microRNAs in the airway epithelium

气道上皮细胞中的 microRNA

• 批准号: 8455022
• 负责人: EDWIN Justin OSTRIN
• 金额: $ 2.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2013-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8455022
• 关键词: 3' Untranslated Regions; Air; Allergens; Asthma; Cell Culture System; Cell Differentiation process; Cell physiology; Cells; Characteristics; Chronic Obstructive Airway Disease; Collaborations; Complex; Cystic Fibrosis; Development; Diagnosis; Disease; Epithelial; Epithelial Cells; Exposure to; Family; Future; Gene Expression; Genetic; Host Defense; Human; In Situ Hybridization; Infectious Agent; Interleukin-13; Liquid substance; Lung; Lung diseases; Mentors; Metaplasia; MicroRNAs; Molecular and Cellular Biology; Monitor; Morbidity - disease rate; Mucous body substance; Patients; Phenotype; Physicians; Play; Process; RNA; Regulator Genes; Repression; Research; Role; Scientist; Secretory Cell; Signal Transduction; Site; Stimulus; System; Testing; Transcript; United States; Up-Regulation; Work; airway epithelium; asthmatic patient; base; cell type; cytokine; experience; falls; human disease; human subject; inhibitor/antagonist; knowledge base; member; mortality; mouse model; notch protein; pollutant; public health relevance; response; sensor; tool

DESCRIPTION (provided by applicant): Changes in the airway epithelium are prominent contributors to a number of human diseases, notably asthma. Our group has shown that the Th2 cytokine interleukin-13 (IL-13) drives characteristic alterations in epithelial gene expression leading to mucous metaplasia in mouse models and in a large subset of human subjects with asthma. I hypothesize that microRNAs (miRNAs) play an important role in these changes. In support of this, we have shown that there are significant differences in expression of miRNAs between healthy and asthmatic subjects. Since joining the Erle lab, I found evidence that many of these changes are driven by IL-13 and developed tools required to monitor, inhibit, or mimic effects of miRNAs in bronchial epithelial cells. In this revised proposal, I focus on testing the involvement of specific epithelial miRNAs in mucous metaplasia. The first aim is to determine if the repression of the miR-449/34 family is necessary and sufficient for IL-13- driven mucous metaplasia. Among the most significantly suppressed miRNAs are members of the miR- 449/34 family, which have known roles in multiciliogenesis through repression of Notch signaling. As Notch signaling also plays a key role in selection of secretory cell fate, I hypothesize that IL-13 repression of miR- 449/34 acts to increase Notch signaling and promote mucous metaplasia. I will test whether miR-449 is necessary and sufficient for IL-13 driven mucous metaplasia using normal human bronchial epithelial cells differentiating in an air-liquid interface culture. I will inhibit miR-449 using miRNA inhibitors and assess for markers of mucous metaplasia. I will use a miR-449 mimic in cells exposed to IL-13 and test for suppression of mucous metaplasia. I will characterize additional targets of this miRNA family that may act in concert with Notch signaling to promote mucous metaplasia. The second aim is to determine how selected miRNAs stimulated by IL-13 and upregulated in asthma are involved in mucous metaplasia and other epithelial abnormalities. I hypothesize that miRNAs upregulated in response to IL-13 have a role in mucous cell differentiation and function. I have identified several miRNAs for further characterization: miR-663a/1908, miR- 1275, miR-765, and miR-1228-5p. These miRNAs have significant upregulation in asthmatic patients versus healthy subjects and are also responsive to IL-13 in our cell culture system. I will first use in situ hybridization to localize the expressionof these miRNAs and use a lentiviral sensor system to determine their activity in mucous cells and other epithelial cells. I will use mimics and inhibitors to define their role in mucous metaplasia.In addition to lab work, I will attend relevant courses and seminars to continue to expand my knowledge base. I will be closely mentored by Dr Erle and a committee comprised of experts in the molecular and cellular biology of lung disease. This proposal will provide me with the tools and experience for future independent work as a research-oriented pulmonary physician-scientist.

描述（由申请人提供）：气道上皮的变化是许多人类疾病的重要贡献者，尤其是哮喘。我们的小组表明，Th2细胞因子白介素13（IL-13）驱动上皮基因表达的特征变化 导致小鼠模型中的粘液化生和哮喘的大部分人类受试者。我假设microRNA（miRNA）在这些变化中起着重要作用。为此，我们已经表明，健康和哮喘受试者之间的miRNA表达存在显着差异。自加入ERLE实验室以来，我发现证据表明，其中许多变化是由IL-13驱动的，并且开发了开发的工具，以监测，抑制或模仿支气管上皮细胞的miRNA效应。在这项修订的建议中，我专注于测试特定上皮miRNA在粘液化生中的参与。第一个目的是确定MiR-449/34家族的抑制是否需要IL-13驱动的粘液质量。在最显着抑制的miRNA中，有mir-449/34家族的成员，该家族通过抑制Notch信号传导在多丝生成中知道了作用。由于Notch信号传导在选择分泌细胞命运中也起着关键作用，因此我假设IL-13 miR-449/34的抑制作用可提高凹口信号传导并促进粘膜化生。我将使用正常的人支气管上皮细胞在空气界面培养物中区分的正常人支气管上皮细胞来测试IL-13驱动的粘液化生型MiR-449是否足以且足够。我将使用miRNA抑制剂抑制miR-449，并评估粘膜化生的标志物。我将在暴露于IL-13的细胞中使用miR-449模拟物并测试抑制粘膜化生。我将表征这个miRNA家族的其他靶标，这些靶标可能会与Notch信号一起起作用以促进粘液化的质量。第二个目的是确定IL-13刺激并在哮喘中上调的选定miRNA如何参与粘液化生和其他上皮异常。我假设对IL-13响应上调的miRNA在粘液细胞分化和功能中起作用。我已经确定了几个miRNA以进一步表征：mir-663a/1908，mir-1275，mir-765和mir-1228-5p。这些miRNA在哮喘患者与健康受试者中具有明显的上调，​​并且在我们的细胞培养系统中对IL-13有反应。我将首先使用原位杂交来定位这些miRNA的表达，并使用慢病毒传感器系统来确定它们在粘液细胞和其他上皮细胞中的活性。我将使用模仿和抑制剂来定义它们在粘液Metaplasia中的作用。除了实验室工作，我还将参加相关课程和研讨会，以继续扩大我的知识库。我将由Erle博士和由肺部疾病分子和细胞生物学专家组成的委员会密切指导。该建议将为我提供作为以研究为导向的肺医生 - 科学家的未来独立工作的工具和经验。