Identifying lincRNAs critical in asthma pathogenesis

描述(申请人提供)：哮喘是一种呼吸道过敏性疾病，已达到流行程度，对健康服务构成重大负担。 全球范围内。哮喘的特征是由Th2细胞启动的一种失调的适应性免疫反应，然而，我们对疾病发生的原因了解有限。如果我们要全面了解哮喘的病因或开发新的治疗方法，阐明调控Th2分化和效应器功能的基因表达程序的机制将是至关重要的。新近发现的大基因间非基因家族 编码RNAs(LincRNAs)已被证明调节核心基因表达程序。数以百计的lincRNAs在免疫系统的细胞内差异表达，突显了lincRNAs在确定免疫细胞的身份和功能以及在哮喘等免疫病理中发挥重要作用的令人兴奋的潜力。然而，对lincRNAs在体内的功能知之甚少，它们在哮喘中的作用也完全不清楚。我们假设Th2细胞表达特定的lincRNAs，这些lincRNAs有助于调节决定Th2分化、Th2效应功能和哮喘易感性的基因表达程序。使用最先进的方法，这项建议试图识别和表征在Th2细胞中特异表达的lincRNAs，并通过使用过敏性呼吸道疾病的小鼠模型来评估它们在体内的生物学功能。目的1鉴定在体外和体内产生的Th2细胞中表达的特定lincRNAs，并确定它们在Th2分化程序的哪个阶段表达，这将揭示最有可能参与Th2分化的lincRNAs。AIM 2将使用基因敲除和过表达研究来确定在体外能够调节Th2细胞因子产生的lincRNAs。同时，高通量RNA测序与稳健的网络和关联计算分析相结合，将被用来为每个lincRNA的功能产生可测试的预测。Aim 3将使用最近开发的Cas9基因打靶技术来产生Th2特异性lincRNAs缺陷的小鼠。通过在这些动物中建立过敏性呼吸道疾病的模型，将首次在体内剖析lincRNAs在哮喘病因中的潜在作用。综上所述，这项建议将测试以前未曾探索过的lincRNAs在哮喘中的功能，并代表了过敏性呼吸道疾病研究的一条新途径。这些研究不仅将确定以前没有研究过的lincRNAs，而且还将提供关于它们在Th2细胞中功能的关键信息。此外，由于大多数小鼠lincRNAs在人类中以同素同源形式存在，本提案中建立的工作最终将成为未来分析哮喘中人类lincRNAs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.