The Role of MicroRNAs in Normal and Diseased Corneal Epithelial Homeostasis

MicroRNA 在正常和患病角膜上皮稳态中的作用

• 批准号: 10737500
• 负责人: Mehrnoosh Saghizadeh Ghiam
• 金额: $ 55.54万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737500
• 关键词: Acute; Adherens Junction; Basal Cell; Biological Process; Blindness; Cell Differentiation process; Cell Fate Control; Cell Maintenance; Cells; Clinical; Cornea; Corneal Diseases; Corneal Injury; Corneal Opacity; Data; Defect; Disease; Epidermal Growth Factor Receptor; Epithelial Cells; Epithelium; Erinaceidae; Exposure to; Functional disorder; Funding; Future; Gene set enrichment analysis; Genes; Goals; Health; Healthcare Systems; Heterogeneity; Homeostasis; Human; In Situ Hybridization; Individual; Inflammation Mediators; Inflammatory; Inflammatory Response; Injury; Interleukin-1; Keratopathy; Maintenance; Mediating; MicroRNAs; Microarray Analysis; Mitotic; Molecular; Natural regeneration; Numbness; Organ Culture Techniques; Pathogenesis; Pathologic; Pathway interactions; Patients; Population; Population Heterogeneity; Proteome; Proteomics; Quality of life; Quantitative Reverse Transcriptase PCR; Receptor Signaling; Recurrence; Regulation; Regulatory Pathway; Research; Resolution; Role; Signal Transduction; Small Interfering RNA; Source; Stimulus; Stress; Stromal Cells; TNF receptor-associated factor 1; TNF receptor-associated factor 6; Techniques; Technology; Therapeutic; Tissues; Transfection; Vision; Visual impairment; Visualization; cell fate specification; cell growth regulation; corneal epithelial wound healing; corneal epithelium; corneal regeneration; cytokine; diabetic; diabetic patient; differential expression; effective therapy; empowerment; epithelial stem cell; epithelial wound; epithelium regeneration; functional gain; inhibitor; innovation; interleukin-1 receptor-associated kinase; limbal; notch protein; single-cell RNA sequencing; small molecule inhibitor; stem cell biomarkers; stem cell homeostasis; stem cells; transcriptome; transcriptome sequencing; translational study; treatment strategy; wound healing

Corneal diseases and injuries leading to visual impairment have significant impact on the quality of life of patients and constitute a major problem for health care system. Corneal diseases are often manifested through limbal epithelial stem cell (LESC) dysfunction and loss, leading to corneal opacity, visual impairment, and blindness. LESC located in the limbal niche are the source of constant corneal epithelial renewal and are required to maintain corneal epithelial homeostasis by adapting properly to internal and external stimuli. However, persistent exposure to pathological stress disrupts the adaptive mechanisms of LESC, leading to serious corneal problems such as altered wound healing, limbal stem cell deficiency (LSCD) and diabetic keratopathy. Therefore, understanding the precise spatial and temporal regulatory pathways and how each LESC/limbal epithelial cells (LECs) responds to various stimuli at the single-cell level governing corneal epithelial regeneration could be key to elucidating the pathogenesis of various corneal diseases and ultimately developing new and effective treatment strategies. Previously, we have shown the important regulatory role of miR-146a in corneal epithelial homeostasis and wound healing via its different targeted pathways such as EGFR, Notch and NF-κB signaling. However, due to the heterogeneity of limbal basal cells and their proximity to limbal stromal cells, it remains to be determined how miRNAs (miRs) regulate maintenance, differentiation, and the active stage of limbal progenitor cells in various corneal cell populations in wounded and unwounded normal and diseased corneas. Recently several studies have shown the heterogeneity and complexity of limbal niche in relation to regeneration and wound healing and identified new putative LESC markers using newly emerged empowering scRNA-seq technology. We propose to investigate the regulatory role and contribution of each miR-146 main targets (EGFR, TRAF6, IRAK1, NUMB and NOTCH-2) in corneal epithelial regeneration in wounded and unwounded states in normal and diabetic corneas using scRNA-seq, proteomics, loss and gain of functional studies. We hypothesize that miR-146a contribute to the molecular regulation of cellular heterogeneity in the limbal niche steady state. We predict that differential miR-146a expression in limbus vs. central corneal epithelium impacts its regulation of limbal niche cells, LECs and limbal stromal cells, and mediates corneal regeneration and wound healing in normal and diseased states. Our Specific Aims are: Specific Aim 1: To identify and characterize downstream targets including EGFR, Notch and inflammatory mediators of miR-146a in distinct populations of heterogeneous limbal stem cells that contribute to homeostatic maintenance and regeneration in human normal unwounded and wounded cornea. Specific Aim 2: To reveal the differential and converged regulatory role of EGFR, inflammatory, and Notch signaling by miR-146a in corneal epithelial homeostasis and wound healing. Specific Aim 3: To investigate miR146a-based therapeutic approach by modulating its targets in human diabetic organ-cultured corneas.

角膜疾病和导致视觉障碍的伤害对生活质量产生重大影响 患者并构成医疗保健系统的主要问题。角膜疾病经常通过 边缘上皮干细胞（LESC）功能障碍和损失，导致角膜不透明，视觉障碍和 失明。位于边缘小众的LESC是恒定角膜上皮更新的来源，是 通过正确调整内部和外部刺激，需要维持角膜上皮稳态。 但是，持续暴露于病理压力会破坏LESC的适应机制，导致 严重的角膜问题，例如改变伤口愈合，边缘干细胞缺乏症（LSCD）和糖尿病 角膜病。因此，了解精确的空间和临时调节途径以及如何 LESC/边缘上皮细胞（LEC）对单细胞水平的各种刺激做出了响应 上皮再生可能是阐明各种角膜疾病的发病机理的关键，最终 制定新的有效的治疗策略。以前，我们已经显示了 miR-146a在角膜上皮稳态中，通过其不同的靶向途径（例如） EGFR，Notch和NF-κB信号传导。但是，由于边缘基本细胞的异质性及其接近性 对于缘缘基质细胞，尚待确定miRNA（miR）如何调节维持，分化， 以及在受伤和未能的各种角膜细胞种群中的边缘祖细胞的活跃阶段 通常和脱离角膜。最近一些研究表明了缘缘的异质性和复杂性 与再生和伤口愈合有关的利基市场，并使用新的假定LESC标记 出现了赋予SCRNA-SEQ技术的能力。我们建议调查监管作用和贡献 每个miR-146主要靶标（EGFR，Traf6，Irak1，Numb和Notch-2）在角膜上皮再生中 使用SCRNA-SEQ，蛋白质组学，损失和增益，在正常和糖尿病角膜下受伤和未能的状态 功能研究。我们假设miR-146a有助于细胞的分子调节 边缘利基稳态的异质性。我们预测，在边缘VS中的miR-146a表达差异。 中央中部上皮影响其调节边缘细胞，LEC和缘缘基质细胞，以及 介导正常和解散状态下的角膜再生和伤口愈合。我们的具体目的是： 特定目的1：识别和表征包括EGFR，Notch和炎症在内的下游目标 MiR-146a的介体在不同种群的异质边缘干细胞中有助于稳态 人类正常未能和受伤的角膜的维持和再生。特定目标2：揭示 miR-146a在 角膜上皮稳态和伤口愈合。特定目的3：研究基于miR146a的治疗 通过调节其在人类糖尿病有组织的角膜中的目标来接近。