MicroRNAs in dendritic cell differentiation and function

描述（由申请人提供）：树突状细胞（DC）是控制先天性和适应性免疫应答的最有效类型的抗原呈递细胞。DC不断从造血祖细胞分化，确保在稳态和免疫挑战期间维持自身与非自身的区分。然而，DC如何在分子水平上发育仍然不完全清楚。microRNAs（miRNAs）是细胞发育、增殖、分化和肿瘤发生的重要调控分子。我们的长期目标是鉴定特定的miRNAs并确定它们在DC发育和功能中的作用。本项目的目的是确定特定的miRNAs是否在人单核细胞来源的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的DC的过程和分化细胞的免疫功能是由特定的miRNA调节。在目标1中，我们将鉴定和验证由特定miRNA调控的靶基因，该miRNA在未成熟和成熟DC中均高度上调。我们的初步数据表明，这种miRNA可能通过反馈机制在调节DC细胞凋亡中发挥作用。为了鉴定miRNA靶标，我们将使用来自过表达或该miRNA表达沉默的未成熟DC的RNA进行mRNA表达微阵列。将在两个实验中分析mRNA的表达，并鉴定降低和增加的mRNA。敲低或过表达候选基因后，将进行分子验证试验（荧光素酶报告基因试验、qPCR、北方和Western印迹）和DC凋亡分析。在目标2中，我们将评估三种特异性miRNA的表达改变对体外人DC发育和功能的影响。我们的初步数据表明，这些miRNA的表达在DC发育过程中发生了显着变化。为了解决这个问题，将在单核细胞和DC中进行沉默或过表达这些miRNA。改变这些miRNA表达水平对DC发育、成熟和生理功能的影响将使用分子和免疫学测定来分析。这项研究具有创新性和重要性，因为miRNA在DC发育和功能中的作用尚未探索。将鉴定在DC发育期间由miRNA差异调节的新靶基因。我们的工作也具有翻译相关性，因为在开发临床应用方面有很大的兴趣，使用miRNAs来调节DC在广泛的疾病中的发育和功能，包括感染、自身免疫、癌症、移植相关的移植物抗宿主病和移植物排斥 公共卫生相关性：我们的研究将确定新的小RNA分子，调节人类树突细胞的发育和功能，树突细胞是保持免疫系统平衡的最有效的白色血细胞类型。我们的研究可能会导致新的小分子的发展，以调节树突状细胞的发育和功能的感染，过敏，癌症，自身免疫和移植。

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.