The Role and Mechanisms of microRNAs in Diabetic Cornea

描述（由申请人提供）：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显著影响。虽然视网膜是糖尿病的主要眼部目标，但50%-70%的糖尿病患者具有角膜异常，这通常会威胁视力。角膜异常包括上皮缺陷和脆性、复发性上皮糜烂、敏感性降低、异常伤口修复， 增加对损伤、溃疡和水肿的敏感性。我们之前发现，除了中央角膜之外，糖尿病还会影响利姆布斯，即角膜和结膜之间容纳角膜上皮干细胞的交界区。目前，有必要更好地了解负责这些异常的机制，并制定一个具体的和有效的治疗。最近发现的称为microRNA的调节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microRNA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。我们的项目旨在确定这些microRNA的水平是否在糖尿病角膜的上皮细胞中改变，方法是使用最先进的深度测序方法测量它们。此外，为了研究它们的作用和作用机制，我们将利用腺病毒驱动的基于microRNA的基因治疗，使用功能验证的miRNA治疗人类器官培养的人类尸检糖尿病角膜。对于治疗，我们将专注于调节伤口愈合的microRNA，并可以影响糖尿病中改变的干细胞标志物的表达。本研究的意义在于：（1）首次将深度测序方法应用于眼内，发现了新的microRNA，建立了角膜上皮细胞microRNA的全面图谱;（2）精确测量了糖尿病角膜中差异产生的microRNA，目前尚无报道;（3）基于microRNA的糖尿病角膜基因治疗的转化发展，其可适用于各种角膜疾病;（4）使用microRNA操作使糖尿病角膜中功能失调的上皮干细胞正常化的新方法。我们将通过单个microRNA或其组合靶向糖尿病角膜上皮细胞，以改善角膜上皮功能。因此，这种策略将增加我们对microRNA在角膜上皮功能以及糖尿病疾病状态中的作用的理解，这可能导致新的治疗方法。我们将在我们的研究中使用人类角膜器官培养。将从国家疾病研究交流中心（NDRI）购买正常和糖尿病人尸检眼睛，NDRI是一家国家供体人体研究组织供应商。所有组织都是在我们实验室获得的，没有捐赠者身份。

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.