MiRNA-18b通过抑制IGF-1信号通路调控糖尿病性视网膜病变中血管内皮细胞功能的研究

Diabetic retinopathy(DR) is the main complication of diabetes mellitus(DM), and is one of the most leading causes of blindness world-wide. So DR is always the major research topics of ophthalmology. As we know, DR is a progressive microangiopathy characterized by small vessel damage and occlusion. The earliest pathologic change is thickening of the capillary endothelial basement membrane and reduction of pericytes. But the mechanism is complicated and not yet fully understood. MicroRNA is a newly discovered regulatory endogenous small RNA molecules which play a negative down-regulation role in functional protein. Several study have been shown that some specific microRNA high express in the lens, cornea and retina of the eye, and more than sixty kinds microRNA have been found in the retina. In our previous research, we have been discovered significant change of some microRNA in DR retinal tissue using gene chips which is along with the progression of DR. Our result under the previous National Natural Foundation useing miRNA microarray to screen the retina of DR model in mice and the endothelial cells by high glucose-stimulated show that the expression of miRNAs significantly changed, which of miRNA-18b(miR-18b) down-regulation significantly. We also found that miR-18b notable reduced 58% under high gulcose conditions compared with normal endothelium. We transfected miR-18b by cultured endothelial cells, and speculated after transfecting endothelial cell could resist damage from high glucose-stimulated. By constructing miR-18b sponge transgenic mice, we found that the mice were more susceptible to the DR injury. But the mechanism is unclear. Bioinformatics predict that insulin-like growth factor -1 (IGF-1) is one of the target genes of miR-18b which have been confirmed by the gene results. IGF-1 is highly expressed at retinal tissue which is involved in DR. This study intends to make the DR models in mice and high glucose-stimulated endothelial cell models. We want to carry out following three areas research below choosing miR-18b as the starting point: (1) Study the relationship between miR-18b and IGF-1 and the downstream signaling pathway IGF1R-PI3K-AKT then analyse the expression and activity of signaling pathway factor in the DR; (2)Using function experiments we want to clarify the mechanism of miR-18b role in IGF-1 and it's related genes; (3) In the DR mice model upwarding or downwarding miR-18b expression, to observe miR-18b effect. We expect to provide the experimental evidence and a new target for early treatment of DR through our works.

糖尿病性视网膜病变(DR)是眼科领域的重大研究课题。MiRNA在DR发生发展中的作用目前受广泛关注。申请者在前项国家自然基金资助下，利用miRNA芯片筛选出小鼠DR模型及高糖刺激内皮细胞模型表达显著改变的多个miRNA，其中miR-18b下调最显著。通过构建miR-18b sponge转基因小鼠，发现此小鼠更易发生DR损伤。生物信息学显示胰岛素样生长因子IGF-1是miR-18b的靶基因，并由荧光素酶报告基因证实。IGF-1在视网膜中表达上调，且密切参与DR发病。本研究拟构建小鼠DR模型及高糖致内皮细胞病变模型，研究miR-18b、IGF-1及下游通路表达和活性变化；通过病毒转染或反义序列特异性上调或下调miR-18b表达，观察该表达变化是否负性调控IGF-1蛋白表达及其下游信号通路;并观察miR-18b对IGF-1通路的调控如何影响内皮细胞功能活动及DR病变。为DR的早期干预提供新靶点。

研究背景：已有研究显示微小RNA与糖尿病视网膜病变相关。我们的前期研究也发现在高糖诱导的大鼠模型内其视网膜内皮细胞中许多微小RNA的表达发生了明显变化。.重要结果：体外高糖培养条件下，人视网膜内皮细胞的增殖明显增加，而miR-18b的表达明显降低。通过过表达miR-18b后可以视网膜内皮细胞的增殖，同时发现miR-18b作用于IGF1，同时抑制IGF1-IGF1R的下游信号通路如PI3K和MAPK信号通路，从而抑制视网膜内皮细胞的增殖以及血管内皮细胞生长因子VEGF的表达。此外，建立了小鼠氧诱导的新生血管病变的小鼠模型和链脲佐菌素诱导的小鼠糖尿病视网膜病变模型，并且证明了过表达miR-18b后可以抑制视网膜血管内皮细胞的增殖。.科学意义：本研究为视网膜病变的发生机制提供了理论基础，为临床上视网膜病变的治疗奠定基础。