The Role and Mechanisms of microRNAs in Diabetic Cornea

描述(申请人提供)：糖尿病是发展中国家的主要健康挑战之一，对包括眼睛在内的不同器官有重大影响。虽然视网膜是糖尿病的主要眼球靶点，但50%-70%的糖尿病患者存在角膜异常，这往往会威胁视力。角膜异常包括上皮缺陷和脆性，反复的上皮侵蚀，敏感度降低，伤口修复异常， 增加对损伤、溃疡和水肿病的易感性。我们之前已经发现，除了中央角膜，糖尿病还会影响角膜缘，角膜和结膜之间的交界区，储存角膜上皮干细胞。目前，有必要更好地了解这些异常的机制，并开发出特异而有效的治疗方法。最近发现的被称为microRNAs的调节性遗传元件被认为对包括角膜上皮细胞在内的细胞调节具有广泛的影响。我们询问这些微小RNA是否参与了角膜的糖尿病状态，以及这种参与的机制是什么。我们的项目试图通过使用新的最先进的深度测序方法来测量糖尿病角膜上皮细胞中这些microRNAs的水平是否发生了变化。此外，为了研究它们的作用和作用机制，我们将使用腺病毒驱动的基于microRNA的基因疗法来使用功能验证的miRNAs来治疗人类器官培养的人尸检糖尿病角膜。对于治疗，我们将专注于调节伤口愈合的microRNAs，并可以影响在糖尿病中改变的干细胞标记物的表达。我们的研究意义如下：(1)首次在眼睛中使用深度测序方法，从而发现了新的microRNA，并在角膜上皮细胞中创建了全面的microRNA图谱；(2)精确测量了糖尿病角膜中差异产生的microRNA，这是尚未见报道的；(3)基于microRNA的糖尿病角膜基因治疗的翻译发展，可能适用于各种角膜疾病；(4)利用microRNA操作使糖尿病角膜功能障碍的上皮干细胞正常化的新方法。我们将通过单个microRNAs或其组合靶向糖尿病角膜上皮细胞，以改善角膜上皮细胞的功能。因此，这一策略将增加我们对microRNA在角膜上皮功能以及糖尿病疾病状态中作用的理解，这可能会导致新的治疗方法。我们将在我们的研究中使用人类角膜器官培养。正常人和糖尿病人的尸检眼睛将从国家疾病研究交换中心(NDRI)购买，NDRI是一家全国性的捐赠人体研究组织的供应商。所有组织都是在我们的实验室获得的，没有捐赠者的身份。

DESCRIPTION (provided by applicant): Diabetes is one of the major health challenges in developing countries, which has significant effects on different organs including eye. Although retina is the main ocular target of diabetes, 50%-70% of diabetic patients have corneal abnormalities, which can often be sight threatening. Corneal abnormalities include epithelial defects and fragility, recurrent epithelial erosions, decreased sensitivity, abnormal wound repair, increased susceptibility to injury, ulcers, and edema. We have previously found that beside central cornea, diabetes affects limbus, the junctional zone between cornea and conjunctiva housing corneal epithelial stem cells. Currently, there is a need to better understand the mechanisms responsible for these abnormalities and develop a specific and efficacious treatment. Recently discovered regulatory genetic elements called microRNAs are thought to have broad influences on cell regulation including corneal epithelial cells. We asked whether these microRNAs are engaged in the diabetic state of the cornea and what are the mechanisms of this engagement. Our project seeks to establish if the levels of these microRNAs are altered in the epithelial cells of diabetic corneas by measuring them using novel state-of-the-art deep sequencing methods. Further, to examine their roles and mechanisms of actions, we will utilize adenovirus-driven microRNA-based gene therapy to treat human organ-cultured human autopsy diabetic corneas using functionally validated miRNAs. For the treatment, we will focus on microRNAs that regulate wound healing and can influence the expression of stem cell markers, altered in diabetes. The significance of our study is as follows: (1) The use of deep sequencing method for the first time in the eye, which allows for a discovery of novel microRNAs and to create a comprehensive microRNA profiles in corneal epithelial cells; (2) Precise measurement of differentially produced microRNAs in diabetic cornea, which has not been reported; (3) Translational development of microRNA-based gene therapy for diabetic cornea, which may apply to various corneal disorders; (4) Novel way of normalizing dysfunctional epithelial stem cell in diabetic corneas using microRNA manipulation. We will target diabetic corneal epithelial cells by single microRNAs or their combinations to improve function of corneal epithelium. As a result, this strategy would increase our understanding of microRNA roles in corneal epithelial function as well as in diabetic disease state, which may lead to novel therapy. We will use human corneal organ culture in our studies. Normal and diabetic human autopsy eyes will be purchased from the National Disease Research Interchange (NDRI), a national supplier of donor human research tissue. All tissues are obtained in our lab without donor identity.