱼氧化损伤基因（ODGs）基因，然后分别在突变体和野生型斑马鱼前房内注射低浓度过氧化氢，筛选出显著差异表达并且靶基因为ODGs的lncRNA。通过慢病毒转染过表达/敲降挑选出的lncRNA，观察其晶状体的分子、病理改变，明确lncRNA对白内障表型的干预。结果发现：在两组中有231个差异表达的lncRNA，其中49个在ARC组中表达下调，182个表达上调；lncRNA PLCD3-OT1、GPX3-AS、H19、NONHSAT143692.2、MEG3分别调节氧化损伤修复基因PLCD3、GPX3、TDG、OGG1、TP53INP1，可能与ARC的发生发展相关；这些lncRNA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14篇，总影响因子49.752，中文文章9篇，参加国家级会议7次。