The Role of MicroRNAs in Corneal Epithelial Homeostasis

项目摘要/摘要 眼睛的前表面起到了阻挡外部环境的作用，保护脆弱的 损伤的潜在结构，部分是通过阐述角膜缘和角膜上皮来实现的。它是好的- 接受角膜缘上皮是角膜上皮干细胞的位置，这对 维持角膜上皮；然而，关于角膜缘如何修复的主要问题仍未解决 上皮细胞是受调节的。MicroRNAs(MiRNAs)是一类主要的调节分子，是 RNAi沉默机器。我们证明miR-184是最丰富的角膜上皮miRNA， 而miR-184具有显著的血管抑制特性，这具有极好的生物学意义，因为 角膜维持无血管状态。令人惊讶的是，鉴于miR-184在角膜缘/角膜上皮中的显著表达 生物学上，人们对其调控知之甚少。我们最近的证据表明miR-184是负调控的 由Ephin-A3(EFNA3)，Eph/Eaffin受体酪氨酸激酶家族的成员。EFNA3，主要是 仅限于角膜缘上皮基底层细胞。有趣的是，EFNA3的过表达显著降低了miR-184 级别。此外，另一种角膜偏好的miRNA miR-210靶向EFNA3，这可能间接地 维持角膜上皮细胞miR-184水平。角膜缘上皮中miR-210的缺失可能是 角膜缘上皮EFNA3的表达。我们的假设是EFNA3/miR-210轴对 MIR-184，支持适当的角膜缘血管，这是干细胞利基的一个重要组成部分；相对 研究不足的地区。为了验证这一假说，我们将调节水下的miRNA和目标蛋白水平。 培养人角膜缘和角膜上皮细胞及人微血管内皮细胞。我们将评估 这种miRNA和蛋白质调节的功能后果结合了生化， 分子生物学、细胞生物学和生理学方法。这项建议的另一个目标是实现 MiRNAs作为影响疾病组织的治疗干预措施的无限潜力。初步数据 表明一种新型的高密度脂蛋白-纳米颗粒(NP)可以将具有功能的miRNAs输送到 人角膜上皮细胞。令人兴奋的是，当局部应用Cy-3-Tage高密度脂蛋白-NP溶液进行休息时 小鼠角膜，Cy-3标记的高密度脂蛋白-纳米粒位于角膜上皮基底部和翼部的细胞质中 细胞。我们建议重点研究miR-HDLNPs如何在体内影响角膜缘/角膜上皮的生物学。至 为此，我们将使用糖尿病小鼠作为受损的角膜上皮伤口愈合和修复的模型 局部使用高密度脂蛋白-NP结合的miR-205，促进迁移的miRNA。我们还将使用缺少 血管紧张素转换酶2(ACE2)作为慢性角膜炎症(如细菌性角膜炎或 干眼症)，并局部使用高密度脂蛋白-NP结合的miR-146a，一种抗炎miRNA。最终我们的 研究将为提供：(I)特定miRNAs或其靶标的抑制剂；或(Ii)miRNAs提供基础 适用于患有影响眼球前上皮的疾病的患者。

PROJECT SUMMARY/ABSTRACT 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. It is well- accepted that the limbal epithelium is the site of the corneal epithelial stem cells, which are crucial for maintaining the corneal epithelium; however, major questions remain unresolved concerning how the limbal epithelium is regulated. microRNAs (miRNAs) are a major class of regulatory molecules that are part of the RNAi silencing machinery. We demonstrated that miR-184 was the most abundant corneal epithelial miRNA, and that miR-184 had marked angiostatic properties, which makes excellent biological sense as it is vital that the cornea maintains avascularity. Surprisingly, given the prominence of miR-184 in limbal/corneal epithelial biology, little is known about its regulation. Our recent evidence suggests that miR-184 is negatively regulated by Ephrin-A3 (EFNA3), a member of the Eph/ephrin receptor tyrosine kinase family. EFNA3, is primarily restricted to limbal epithelial basal cells. Interestingly, overexpression of EFNA3 significantly reduced miR-184 levels. Furthermore, another corneal-preferred miRNA, miR-210, targets EFNA3, which could indirectly maintain miR-184 levels in the corneal epithelium. The lack of miR-210 in the limbal epithelium likely accounts for limbal epithelial EFNA3 expression. Our hypothesis is that a EFNA3/miR-210 axis negatively regulates miR-184, which enables proper limbal vascularity, an essential component of the stem cell niche; a relatively understudied area. To test this hypothesis, we will modulate miRNA and target protein levels in submerged cultures of human limbal and corneal epithelial cells and human microvascular endothelial cells. We will assess the functional consequences of such miRNA and protein modulations with a combination of biochemical, molecular biological, cell biological and physiological approaches. Another goal of this proposal is to realize the unlimited potential of miRNAs as therapeutic interventions to affect diseased tissues. Preliminary data indicates that a novel high density lipoprotein (HDL)-nanoparticle (NP) can deliver functional miRNAs into human corneal epithelial cells. Excitingly, when a Cy-3-taged HDL-NP solution was applied topically to resting mouse corneas, the Cy-3-tagged HDL-NPs were detected in the cytoplasm of corneal epithelial basal and wing cells. We propose to focus on how miR-HDL-NPs affect the biology of limbal/corneal epithelia in vivo. To accomplish this, we will use diabetic mice as a model of compromised corneal epithelial wound healing and topically treat with a HDL-NP conjugated miR-205, a pro-migration miRNA. We will also use mice lacking angiotensin converting enzyme 2 (ACE2) as a model of chronic corneal inflammation (e.g., bacterial keratitis or dry eye) and topically treat with a HDL-NP conjugated miR-146a, an anti-inflammatory miRNA. Ultimately our studies will provide a foundation for delivery of: (i) inhibitors of specific miRNAs or their targets; or (ii) miRNAs to patients with diseases that affect the ocular anterior epithelia.