Investigating the Function of a Highly Expressed lncRNA in Airway Epithelium

项目摘要/摘要 哮喘是一种复杂的肺部炎症性疾病，在美国的患病率持续上升， 每十二个人中就有一个患有此病。尽管吸入性皮质类固醇和β-2激动剂一直是主流 在治疗方案中，这些疗法在不同的患者群体中具有不同的疗效，并且 建议以缓解和控制症状为基础，而不是针对致病机制。 过敏性哮喘患者最常见，以呼吸道上皮细胞过敏为特征。 吸入过敏原，导致2型获得性免疫反应。免疫细胞释放2型细胞因子， 它们通过STAT的成员(信号转导和激活因子)触发呼吸道上皮细胞 转录)家族，导致哮喘的许多临床病理特征。因此，呼吸道上皮细胞 是哮喘发病过程中的一个关键环节。尽管如此，中国对下游赛事的监管 2型细胞因子刺激后的呼吸道上皮细胞还不是很清楚。虽然许多研究都有 阐明编码基因组在过敏性哮喘发生发展中的作用，由非编码调控 基因组没有得到很好的描述。长的非编码RNA(LncRNAs)越来越被认为是重要的 调节免疫反应和细胞特性的功能分子。我们发现了lncRNA WFDC21P在IL-13刺激后在原代人支气管上皮细胞中高表达。WFDC21P 先前被发现调节树突状细胞的分化和功能，然而它在呼吸道上皮细胞中的作用 细胞还没有被研究过。我们的初步数据表明WFDC21P指导呼吸道上皮细胞的分化 通过调节STAT3的磷酸化水平影响细胞的功能。为了检验这一假设，我们提出了一个组合 先进的基因组和分子工具研究WFDC21P对基因组、细胞和分子的影响 水平。首先，我们将确定WFDC21P在IL-13诱导后的细胞类型特异性，并确定其 细胞和分子对呼吸道上皮形态和STAT信号的影响。这些研究将是 通过单细胞RNA测序、组织学、免疫荧光和流式细胞术进行。后来， 我们将使用RNA干扰、ATAC测序和RNA-DNA分析来评估WFDC21P对基因组的影响。 排序之后进行了一系列的计算分析。完成这些研究将提供关键 呼吸道上皮细胞中STAT信号的精确下游调控及其机制的生物学洞察力 LncRNAs参与了呼吸道生物学和2型免疫反应。因此，这些研究将提供新的 用于未来哮喘治疗的途径和调控靶点。

PROJECT SUMMARY / ABSTRACT Asthma is a complex, inflammatory disease of the lung whose prevalence continues to rise in the United States, with one in twelve individuals afflicted. Although inhaled corticosteroids and β2-agonists have been the mainstay of treatment regimens, these therapies have varying efficacy in a heterogeneous patient population and are recommended based on symptom alleviation and control, rather than targeting pathogenic mechanisms. Individuals most commonly suffer from allergic asthma, characterized by hypersensitivity of airway epithelial cells to inhaled allergens, leading to a type 2 adaptive immune response. Immune cells release type 2 cytokines, which trigger airway epithelial cells through members of the STAT (signal transducer and activator of transcription) family, causing many of the clinicopathologic features of asthma. Thus, airway epithelial cells represent a critical interface in the development of asthma. Despite this, the regulation of downstream events in airway epithelium following type 2 cytokine stimulation is not well understood. While many studies have elucidated the role of the coding genome in the development of allergic asthma, regulation by the non-coding genome is not well described. Long noncoding RNAs (lncRNAs) have increasingly been identified as important functional molecules in regulating an immune response and cellular identity. We discovered the lncRNA WFDC21P is highly expressed following IL-13 stimulation in primary human bronchial epithelial cells. WFDC21P was previously found to regulate dendritic cell differentiation and function, however its role in airway epithelial cells has not yet been studied. Our preliminary data suggest WFDC21P directs differentiation of airway epithelial cells via regulating the level of STAT3 phosphorylation. To test this hypothesis, we propose a combination of advanced genomic and molecular tools to study the effects of WFDC21P on a genomic, cellular, and molecular level. First, we will determine the cell-type specificity of WFDC21P following IL-13 induction and determine its cellular and molecular effects on airway epithelial morphology and STAT signaling. These studies will be performed via single-cell RNA sequencing, histology, immunofluorescence, and flow cytometry. Subsequently, we will assess the effects of WFDC21P on the genome using RNA interference, ATAC-sequencing, and RNA- sequencing followed by a series of computational analyses. Completion of these studies will provide key biological insight into the precise downstream regulation of STAT signaling in airway epithelial cells and how lncRNAs contribute to airway biology and the type 2 immune response. These studies will thus provide novel pathways and regulatory targets to be used for future therapeutics in the treatment of asthma.