microRNA-194通过组蛋白修饰对角膜内皮早衰的调控机制

The corneal chronic allograft dysfunction (CCAD) induced by corneal endothelial cell stress-induced premature senescence (SIPS) has become the predominant cause of late graft failure. At present, we have no effective interventions to deal with it. Furthermore, CCAD is also one of the most important scientific problems demanding prompt solution after corneal transplantation. How to clarify the molecular mechanism of CCAD and how to improve the long-term survival rate of corneal graft are still the key issues in studying the pathogenesis of keratopathy. MicroRNAs are small non-coding RNAs that play important roles in the regulation of target genes. These miRNAs have been correlated with a number of diseases, such as diabetes, neurodegenerative diseases and heart failure. They can bind to complementary regions of messenger transcripts to either repress translation or regulate degradation. In the preliminary experiments, we found that the expression of microRNA-194 was down-regulation in corneal endothelial cells of old mice using miRNA expression profiling tools and the whole histone acetylation level also decreased in corneal endothelia of old mice. Therefore, we have a hypothesis that the pathway of miR-194 mediated histone modification is critical for corneal endothelial cell premature senescence. In this study, we will use the SIPS model in vitro and the mice corneal transplantation model in vivo to validate our hypothesis. The first purpose of this study is to find the change of whole histone acetylation and methylation during premature senescence of corneal endothelial cells. And the second purpose is to investigate the key target genes of miR-194 during premature senescence of corneal endothelial cells. Also, we will find the down-stream regulational genes and pathways of these key target genes using methods of ChIP-Seq, reporter gene system, real time PCR, western blot, and adenovirus -gene transfer system. Finally, we will use miR-194 agomir to treat the CCAD mice model. The results of this study will provide a theoretical basis for improving the long-term survival and maintaining long-term transparency of corneal graft.

项目组前期工作表明内皮细胞应激性早衰导致的植片慢性失功（CCAD）是角膜移植术后远期植片失败最主要的原因，目前缺乏有效的干预手段。近年，miRNAs 通过对组蛋白修饰精确调控细胞衰老引起了广泛关注。本项目在结合文献及预实验的基础上提出miRNA-194通过组蛋白修饰是调控角膜内皮细胞衰老的关键途径的假设。拟从体外细胞应激早衰模型以及体内小鼠CCAD模型，采用报告基因、ChIP-Seq分析、腺病毒介导的基因转染、Western blot与定量PCR等技术研究：(1）miR-194调控角膜内皮细胞早衰组蛋白乙酰化与甲基化修饰的关键靶基因；(2）miR-194靶基因的关键下游调控基因及其信号通路；(3）miR-194体内对小鼠角膜移植后内皮细胞早衰的调控与干预作用。本研究从一个全新角度探讨角膜内皮细胞衰老机制，以期为延缓植片角膜内皮早衰，提高植片的远期存活率提供新理论依据与新途径。

项目组前期工作表明内皮细胞应激性早衰导致的植片慢性失功（CCAD）是角膜移植术后远期植片失败最主要的原因，目前缺乏有效的干预手段。近年，miRNAs 通过对组蛋白修饰精确调控细胞衰老引起了广泛关注。我们在慢性失功的角膜植片内皮细胞上检测到了活性氧自由基的积累与内皮细胞的早衰，提示植片内皮细胞处于应激诱导早衰的状态。我们首次发现角膜内皮衰老与p16的高表达以及Bmi1的低表达有关。并阐明了衰老小鼠角膜内皮与幼年小鼠角膜内皮的差异表达miRNAs。. 糖尿病是多病因引起的慢性代谢性疾病，目前糖尿病眼部并发症的研究主要集中于糖尿病性视网膜病变，但近一半的糖尿病患者会出现原发性角膜病变，临床特征主要表现为角膜内神经密度和神经营养功能下降引起的角膜上皮愈合延迟、神经营养性角膜溃疡，最终导致角膜溃疡甚至穿孔，患者的经济和精神负担严重。角膜含有丰富的神经纤维，是人体内神经最密集的组织之一。角膜神经由三叉神经眼支发出，在维持角膜上皮功能和解剖完整性等方面起重要作用。.申请人取得了如下成果。.（1）首次发现Sirt1在糖尿病小鼠角膜的表达水平显著低于正常对照小鼠（2012年，王晔等，中华实验眼科杂志）；.（2）全面而系统的综述了糖尿病角膜病变的研究进展（2014年，王晔等，中华眼科杂志，优先发表）；.（3）过量表达SIRT1后显著促进糖尿病角膜上皮的损伤修复过程（2013年，王晔等，眼科基础研究权威杂志IOVS，优先发表）。.（4）微小RNA，miR-204-5p显著促进糖尿病角膜上皮细胞周期的循环，其作用是通过靶向Sirt1进行的（2015年，王晔等，眼科基础研究权威杂志IOVS）。.（5）微小RNA，miR-182促进糖尿病角膜神经的再生。这部分工作已经发表于糖尿病研究领域权威杂志Diabetes（影响因子IF=8.8）。上述研究结果连续三次受邀在美国眼科年后上作大会发言，并获得Travel Grant资助。