miR-183/96敲除对小鼠视网膜结构和功能的影响及其作用机制研究

MicroRNAs are a class of non-coding regulator small RNAs associated with various human diseases. miR-183 cluster includes miR-183/96/182 which are located in the nearby genomic position and have very similar root sequence regulating target genes, and are highly expressed in the retina. Previous studies suggested that the miR-183/96/182 triple knockout lead to severe retinal degeneration in mice. However, our previous study implicated that targeted deletion of miR-182 did not lead to obvious retinal degeneration, indicating that miR-183/96 play an important role in maintaining the structure and function of retina. Based on these evidences, we generated miR-183/96 double knockout mouse model and the purpose of this study is to elucidate whether and to what extent miR-183/96 knockout mice display retinal degeneration, to identify the direct target of miR-183/96 in retina and to rescue the diseased retina. Our studies will elucidated the mechanism of miR-183/96 in retina and provide new insights miRNA-related retinal degeneration.

微小RNA是一类内源性的具有调控功能的非编码小RNA，与疾病的发生发展密切相关。miR-183簇包含miR-183/96/182三种小RNA，它们在基因组上位置邻近，并且调控序列相似，同时均在视网膜高丰度表达，其表达异常会导致明确的视网膜变性。既往研究表明miR-183/96/182三联敲除会引起严重的视网膜变性。我们前期研究发现，单独敲除miR-182不会引起明显的视网膜病变。这提示miR-183/96可能对维持视网膜结构与功能十分关键，因而构建了miR-183/96双重敲除小鼠。本项目拟通过监测miR-183/96敲除小鼠在不同发育阶段中视网膜结构和功能的变化；寻找并验证miR-183/96对视网膜进行调控的直接靶基因及逆转由miR-183/96敲除导致的视网膜病变。进一步阐明miR-183/96在视网膜中的调控机制，为miRNA在视网膜变性疾病中的分子致病机理提供一种新的科学依据。

miR-183簇（miR-183/96/182）在视网膜发育和功能中起着重要的调控作用。miR-182敲除小鼠并没有表现出明显的视网膜形态和功能受损，而单纯的miR-183/96是否对视网膜结构和功能的影响目前还未曾报道。本课题利用miR-183/96敲除小鼠研究了其对视网膜的调控作用。结果表明miR-183/96敲除小鼠光感受器细胞的成熟及其功能受到了明显损伤。首先，我们可以看到在敲除小鼠中视锥细胞核迁移受到影响；光感受器细胞的外节变短核层数减少；明场视网膜电波ERG呈熄灭状，而暗场的视网膜电波ERG呈明显的降低趋势；其次，我们预测并验证出miR-183和miR-96的共同靶点Slc6a6；与野生小鼠相比，敲除小鼠中slc6a6的蛋白水平明显升高；同时，slc6a6蛋白水平在通过AAV注射过表达miR-183或miR-96的小鼠中明显降低。最后我们发现过表达或降低slc6a6的表达均会对小鼠视网膜光感受器细胞产生毒性，使其视网膜电波ERG明显降低。我们证实了slc6a6对于维持视网膜的发育和功能是不可或缺的，进一步证实了作为重要的表观调控因子，miR-183和miR-96在视网膜中的重要调控作用。