Identifying lincRNAs critical in asthma pathogenesis

描述（由申请人提供）：哮喘是一种气道过敏性疾病，已达到流行病的比例，并代表了卫生服务的重大负担 在全球 哮喘的特征是由Th2细胞启动的适应性免疫反应失调，然而，我们对疾病发展的理解是有限的。如果我们要充分了解哮喘的病因或开发新的治疗方法，那么阐明调节Th2分化和效应功能的基因表达程序的机制将是至关重要的。 最近发现的大基因间非 编码RNA（lincRNA）已显示调节核心基因表达程序。 数百种lincRNA在免疫系统的细胞内差异表达，突出了lincRNA在定义免疫细胞身份和功能以及促进免疫病理学如哮喘中的重要作用的令人兴奋的潜力。然而，关于lincRNA在体内的功能知之甚少，并且它们在哮喘中的作用完全未知。我们假设Th2细胞表达特异性lincRNA，其有助于调节决定Th2分化、Th2效应子功能和哮喘易感性的基因表达程序。使用国家的最先进的方法，该建议旨在确定和表征lincRNA特异性表达的Th2细胞，并评估其生物学功能，在体内通过使用过敏性气道疾病的小鼠模型。目的1将鉴定在体外和体内产生的Th2细胞中表达的特异性lincRNA，并确定它们在Th2分化程序的哪个阶段表达，这将揭示lincRNA最有可能参与指导Th2分化。 目的2将使用敲低和过表达研究来定义具有体外调节Th2细胞因子产生的能力的lincRNA。同时，将利用高通量RNA测序结合稳健的网络和关联内疚计算分析来生成每个lincRNA功能的可测试预测。目的3将使用最近开发的Cas9基因靶向技术来产生Th2特异性lincRNA缺陷的小鼠。通过在这些动物中模拟过敏性气道疾病，lincRNA在哮喘病因学中的潜在作用将首次在体内被剖析。总之，该提案将测试lincRNA在哮喘中先前未探索的功能，并代表过敏性气道疾病研究的新途径。 这些研究不仅将鉴定先前未研究的lincRNA，而且阿索将提供关于它们在Th2细胞中的功能的关键信息。 此外，由于大多数鼠lincRNA在人类中作为同线直系同源物存在，因此本提案中建立的工作最终将作为未来分析哮喘中人类lincRNA的平台。

DESCRIPTION (provided by applicant): Asthma is an allergic disease of the airways that has reached epidemic proportions and represents a significant burden on health services globally. Asthma is characterized by a dysregulated adaptive immune response initiated by Th2 cells~ however, our understanding of why the disease develops is limited. Delineating the mechanisms which regulate the gene expression programs that define Th2 differentiation and effector function will be essential if we are to fully understand the etiology of asthma or develop new therapies. The recently identified family of large intergenic non coding RNAs (lincRNAs) has been shown to regulate core gene expression programs. Hundreds of lincRNAs are differentially expressed within cells of the immune system highlighting the exciting potential for an important role for lincRNAs in defining immune cell identity and function, as well as contributing to immunopathologies such as asthma. However, very little is known about the function of lincRNAs in vivo and their role in asthma is completely unknown. We hypothesize that Th2 cells express specific lincRNAs that are instrumental in regulating the gene expression programs that determine Th2 differentiation, Th2 effector function and susceptibility to asthma. Using state-of-the-art methodologies this proposal seeks to identify and characterize lincRNAs specifically expressed in Th2 cells and evaluate their biological function in vivo by using a murine model of allergic airway disease. Aim 1 will identify specific lincRNAs expressed in Th2 cells generated in vitro and in vivo and determine at which stage in the Th2 differentiation program they are expressed~ this will reveal lincRNAs most likely to be involved in directing Th2 differentiation. Aim 2 will use knockdown and overexpression studies to define lincRNAs with the ability to modulate Th2 cytokine production in vitro. In parallel, high-throughput RNA-sequencing combined with robust network and guilt-by-association computational analyses will be utilized to generate testable predictions for the function of each lincRNA. Aim 3 will use the recently developed Cas9 gene targeting technology to generate mice deficient in Th2 specific lincRNAs. By modeling allergic airway disease in these animals the potential role for lincRNAs in the etiology of asthma will be dissected in vivo for the first time. In summary this proposal will test the previously unexplored function of lincRNAs in asthma and represents a novel avenue in allergic airway disease research. These studies will not only identify previously unstudied lincRNAs but will aso provide critical information on their function in Th2 cells. Furthermore, as the majoriy of murine lincRNAs exist as syntenic orthologues in humans the work established in this proposal will ultimately serve as a platform for future analysis of human lincRNAs in asthma.