The role of circular RNA AEBP2 in dendritic cells

环状RNA AEBP2在树突状细胞中的作用

• 批准号: RGPIN-2019-04545
• 负责人: Zheng, Xiufen
• 金额: $ 2.33万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=684460
• 关键词: role; circular; RNA; AEBP2; dendritic

Circular RNAs (circRNAs) are a new class of endogenously expressed non-coding RNAs that are produced from back splicing and form a covalently closed loop. Emerging evidence is showing that circRNAs regulate gene expression and play an important role in many physiological processes. However, it remains unknown whether circRNAs participate in the development of dendritic cells (DCs) that are the centre of the immune system. ******DCs are unique professional antigen presenting cells capable of bridging the innate immune system and the adaptive immune system. New molecular regulators involved in DC development wait to be unveiled to fully understand the development and function of DCs despite advancement made in understanding DC development and function. Our recent results from circRNA microassay assays suggest that circRNA may be a new interesting regulators of DCs, as our preliminary data show that circRNAs are abundantly expressed in DCs and the expression profiles of circRNAs are significantly different between mature DCs (mDCs) and immature DCs (imDCs). circular RNA AEBP2 (circAEBP2) is one of these altered circRNAs and signifcantly up-regulated in mDCs. Additionally, treatment with TGF beta induces imDCs and downregulates circAEBP2. Bioinformatics analyses and RNA immunoprecipitate assays (RIP) suggest that circAEBP2 may bind to RelB and STAT1. Therefore, we hypothesize that circAEBP2 is required for DCs to develop and differentate towards mature and immuneogenic DCs through interaction with RelB and STAT1. There are two specific objectives to test our hypothesis: ******Aim1: To investigate the role of circAEBP2 in DCs. We will culture bone marrow derivd DCs, transfect DCs with circAEBP2 siRNA to knockdown circAEBP2 or with circAEBP2 expression plasmids to upregulate circAEBP2, then assess the maturation status and function of DCs (to activate and polarize T cells and induce regulatory T cells and exhausted T cells) in mixed lymphocyte reactions (MLR) .***Aim 2: To determine the molecular mechanisms by which circAEBP2 regulates DCs. We will peroform a series of RIP assays on circAEBP2 overe-expressed DCs to pull down circAEBP2 bound proteins using circAEBP probes, and to pull down bound circRNAs using antibodies against RelB and STAT1 protein, followed by Western blotting and qRT-PCR, to determine the interaction of circAEBP2 and those proteins. ******The goals of the present study are to explore a new role of circRNA and to discover new molecular regulators determining DC development, differentiation and function, which are the fundamental basic scientific questions. The proposed research is at the cutting-edge in the field of circRNA and immunology. It will offer an excellent training environment for trainees. ******The success of the study will provide insights into new molecular regulators in DC development and impact of circAEBP2 on DCs. This study will advance our knowledge on circRNA, post-transcriptional regulation and DCs.*****

环状RNA（CircRNA）是一类新的内源性表达的非编码RNA，由反向剪接产生，形成共价闭合环。越来越多的证据表明，circRNA调节基因表达，并在许多生理过程中发挥重要作用。然而，circRNA是否参与作为免疫系统中心的树突状细胞（DC）的发育仍然是未知的。** DC是独特的专职抗原呈递细胞，能够桥接先天免疫系统和适应性免疫系统。尽管对DC发育和功能的理解取得了进展，但参与DC发育的新分子调控因子有待于揭示，以充分了解DC的发育和功能。我们最近的结果从circRNA微量分析表明，circRNA可能是一个新的有趣的调节DCs，因为我们的初步数据显示，circRNA在DCs中大量表达，并且circRNA的表达谱在成熟DCs（mDCs）和未成熟DCs（imDCs）之间存在显著差异。 环状RNA AEBP 2（circAEBP 2）是这些改变的circRNA之一，并且在mDC中显著上调。此外，用TGF β处理诱导imDC并下调circAEBP 2。生物信息学分析和RNA免疫沉淀分析（RIP）表明circAEBP 2可能与RelB和STAT 1结合。因此，我们假设circAEBP 2是DC通过与RelB和STAT 1相互作用而向成熟和免疫原性DC发育和分化所必需的。有两个具体目标来检验我们的假设：** 目的1：研究circAEBP 2在DC中的作用。我们将培养骨髓来源的DC，用circAEBP 2 siRNA敲低circAEBP 2或用circAEBP 2表达质粒上调circAEBP 2，然后在混合淋巴细胞反应（MLR）中评估DC的成熟状态和功能（激活和活化T细胞，诱导调节性T细胞和耗竭性T细胞）。目的2：探讨circAEBP 2调控DC的分子机制。 我们将在circAEBP 2过表达的DC上进行一系列RIP测定，以使用circAEBP探针拉下circAEBP 2结合的蛋白，并使用针对RelB和STAT 1蛋白的抗体拉下结合的circRNA，随后进行Western印迹和qRT-PCR，以确定circAEBP 2和这些蛋白的相互作用。** 本研究的目标是探索circRNA的新作用，并发现决定DC发育，分化和功能的新分子调节剂，这是基本的基础科学问题。这项研究处于circRNA和免疫学领域的前沿。它将为学员提供一个良好的培训环境。** 这项研究的成功将为DC发育中的新分子调节剂以及circAEBP 2对DC的影响提供深入了解。这项研究将推进我们对circRNA，转录后调控和DC的认识。