microRNAs及其靶基因遗传变异与湿性老年黄斑变性易感性的分子流行病学研究

Wet age-related macular degeneration (AMD) is a common blinding eye disease which severely compromises life quality of middle aged and elderly people. Previously,we predicted some wet AMD susceptibility genes are regulated by microRNA (miRNAs), using bioinformatics approach; and then found that 55 miRNAs were different expressed between wet AMD model cells and controls using Solexa sequencing technology, which indicates miRNAs might be involved in the disease. In this project, we intend to study the expression profiles of miRNA and mRNA in vitro and in vivo using Solexa sequencing technology. In combination with the results of bioinformatics analysis and dual-luciferase reporter assay system, candidate miRNAs and their target genes are obtained. A population-based case-control study is conducted, and the relationships between polymorphisms of the candidate miRNAs and their target genes and genetic susceptibility of wet AMD are analyzed after genotyping using matrix-assisted laser desorption/ionization time of flight mass spectrometry; and the key miRNAs and their target genes are determined. After establishing cell and animal models, we discuss the regulatory mechanism of the key miRNAs and their target genes for wet AMD using technologies of RNAi and over expression. This study will reveal the influence and the molecular mechanism of the key miRNAs and their target genes on the risk of wet AMD from multi-aspect, so as to provide theoretical and experimental basis for early diagnosis and individualized treatment for wet AMD, and the establishment of effective prevention strategies.

湿性老年黄斑变性（AMD）是一种严重影响中老年人群生活质量的常见致盲眼病。课题组前期经生物信息学发现一些已知湿性AMD易感基因受miRNAs调控；采用测序获得55个miRNAs在湿性AMD模型细胞中差异表达，提示其可能参与该病发生。本项目通过Solexa测序绘制离体-在体miRNA和mRNA表达谱，结合生物信息学分析和双荧光素酶报告基因系统验证，获得候选miRNAs及其靶基因；采用病例对照设计，通过飞行时间质谱进行基因分型，分析候选miRNAs及其靶基因多态性与湿性AMD遗传易感性的关联，确定该病关键miRNAs及其靶基因；应用RNAi和过表达载体，在细胞及动物水平探讨关键miRNAs及其靶基因在湿性AMD发生中的调控机制。本研究从不同层面系统揭示关键miRNAs及其靶基因对罹患湿性AMD风险的影响及分子机制，为湿性AMD的早期诊断、个体化治疗和有效预防策略的建立提供理论依据和实验基础。

作为一种多因素复杂疾病，AMD 的分子发病机制尚未阐明。目前研究表明 AMD 的发生与细胞信号网络调控紊乱密切相关，通过影响 AMD 相关信号通路中某些关键基因的表达，可能对降低该病罹患风险和减少其带来的危害具有重要意义；而 DNA 水平的基因突变和转录后水平的 miRNA 调控是影响基因表达的重要因素。然而目前针对 miRNAs 及其靶基因遗传变异对罹患AMD 风险的影响及分子调控机制尚不清楚。本研究发现：1）发现 miR-4443及 SREBP2、LDLR 等胆固醇代谢网络相关靶基因和 miR-195-5p 及其 PEX13、FGF2、WNT3A 等衰老相关靶基因可能参与 AMD 发生；2）明确胆固醇代谢网络相关基因的 ABCA1 rs1883025、LIPC rs10468017、LIPC rs493258、CETP rs3764261 多态性与 AMD 罹患风险相关，提示胆固醇代谢网络关键基因遗传变异可能与 AMD 发生有关。3）在细胞及动物水平，发现在AMD 发生过程中，SREBP2 的表达上升，调控的下游合成胆固醇的靶基因 LDLR、HMG-COA reductase、HMG-COA synthetase 改变，激活 NLRP3炎症小体，增加炎症因子的分泌，从而引起AMD的发生，从而阐明 miR-4443 及其靶基因 SREBP2、LDLR 等基因在 AMD 发生中的调控机制。本研究为ARM 治疗药物的研发提供理论和实验依据，为 AMD 干预策略与防治方案的建立提供新思路。相关研究成果以论文的形式发表 12 篇 SCI 收录期刊论文，研究期间指导博士后 2 名、博士 2 名和硕士生 13 名。