The Role of MicroRNAs in Corneal Epithelial Homeostasis

描述（由申请人提供）：眼睛的前表面作为外部环境的屏障，保护脆弱的底层结构免受伤害，部分是通过精致的角膜缘和角膜上皮。作为自我更新的组织，这些上皮细胞由干细胞控制，在组织稳态、再生、移植、基因治疗和一些眼前表疾病的发病机制中起着至关重要的作用。对正常视力同样重要的是角膜透明度的需要，这是通过无血管来实现的。人们普遍认为角膜缘上皮是角膜上皮干细胞的所在地；然而，关于角膜缘上皮是如何调控的主要问题仍未解决。同样，我们对控制血管生成的因素的理解也是不完整的。microRNAS （miRNAs）是一类主要的调控分子，是RNAi沉默机制的一部分。虽然一些研究旨在破译miRNA在角膜上皮中的作用，但对富含干细胞的角膜缘上皮中的miRNA特征知之甚少。我们最近发现mir -103/107是边缘优先的。此外，我们有证据表明，miRs-103/107能够确保角膜缘上皮细胞的正常接触、自噬和影响细胞周期静止。直到最近，人们才认为，最丰富的角膜上皮miRNA miR-184可以减弱miR-205的作用，从而保证细胞的正常迁移和存活。我们现在有证据表明miR-184可能直接阻止角膜上皮血管生成。正常的视力需要稳定的角膜缘上皮和角膜清晰度；因此，我们建议关注：(1)mir -103/107在确保角膜缘上皮完整性中的作用；(2)miR-184如何维持角膜血管的功能。为了实现这些目标，我们将利用我们的能力来阐明miRNA靶蛋白，并在人角膜缘和角膜上皮角质形成细胞以及人微血管内皮细胞的深层培养中调节miRNA和靶蛋白水平。我们将操纵这些培养的细胞形成三维器官型筏，或内皮管，模仿体内组织。我们将结合生物化学、分子生物学、细胞生物学和生理学的方法来评估这种miRNA和蛋白质调节的功能后果。通过关注这些mirna及其靶蛋白的生物学，我们的建议代表了一种新的方法来理解：(i)角膜缘上皮是如何调节的；(2)是什么维持了角膜的血管畅通。这些知识与干细胞生物学和临床离体角膜上皮移植相关。这一建议也具有临床意义，而不仅仅是角膜血管，并可能影响病理性视网膜血管生成。最终，我们的研究将为创新治疗方案的开发提供依据，重点是：(i)特定mirna或其靶点的抑制剂；或（ii）影响眼前节上皮的疾病患者的mirna递送。

DESCRIPTION (provided by applicant): The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying structures from injury, in part, through the elaboration of the limbal and corneal epithelia. As self-renewing tissues, these epithelia are governed by stem cells, which play a crucial role in tissue homeostasis, regeneration, transplantation, gene therapy and in the pathogenesis of several anterior ocular surface diseases. Equally important for proper vision is the need for corneal transparency, which is achieved through avascularity. It is well-accepted that the limbal epithelium is the site f the corneal epithelial stem cells; however, major questions remain unresolved concerning how the limbal epithelium is regulated. Likewise, our understanding of factors that control angiogenesis is incomplete. microRNAS (miRNAs) are a major class of regulatory molecules that are part of the RNAi silencing machinery. While some studies have been directed towards deciphering the roles of miRNAs in the corneal epithelium, little is known about the miRNA signature in the stem cell-enriched limbal epithelium. We have recently discovered that miRs-103/107 are limbal-preferred. Furthermore, we have evidence that miRs-103/107 function to insure proper limbal epithelial cell-cell contact, autophagy and impact on cell cycle quiescence. Until recently, it was believed that miR-184, the most abundant corneal epithelial miRNA, functioned to attenuate miR-205, which insured proper cell migration and cell survival. We now have evidence that miR-184 may directly prevent corneal epithelial angiogenesis. Proper vision requires both a stabile limbal epithelial and corneal clarity; therefore we propose to focus on: (1 The roles of miRs-103/107 in assuring the integrity of the limbal epithelium and (2) how miR-184 functions to maintain corneal avascularity. To accomplish these goals, we will capitalize on our ability to elucidate miRNA target proteins and modulate miRNA and target protein levels in submerged cultures of human limbal and corneal epithelial keratinocytes and human microvascular endothelial cells. We will manipulate these cultured cells to form either 3-D organotypic rafts, or endothelial tubes, which mimic the in vivo tissues. We will assess the functional consequences of such miRNA and protein modulations with a combination of biochemical, molecular biological, cell biological and physiological approaches. By focusing on the biology of these miRNAs and their target proteins, our proposal represents a novel approach to understand: (i) how the limbal epithelium is regulated; and (ii) what contributes to the maintenance of corneal avascularity. Such knowledge has relevance to stem cell biology and clinically to ex vivo corneal epithelial transplantation. This proposal also has clinical implicatins beyond just corneal avascularity and may impact on pathological retinal angiogenesis. Ultimately our studies will provide rationales for the development of innovative treatment regimens focused on the use of either: (i) inhibitors of specific miRNAs or their targets; or (ii) delivery of miRNAs in patients with diseases that affect the ocular anterior segmental epithelia.