The Role of MicroRNAs in Corneal Epithelial Homeostasis

描述(申请人提供)：眼睛的前表面起到了对外部环境的屏障作用，并保护脆弱的潜在结构免受伤害，部分是通过对角膜缘和角膜上皮的精致加工。作为自我更新的组织，这些上皮细胞由干细胞调控，在组织内稳态、再生、移植、基因治疗以及几种前眼表疾病的发病机制中起着至关重要的作用。对于正常的视力来说，同样重要的是角膜的透明度，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部位，然而，关于角膜缘上皮如何调节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不完整。MicroRNAs(MiRNAs)是RNAi沉默机制中的一类主要调节分子。虽然一些研究针对的是破译miRNAs在角膜上皮中的作用，但对干细胞丰富的角膜缘上皮中的miRNA签名知之甚少。我们最近发现miRs-103/107是角膜缘首选的。此外，我们有证据表明miRs-103/107能够确保角膜缘上皮细胞与细胞的正确接触、自噬以及对细胞周期停滞的影响。直到最近，人们还认为miR-184是最丰富的角膜上皮miRNA，其功能是减弱miR-205，从而确保细胞的正常迁移和细胞存活。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。正确的视力需要稳定的角膜缘上皮和透明的角膜；因此，我们建议重点研究：(1)miRs-103/107在确保角膜缘上皮完整性方面的作用；(2)miR-184如何发挥作用以维持角膜无血管。为了实现这些目标，我们将利用我们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培养中阐明miRNA目标蛋白和调节miRNA和目标蛋白水平的能力。我们将操纵这些培养的细胞形成3D器官型木筏或内皮管，以模拟体内组织。我们将结合生化、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。通过专注于这些miRNAs及其靶蛋白的生物学研究，我们的提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；以及(Ii)是什么有助于维持角膜的无血管。这些知识与干细胞生物学和临床上的体外角膜上皮移植相关。这一建议还具有临床意义，不仅仅是角膜无血管，还可能影响病理性视网膜血管生成。最终，我们的研究将为开发创新的治疗方案提供理论基础，重点是使用：(I)特定miRNAs的抑制剂或其靶点；或(Ii)在患有影响眼前段上皮细胞的疾病的患者中传递miRNAs。

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.