MICRORNA REGULATION OF NK CELL DEVELOPMENT AND FUNCTION

微小RNA对NK细胞发育和功能的调节

• 批准号: 8890768
• 负责人: TODD A FEHNIGER
• 金额: $ 38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-06 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890768
• 关键词: Activities of Daily Living; Acute Myelocytic Leukemia; Allogenic; Bioinformatics; Biological Models; Cell physiology; Cells; Cellular biology; Cessation of life; Complement; Cytokine Activation; Cytomegalovirus Infections; Data; Development; Disease; Event; Exhibits; Future; Genes; Goals; Granzyme; Health; Herpesviridae Infections; Host Defense; Human; Immune; Immune response; Immunity; Immunoprecipitation; Immunotherapeutic agent; In Vitro; Individual; Infection; Inflammatory; Interferon Type II; Knock-out; Laboratories; Lead; Life; Link; Listeria monocytogenes; Lymphocyte; Maintenance; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Modeling; Molecular; Murid herpesvirus 1; Mus; NK Cell Activation; Natural Killer Cells; Natural killer triggering receptor; Pathway interactions; Patients; Physiological; Production; Proteins; Publishing; Regulation; Research; Rest; Role; Subfamily lentivirinae; Testing; Translations; Virus; base; cancer immunotherapy; cytokine; cytotoxicity; effective therapy; functional status; in vivo; insight; killings; loss of function; mRNA Stability; mouse model; neoplastic; neoplastic cell; next generation sequencing; novel; novel strategies; overexpression; pathogen; perforin; receptor; research study; response; tumor

DESCRIPTION (provided by applicant): The long term goals of this application are to define molecular events that regulate NK cell development and function, thereby providing insight into pathways that may be manipulated to promote or inhibit these aspects of NK cell biology. NK cells are innate immune lymphocytes that are important during host defense against infections and mediate anti-neoplastic immune responses. This is exemplified by rare patients with selective NK cell deficiencies that succumb to fatal herpesvirus infections early in life, demonstrating the critical importance of these cells for human health. Moreover, allogeneic NK cells can be an effective treatment for acute myeloid leukemia patients, and thus NK cells hold promise as a cancer immunotherapy approach. Currently, we have an incomplete understanding of the molecular mechanisms responsible for regulating NK cell functionality (cytokine production, cytotoxicity, proliferation). MicroRNAs (miRNAs) are small regulatory RNAs that target mRNA stability and/or limit protein translation, consequently regulating critical cellular processes. Recent studies have identified miRNAs expressed in resting and activated mouse and human NK cells, and have shown that global miRNA deficiency results in altered mature NK cell functional responses in vitro and in vivo. However, our understanding of how individual miRNAs regulate NK cell biology is limited. Of multiple candidate miRNAs that are expressed and have a rationale for regulating NK cell function, miR-155 was prioritized at the highest for in depth study. This application describes a 5 year plan to study the regulatory role o miR-155 on NK cell function. We hypothesize that miR-155 regulates key aspects of NK cell functionality, including effector cytokine (e.g., IFN-γ) production, cytotoxicity, and intracellula pathways important for promoting or inhibiting NK cell activation. Our approach utilizes NK cell-specific (Ncr1-iCre) conditional gain (via loxP-STOP-loxP overexpression) and loss (floxed) of function models for miR-155. In Aim 1 miR-155 mouse models will be used to define the regulatory contribution of miR-155 to NK cell function, including development/maturation, IFN-γ production, and cytotoxicity. In Aim 2 we will elucidate the regulatory mechanism(s) of miR-155 by defining their target mRNAs in NK cells using bioinformatics, mRNA profiling, and Argonaute-immunoprecipitation followed by next-generation sequencing. These targets will in turn be assessed in functional experiments, described in Aims 1 and 3, and confirmed in human NK cells. In Aim 3 we will define the in vivo significance of miR-155 regulation on NK cell function using model pathogens. There is a high level of integration between Aim 2 and Aims 1 and 3, to deeply define mechanisms whereby miR-155 targets specific mRNAs, which in turn regulate NK cell function. Thus, these studies will provide novel information about how miR-155 regulates NK cell activation, their mechanism of action by identifying mRNA targets, and the importance of miR-155 to NK cell-mediated host defense in vivo. These studies may therefore inform future strategies to augment NK cell functionality as an immunotherapeutic strategy.

描述（由申请人提供）：本申请的长期目标是定义调节NK细胞发育和功能的分子事件，从而深入了解可能被操纵以促进或抑制NK细胞生物学这些方面的途径。NK细胞是先天免疫淋巴细胞，在宿主防御感染和介导抗肿瘤免疫反应中起重要作用。罕见的选择性NK细胞缺陷患者在生命早期死于致命的疱疹病毒感染，证明了这些细胞对人类健康的至关重要性。此外，同种异体NK细胞可以有效治疗急性髓性白血病患者，因此NK细胞有望成为一种癌症免疫治疗方法。目前，我们对调节NK细胞功能（细胞因子产生、细胞毒性、增殖）的分子机制还不完全了解。MicroRNAs （miRNAs）是一种小的调控rna，其目标是mRNA的稳定性和/或限制蛋白质的翻译，从而调节关键的细胞过程。最近的研究已经确定了静息和激活的小鼠和人类NK细胞中表达的miRNA，并表明在体外和体内，全球miRNA缺乏会导致成熟NK细胞功能反应的改变。然而，我们对单个mirna如何调节NK细胞生物学的理解是有限的。在表达的多个候选mirna中，miR-155在深入研究中被优先考虑。该申请描述了研究miR-155对NK细胞功能调节作用的5年计划。我们假设miR-155调节NK细胞功能的关键方面，包括效应细胞因子（如IFN-γ）的产生、细胞毒性和促进或抑制NK细胞活化的重要细胞内通路。我们的方法利用NK细胞特异性（Ncr1-iCre）条件增益（通过loxP-STOP-loxP过表达）和miR-155功能模型的损失（floxed）。在Aim 1中，miR-155小鼠模型将用于定义miR-155对NK细胞功能的调节作用，包括发育/成熟、IFN-γ产生和细胞毒性。在Aim 2中，我们将通过生物信息学、mRNA谱分析和argonaute免疫沉淀以及下一代测序来确定NK细胞中miR-155的靶mRNA，从而阐明miR-155的调控机制。这些靶点将在功能实验中进行评估，在目标1和3中描述，并在人类NK细胞中得到证实。在Aim 3中，我们将使用模型病原体定义miR-155调控NK细胞功能的体内意义。Aims 2与Aims 1和Aims 3之间存在高度整合，以深入定义miR-155靶向特定mrna的机制，从而调节NK细胞功能。因此，这些研究将提供关于miR-155如何调节NK细胞活化，其通过识别mRNA靶点的作用机制，以及miR-155在体内对NK细胞介导的宿主防御的重要性的新信息。因此，这些研究可能为未来增强NK细胞功能作为免疫治疗策略提供信息。