MicroRNAs in dendritic cell differentiation and function

描述(申请人提供)：树突状细胞(DC)是控制先天免疫反应和获得性免疫反应的最有效的抗原提呈细胞。DC不断地与造血祖细胞区分开来，确保在稳定状态和免疫挑战期间保持自我与非自我的区别。然而，树突状细胞是如何在分子水平上发育的，目前还不完全清楚。MicroRNAs(MiRNAs)是发育、细胞增殖、分化和肿瘤发生过程中的关键调控分子之一。我们的长期目标是识别特定的miRNAs，并确定它们在DC发展和功能中的角色。该项目的目的是确定特定的miRNAs是否在人类单核细胞来源的DC分化和功能中起关键作用。我们的假设是，单核细胞分化为不同的未成熟和成熟DC的过程以及分化细胞的免疫功能受特定miRNAs的调控。在目标1中，我们将识别并验证由特定的miRNA调控的靶基因，该miRNA在未成熟和成熟的DC中都高度上调。我们的初步数据表明，该miRNA可能通过反馈机制在调节DC凋亡中发挥作用。为了确定miRNA靶点，我们将使用来自过表达或沉默miRNA表达的未成熟DC的RNA进行mRNA表达微阵列。在这两个实验中都将对mRNA的表达进行分析，并将识别相交的降低和增加的mRNA。将进行分子验证分析(荧光素酶报告实验、qPCR、Northern和Western印迹)以及敲除或过度表达候选基因后的DC细胞凋亡分析。在目标2中，我们将评估三种特定miRNAs的表达变化对体外培养的人DC发育和功能的影响。我们的初步数据表明，这些miRNAs的表达在DC发育过程中发生了显著变化。为了解决这个问题，将在单核细胞和DC中进行沉默或过度表达这些miRNAs。改变这些miRNA表达水平对DC发育、成熟和生理功能的影响将通过分子和免疫学分析。这项拟议的研究具有创新性和重要意义，因为miRNA在DC发育和功能中的作用仍未被探索。将发现在DC发育过程中受miRNAs差异调控的新靶基因。我们的工作也具有翻译相关性，因为人们对开发使用miRNAs来调节DC的发育和功能的临床应用非常感兴趣，这些疾病包括感染、自身免疫、癌症、与移植相关的移植物抗宿主病和移植物排斥反应。 与公共健康相关：我们的研究将确定调节人类树突状细胞的发育和功能的新型小RNA分子，树突状细胞是维持免疫系统平衡的最有效的白细胞类型。我们的研究可能导致开发新的小分子来调节树突状细胞的发育和在感染、过敏、癌症、自身免疫和移植中的功能。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) are the most potent type of antigen-presenting cells controlling both innate and adaptive immune responses. DCs are continually differentiated from hematopoietic progenitors, ensuring that self versus non-self discrimination is maintained in the steady-state and during immunological challenges. However, how DCs are developed at the molecular level remains incompletely understood. MicroRNAs (miRNAs) have emerged as one of the critical regulatory molecules in development, cell proliferation, differentiation and tumorigenesis. Our long-term goal is to identify specific miRNAs and define their roles in DC development and function. The objective of this project is to determine whether specific miRNAs are critical in human monocyte-derived DC differentiation and function. Our hypothesis is that the process of monocyte differentiation into distinct immature and mature DCs and the immunological functions of differentiated cells are regulated by specific miRNAs. In Aim 1, we will identify and validate the target genes regulated by a specific miRNA that is highly up-regulated in both immature and mature DCs. Our preliminary data suggest that this miRNA may play a role in modulating DC apoptosis through a feedback mechanism. To identify the miRNA targets, we will perform an mRNA expression microarray using RNA from immature DCs that are either over- expressing or have this miRNA expression silenced. Expression of mRNA will be profiled in both experiments, and the intersected mRNA that are decreased and increased will be identified. Molecular validation assays (a luciferase reporter assay, qPCR, Northern and Western blot) and DC apoptosis analysis after knocking down or over-expressing the candidate gene will be carried out. In Aim 2, we will evaluate the impact of altered expression of three specific miRNAs on human DC development and function in vitro. Our preliminary data demonstrate that expression of these miRNAs is significantly altered during DC development. To address this question, silencing or over-expressing these miRNAs will be performed in monocytes and DCs. The effects of altering these miRNA expression levels on DC development, maturation and physiological functions will be analyzed using both molecular and immunological assays. The proposed research is innovative and significant because the role of miRNA in DC development and function remains unexplored. New target genes that are differentially regulated by miRNAs during DC development will be identified. Our work also has translational relevance, as there is a great deal of interest in developing clinical applications using miRNAs to modulate DC development and functions in a broad spectrum of diseases including infections, autoimmunity, cancer, transplantation-related graft versus host disease, and graft rejection PUBLIC HEALTH RELEVANCE: Our research will identify novel small RNA molecules that regulate the development and function of human dendritic cells, the most potent type of white blood cells that keep the immune system in balance. Our research may lead to the development of new small molecules to modulate dendritic cell development and function in infection, allergy, cancer, autoimmunity, and transplantation.