The Role of MicroRNAs in Corneal Epithelial Homeostasis

描述（由申请人提供）：该项目的长期目标是了解microRNAs （miRNAs）在调节角膜和角膜缘上皮中的作用。mirna是一类内源性小rna，作为翻译后抑制因子，已被证明在细胞发育、分化、干细胞调控和癌症进展中发挥作用。尽管这些调控rna很重要，但很少有人关注哺乳动物眼前段的mirna特征。我们已经证明microRNA- 184 （miR-184）是特殊的，因为它是角膜上皮中最丰富的miRNA，而在角膜缘或结膜上皮中不表达。MiR-205是角膜上皮中第二丰富的miRNA，在整个前节上皮以及许多其他分层鳞状上皮中均有广泛的表达。最重要的是，我们的初步数据表明，miR-205通过与角膜首选的miR-184相互作用，经历了一种独特的调节形式。这是第一次证明一个miRNA作为另一个miRNA的拮抗剂发挥主要作用，并突出了角膜上皮的独特性。我们成功地鉴定了脂质磷酸酶SHIP2是miR-205的靶标之一，并观察到miR-205的功能可以增强细胞存活并改变细胞骨架组织。鉴于这两个过程对角膜上皮稳态的重要性，我们建议研究miR-205在角膜上皮中的独特调控的功能意义。我们还将验证我们的假设，即干细胞富集的角膜缘基底细胞具有独特的miRNA特征。为此，我们重点研究了调节角膜上皮细胞中miR-184和miR-205的水平将如何：(i)影响它们的生存和生长能力，并确定这些事件是否通过SHIP2起作用；(2)影响其粘附和迁移能力。我们还将定义唇缘基底细胞的miRNA表达模式，并确定那些唇缘上皮独有的miRNA的靶标。为了实现这些目标，我们将利用我们使用拮抗剂下调特定miRNA的能力，并结合生化、分子生物学和细胞生物学方法评估这种miRNA调节的功能后果。利用激光捕获显微解剖，我们将分离相对纯净的角膜缘和角膜上皮基底细胞群体，并通过微阵列分析、qRT-PCR和原位杂交确定其miRNA表达模式。这些研究的信息将为深入了解角膜富集mirna如何影响细胞存活、生长、迁移和粘附提供信息。这些事件对角膜上皮的稳态和伤口的适当修复至关重要。我们提出的研究也将有助于更好地理解控制干细胞维持的调控mirna。

DESCRIPTION (provided by applicant): The long term goal of this project is to understand how microRNAs (miRNAs) function in regulating the corneal and limbal epithelia. MiRNAs are a family of endogenous, small RNAs that acting as post-translational repressors, have been shown to function in cell development, differentiation, stem cell regulation and the progression of cancer. Despite the importance of these regulatory RNAs, little attention has been directed towards characterizing miRNAs in the mammalian ocular anterior segment. We have shown that microRNA- 184 (miR-184) is special in that it is the most abundant miRNA in the corneal epithelium and is not expressed in the limbal or conjunctival epithelia. MiR-205, the second most abundant miRNA in the corneal epithelium exhibits a broad range of expression throughout the anterior segmental epithelia as well as in many other stratified squamous epithelia. Most importantly, our preliminary data indicates that miR-205 undergoes a unique form of regulation through an interaction with the corneal-preferred miR-184. This is the first demonstration that one miRNA plays a major role as an antagonist of another and highlights the distinctiveness of the corneal epithelium. We successfully identified the lipid phosphatase SHIP2 as one of the targets of miR-205 and observed that miR-205 functions to enhance cell survival and alter cytoskeletal organization. Given the importance of these two processes to corneal epithelial homeostasis, we propose to investigate the functional significance of the unique regulation of miR-205 in corneal epithelium. We will also test our hypothesis that the stem cell-enriched limbal basal cells have a distinct miRNA signature. Towards these ends, we focus on how modulating miR-184 and miR-205 levels in corneal epithelial cells will: (i) affect their survival and growth capabilities and determine whether these events are acting through SHIP2; and (ii) affect their adhesive and migratory abilities. We will also define the miRNA expression patterns of the limbal basal cells and identify targets of those miRNAs that are unique to the limbal epithelium. To accomplish these goals we will capitalize on our ability to downregulate specific miRNAs with antagomirs and assess the functional consequences of such miRNA modulation with a combination of biochemical, molecular biological and cell biological approaches. Using laser capture microdissection, we will isolate relatively pure populations of limbal and corneal epithelial basal cells and define their miRNA expression patterns by microarray profiling, qRT-PCR and in situ hybridization. Information from these studies will provide insight into the how the corneal- enriched miRNAs affect cell survival, growth, migration and adhesion. These events are critical for corneal epithelial homeostasis as well as proper wound repair. Our proposed studies will also lead to a better understanding of the regulatory miRNAs that govern stem cell maintenance.