MIR182 and Ocular Hypertension.

摘要 尽管microRNAs(MiRNAs)在青光眼的发病机制方面做出了重大的研究努力，但他们的 房水流出途径中的具体作用尚不清楚。降低眼压是唯一的方法 原发性开角型青光眼的临床治疗，无论患者是否有高眼压(IOP&GT；21 或正常眼压性青光眼(眼压≤为21毫米汞柱)。取得了重大成就。 了解眼压的调节规律，确定降眼压的治疗靶点。我们和其他人有 鉴定了MIR182基因rs76481776与POAG的相关性。然而，目前还不清楚是如何做到的 MIR-182-5P增加了POAG风险。如果没有这样的知识，基于miRNA的青光眼的发展 治疗可能仍将是困难的。我们的总体目标是确定miR-182-5p的表达上调 导致高眼压的POAG。与POAG相关的rs76481776与POAG相关的风险等位基因A 报道称在体外增加了成熟miR-182-5p的表达，提示miR-182-5p的表达增加 可能对POAG有贡献。我们发现在青光眼房水中miR-182-5p的表达增加了2倍 与对照组相比。我们的中心假设是miR-182-5p的高表达通过调节眼压来影响眼压 人眼小梁网(TM)和Schlemm管(SC)内皮细胞的细胞功能。人类 诱导早衰的TM细胞miR-182-5p的表达增加7-9倍，并且过表达 这些HTM细胞中miR-182-5p的缺失导致部分细胞衰老。MIR-182-5P已被显示为目标 许多途径中的基因，如CHEK2、FOXO1、MTSS1和CyLD。MiR-182-5p的表达可 经转化生长因子-β诱导的癌细胞，导致核因子-κB活化时间延长，而转化生长因子-β相关 通路已被证明在POAG和纤维化中起着重要作用。这项提议的理由是， 一旦我们确定miR-182-5p的高表达如何影响人类TM和SC的细胞功能， 某些基因或miRNAs可以在药理学上被上下操纵，从而产生新的和 降低眼压和延缓/预防青光眼进展的创新方法。在目标1中，我们将确定 MiR-182-5p在原代人TM细胞中的分子靶点。人类TM细胞将受到循环的影响 机械拉伸和组织培养采用单轴组织拉伸进行三维细胞培养。我们将进行完整的蛋白质组学 对miR-182-5p靶基因进行图谱分析和链特异性RNA-Seq分析。在目标2中，我们将确定 MiR-182-5p在TM和SC过表达对流出功能和眼压水平的影响 转基因小鼠。成功完成后，我们预计将确定miR-182的特定分子靶点- 5P可更有效地降低青光眼患者的眼压。

Abstract Despite the significant research effort of microRNAs (miRNAs) in the pathogenesis of glaucoma, their specific roles in the aqueous outflow pathway remain unclear. Lowering intraocular pressure (IOP) is the only clinical treatment for primary open-angle glaucoma (POAG), no matter whether the patients have high (IOP > 21 mmHg) or normal-tension glaucoma (IOP ≤ 21 mmHg). Significant achievements have been made in understanding the regulation of IOP and identifying therapeutic targets to lower IOP. We and others have identified the association of rs76481776 in the MIR182 gene with POAG. However, it remains unknown how miR-182-5p contributes to POAG risk. Without such knowledge, the development of miRNA-based glaucoma therapy will likely remain difficult. Our overall objective is to determine how elevated expression of miR-182-5p contributes to POAG with high IOP. The risk allele A of POAG-associated rs76481776 with POAG has been reported to increase mature miR-182-5p expression in vitro, suggesting that elevated miR-182-5p expression may contribute to POAG. We identified 2-fold higher miR-182-5p expression in glaucomatous aqueous humor compared to controls. Our central hypothesis is that elevated expression of miR-182-5p affects IOP by regulating the cellular functions of human trabecular meshwork (TM) and Schlemm's canal (SC) endothelial cells. Human TM cells with induced premature senescence have 7-9 fold higher miR-182-5p expression, and overexpression of miR-182-5p in these HTM cells leads to partial cellular senescence. miR-182-5p has been shown to target genes in many pathways, such as CHEK2, FOXO1, MTSS1, and CYLD. The expression of miR-182-5p could be induced by TGF-β treatment in cancer cells, leading to prolonged NF-κB activation, while TGF-β related pathways have been shown to play essential roles in POAG and fibrosis. The rationale for this proposal is that, once we determine how elevated miR-182-5p expression affects the cellular functions of human TM and SC, certain genes or miRNAs could be manipulated either up or down pharmacologically, resulting in new and innovative approaches to lower IOP and to delay/prevent the progression of glaucoma. In Aim 1, we will identify molecular targets of miR-182-5p in primary human TM cells in vitro. Human TM cells will be subject to cyclic mechanical stretch and tissue train 3D cell culture with uniaxial tissue stretch. We will perform whole proteomics profiling and strand-specific RNA-Seq analysis to identify miR-182-5p target genes. In Aim 2, we will determine the impact of miR-182-5p overexpression in the TM and SC on outflow facility and IOP levels in vivo using transgenic mice. Upon successful completion, we expect to identify the specific molecular targets of miR-182- 5p in human TM and SC to lower IOP more effectively in glaucoma patients.