MiR-212/203调控糖网病神经节细胞凋亡的作用及机制研究

MiRNAs consist of small, noncoding RNAs, inducing translational repression/ degradation of mRNA and attenuating protein translation. Evidence has uncovered the critical role of miRNAs in apoptosis of retinal ganglion cells (RGCs) in diabetic retinopathy (DR). In our preliminary experiment, there is evidence that miR-212 and miR-203 target specific mRNAs for regulating the progression of DR. The functionality of miR-212 and miR-203 lies in its contribution to regulating apoptosis in several diseases. To define the role of miR-212 and miR-203 in apoptosis of RGCs in DR and to understand the molecular mechanism further, we will give an analysis from the following aspects through Dual-luciferase assay system and transgenic technology: first, we will investigate whether miR-212 and miR-203 could regulate apoptosis of RGCs in DR; second, we will investigate whether the expression of target gene p300, slit2 and p300/p53, PI3K/Akt, slit-robo signalling pathway are involved in miR-212's suspension or abrogation of apoptosis in RGCs, meanwhile, we will focus on mechanisms of brain derived neurotrophic factor in DR, which select the upstream signaling pathway regulating the expression of miR-212 as the cut in point; third, we will investigate whether miR-203 could mediate the processe of apoptosis in RGCs by controling the expression of target gene socs3, socs6 and STAT3, PI3K/Akt signalling pathway. This project will illuminate molecular mechanism of miR-212 and miR-203 in the pathogenesis of DR by biological methods and provide experimental evidences for potential value in prevention and treatment of DR.

MiRNAs是一种非编码小RNA，多个miRNAs可能参与糖尿病视网膜病变（DR）神经节细胞（RGC）凋亡调控。我们前期研究发现miR-212和-203在DR发病过程中有显著差异表达。MiR-212和-203在众多疾病中有凋亡调控作用，但对DR的RGC凋亡调控及机制尚不清楚。本课题拟应用转基因及荧光素酶报告等分子生物学技术研究:1.验证miR-212和-203是否对DR的RGC具有凋亡调控作用；2.miR-212是否通过靶基因p300、slit2及p300/p53、PI3K/Akt、slit-robo信号通路调控DR的RGC凋亡并观察BDNF是否通过miR-212对DR的RGC有保护作用；3.miR-203是否通过靶基因SOCS3和SOCS6分别经JAK/STAT3及PI3K/Akt信号通路调控DR的RGC凋亡。本课题拟从体内外揭示单个miRNA的分子机理，为临床治疗寻找有价值的作用靶点。

糖尿病性视网膜病变(diabetic retinopathy，DR）是糖尿病的主要微血管并发症，也是成年人群致盲及视力损害的世界性首要眼病。随着近年的研究不断深入，目前研究发现视网膜神经退行性病变是DR早期重要的病理改变。本研究前期研究中利用microRNA芯片技术比较STZ和正常对照大鼠组中microRNA表达的差异，发现17个差异microRNA，通过RT-PCR体外验证STZ组中miR34、miR203、miR410、miR216上调，miR212下调。有研究表明miR212/203在众多疾病中有凋亡作用，但在DR中的凋亡调控及机制尚不清楚。本研究通过氯化钴刺激原代培养 RGCs 细胞及RPE细胞及STZ鼠和OIR大鼠模拟糖尿病的体内体外缺血缺氧环境，发现糖尿病环境下RGCs 上的miRNA212表达减低，RGCs 和RPE上的miR203表达上调，RGCs中表达降低的miRNA212可通过结合其靶基因annexin A4调控下游NF-κB信号通路增加细胞的凋亡；而RPE中表达增加的miR-203可通过结合其靶基因SOCS-3调控RPE的凋亡，本研究揭示了 miR212/203在 DR 发病中的作用机制，为DR的临床治疗提供一定的理论依据。