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)如何在调节角膜和角膜缘上皮中发挥作用。MiRNAs是一个内源性小RNA家族，作为翻译后抑制因子，已被证明在细胞发育、分化、干细胞调节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中miRNAs的特征。我们已经证明了microRNA-184(miR-184)的特殊之处在于它是角膜上皮中含量最丰富的miRNA，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，在前段上皮和许多其他复层鳞状上皮中都有广泛的表达。最重要的是，我们的初步数据表明，miR-205通过与角膜偏好的miR-184相互作用，经历了一种独特的调节形式。这是第一次证明一种miRNA作为另一种miRNA的拮抗剂发挥了重要作用，并突出了角膜上皮的特殊性。我们成功地将脂磷酸酶SHIP2确定为miR-205的靶标之一，并观察到miR-205具有提高细胞存活和改变细胞骨架结构的功能。鉴于这两个过程对角膜上皮细胞动态平衡的重要性，我们建议研究miR-205在角膜上皮细胞中独特调节的功能意义。我们还将测试我们的假设，即富含干细胞的角膜缘基底细胞具有明显的miRNA签名。为此，我们重点研究调节角膜上皮细胞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；以及(Ii)影响它们的黏附和迁移能力。我们还将定义角膜缘基底细胞的miRNA表达模式，并确定这些miRNAs的靶点是角膜缘上皮所特有的。为了实现这些目标，我们将利用我们通过反交配子下调特定miRNAs的能力，并结合生化、分子生物学和细胞生物学的方法来评估这种miRNA调控的功能后果。利用激光捕获显微切割技术，我们将分离相对纯净的角膜缘和角膜上皮基底层细胞群体，并通过基因芯片图谱、qRT-PCR和原位杂交来确定它们的miRNA表达模式。来自这些研究的信息将为深入了解富含角膜的miRNAs如何影响细胞生存、生长、迁移和黏附提供信息。这些事件对于角膜上皮细胞的动态平衡以及正确的伤口修复至关重要。我们提出的研究还将使我们更好地理解管理干细胞维护的调控miRNAs。 公共卫生相关性：这个项目的信息将影响我们对microRNAs如何调控重要细胞过程的理解，如角膜上皮细胞的存活、生长、黏附和迁移。这项研究的一个好处是使用microRNA抑制剂作为创新的治疗策略来促进损伤后的角膜上皮修复。这些研究还将有助于更好地了解调控角膜缘上皮干细胞行为的调节蛋白，从而最终有助于我们了解这些前体细胞如何维持角膜上皮。

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. PUBLIC HEALTH RELEVANCE: Information from this project will impact on our understanding of how microRNAs regulate vital cellular processes such as survival, growth, adhesion and migration in the corneal epithelium. A benefit from this research will be the use of microRNA inhibitors as innovative therapeutic strategies to promote corneal epithelial repair following injury. These studies will also lead to a better appreciation of the regulatory proteins that govern limbal epithelial stem cell behavior and thus ultimately contribute to our knowledge of how these progenitor cells maintain the corneal epithelium.