MIR182 and Ocular Hypertension.

摘要 尽管对microRNAs（miRNAs）在青光眼发病机制中的重要研究努力，但它们在青光眼发病机制中的作用仍有待进一步研究。 在房水流出途径中的具体作用仍不清楚。降低眼内压（IOP）是唯一 原发性开角型青光眼（primary open-angle glaucoma，POAG）的临床治疗，无论患者是否有高眼压（IOP > 21 正常眼压性青光眼（IOP ≤ 21 mmHg）。取得重大成就 了解IOP的调节并确定降低IOP的治疗靶点。我们和其他人已经 鉴定了MIR 182基因中的rs76481776与POAG的关联。然而，目前尚不清楚如何 miR-182- 5 p与POAG风险有关。如果没有这些知识，基于miRNA的青光眼的发展 治疗可能仍然很困难。我们的总体目标是确定miR-182- 5 p的表达升高是如何影响miR-182- 5 p的表达的。 导致POAG伴高IOP。POAG相关rs76481776的风险等位基因A与POAG相关， 据报道，在体外增加成熟的miR-182- 5 p表达，这表明miR-182- 5 p表达的升高 可能导致POAG。我们发现在青光眼的房水中miR-182- 5 p的表达高2倍， 与对照相比。我们的中心假设是miR-182- 5 p的高表达通过调节眼压而影响眼压。 人小梁网（TM）和施累姆氏管（SC）内皮细胞的细胞功能。人类 具有诱导性早衰的TM细胞具有高7-9倍的miR-182- 5 p表达，并且过表达 miR-182- 5 p在这些HTM细胞中的表达导致部分细胞衰老。miR-182- 5 p已经显示靶向 许多途径中的基因，如CHEK 2，FOXO 1，MTSS 1和CYLD。miR-182- 5 p的表达可 在癌细胞中，TGF-β处理可诱导NF-κB活化延长，而TGF-β相关 已经显示，这些通路在POAG和纤维化中起重要作用。这项建议的理由是， 一旦我们确定升高的miR-182- 5 p表达如何影响人TM和SC的细胞功能， 某些基因或miRNAs可以被向上或向下操纵，从而产生新的， 降低IOP和延迟/预防青光眼进展的创新方法。在目标1中，我们将确定 体外原代人TM细胞中miR-182- 5 p的分子靶点。人TM细胞将经历周期性的 机械拉伸和具有单轴组织拉伸的组织训练3D细胞培养。我们将进行完整的蛋白质组学 分析和链特异性RNA-Seq分析以鉴定miR-182- 5 p靶基因。在目标2中，我们将确定 在TM和SC中miR-182- 5 p过表达对流出功能和IOP水平的影响， 转基因小鼠在成功完成后，我们希望确定miR-182的特异性分子靶点， 5 p在人TM和SC中更有效地降低青光眼患者的IOP。

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.