DNA甲基化介导的miR-675、193a、485表达改变在年龄相关性白内障发病中的作用机制研究

Age-related cataract (ARC) is the leading cause of blindness in the world.The study of epigenetic in the pathogenesis of ARC is just start. Our previous study showed that DNA methylation of promoter of microRNA (miR)-675、193a、485 genes detected by HumanMethylation450 Beadchip (Illumina) was significantly decreased in the lens epithelia of ARC patients compared to age matched controls. The expression of miR-675, 193a, 485 was significantly increased in the lens epithelia of ARC patients compared to controls. All the three miRNA tageted CRYAA gene. The CRYAA gene was demonstrated by our previous work to be of great importance in the pathogenesis of ARC. Accordingly, we proposed the hypothesis that "With aging, DNA methylation of promoter of miR-675, 193a, 485 genes is decreased and the binding of the transcription factors is increased in the lens epithelia of ARC cases vs. age matched controls. These lead to the up-expression of these miRNAs and down-expression of their target gene CRYAA. DNA methylation mediated expression change of miR-675, 193a, 485 plays an important role in the pathogenesis of ARC." This study will use pyrosequencing and qRT-PCR technique for verification in large numbers of clinical samples. Furthermore,cell line and tissue model over-expressing or down-expressing miRNA will be constructed. Influence of promoter DNA methylation on promoter efficiency and transcription factor binding efficiency will be studied in transcriptional level by Dual Luciferase Reporter Gene Assay and Electrophoretic Mobility Shift Assay (EMSA). Biological phenotype of lens epithelial cells and expression of target CRYAA gene of miR-675、193a、485 will be detected in molecular level, cellular level and tissue level. The aim of this study is to detect the association between DNA methylation-mediated expression change of miR-675, 193a, 485 and the risk of ARC, to clarify the function of those miRNAs, and to provide a theoretical basis of the epigenetic pathogenesis of ARC.

年龄相关性白内障（ARC）是全球首位致盲眼病。本项目组前期芯片筛选发现在ARC发病中microRNA(miR)-675、193a、485的表达增加伴随DNA甲基化程度降低；同时这3个miRNA均靶向调节CRYAA基因；而CRYAA基因在ARC的发病中发挥着重要作用。据此提出"随着年龄增长，miR-675、193a、485基因启动子区DNA甲基化程度降低，转录因子结合增加，导致这些miRNA表达增加，靶基因CRYAA表达下降，出现白内障"的假说。本研究拟进行大样本临床验证，通过荧光素酶报告基因和凝胶电泳迁移实验研究DNA甲基化介导miRNA表达改变的机制，利用细胞和组织模型检测miRNA表达量改变对靶基因及抗氧化应激和抗凋亡等生物学表型的影响。以阐明DNA甲基化介导的miR-675、193a、485表达改变在ARC发生发展中的作用，为深入研究ARC的表观遗传发病机制提供新的思路和实验依据。

年龄相关性白内障（Age related cataract, ARC）是世界范围内的首位致盲性眼病，手术是其唯一有效的治疗途径。ARC的发病机制尚未明确，因此深入研究其发病机理，具有重要意义。本项目组深入研究了受DNA甲基化调控的miR-675及miR-34a通过靶向关键基因，调控人晶状体上皮细胞（Human lens epithelial cells, HLECs）的凋亡、增殖、迁移等生物学表型，并揭示了长链非编码RNA在其中的重要作用，从而介导ARC的发生发展。首先研究了受DNA甲基化调控的microRNA的表达改变和功能验证，结果表明人晶状体上皮细胞中miR-34a是通过与Notch1和Notch2 3'UTR结合，靶向抑制其转录，Notch2和N2-ICD的降低引发线粒体功能障碍和氧化应激，导致HLECs的凋亡，参与ARC的发病机制。第二，在转录水平进行了DNA甲基化介导的CRYAA基因在核性ARC中作用机制研究。CRYAA基因启动子区的DNA高甲基化影响了基因mRNA及蛋白水平的表达，CRYAA基因的DNA甲基化通过直接影响基因与转录因子SP1的结合而抑制基因转录, DNA去甲基化药物Zebularine在体外可以有效上调HLECs中CRYAA基因的表达，并且呈现时间和浓度依赖性改变。第三，在原课题假说基础上创新性地研究了H19/ miR-675/ CRYAA作用环路在核性ARC中的作用。最新研究进展表明，长链非编码RNA（long non-coding RNAs, lncRNAs）可与microRNA发挥协同作用调控ARC的发生发展，通过筛选和临床验证，发现lncRNA H19在核性ARC中的表达显著升高，且H19的表达水平与核性ARC的严重程度分级正相关。H19下调后调控了HLECs在氧化应激下的凋亡、增殖和迁移。H19的外显子1编码miR675，而miR675靶向关键的CRYAA基因，H19/ miR-675可下调CRYAA的表达，miR-675还可调控HLECs的功能，说明H19/ miR-675/ CRYAA调控环路在ARC发生发展中发挥了重要作用。该项目的施行，为深入研究ARC的表观遗传学发病机制提供新的思路和实验依据。