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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761957
• 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）是一类新的内源表达非编码RNA，由反向剪接产生并形成共价闭环。新的证据表明，circRNA 调节基因表达并在许多生理过程中发挥重要作用。然而，目前尚不清楚 circRNA 是否参与作为免疫系统中心的树突状细胞 (DC) 的发育。 DC 是独特的专业抗原呈递细胞，能够桥接先天免疫系统和适应性免疫系统。尽管在了解 DC 发育和功能方面取得了进展，但参与 DC 发育的新分子调节剂仍有待揭晓，以充分了解 DC 的发育和功能。我们最近的 circRNA 微量测定结果表明，circRNA 可能是 DC 的一种新的有趣的调节因子，因为我们的初步数据表明 circRNA 在 DC 中大量表达，并且 circRNA 的表达谱在成熟 DC (mDC) 和未成熟 DC (imDC) 之间显着不同。环状 RNA AEBP2 (circAEBP2) 是这些改变的 circRNA 之一，并且在 mDC 中显着上调。此外，用 TGF beta 治疗可诱导 imDC 并下调 circAEBP2。生物信息学分析和 RNA 免疫沉淀分析 (RIP) 表明 circAEBP2 可能与 RelB 和 STAT1 结合。因此，我们假设 circAEBP2 是 DC 发育和通过与 RelB 和 STAT1 相互作用分化为成熟和免疫原性 DC 所必需的。有两个具体目标来检验我们的假设： 目标 1：研究 circAEBP2 在 DC 中的作用。我们将培养骨髓衍生DC，用circAEBP2 siRNA转染DC以敲低circAEBP2或用circAEBP2表达质粒转染上调circAEBP2，然后在混合淋巴细胞反应（MLR）中评估DC的成熟状态和功能（激活和极化T细胞并诱导调节性T细胞和耗竭T细胞）。目标 2：确定 circAEBP2 调节 DC 的分子机制。我们将对 circAEBP2 过度表达的 DC 进行一系列 RIP 测定，以使用 circAEBP 探针下拉 circAEBP2 结合蛋白，并使用抗 RelB 和 STAT1 蛋白的抗体下拉结合的 circRNA，然后进行蛋白质印迹和 qRT-PCR，以确定 circAEBP2 和这些蛋白的相互作用。 本研究的目标是探索circRNA的新作用，发现决定DC发育、分化和功能的新分子调节因子，这是基本的科学问题。拟议的研究处于circRNA和免疫学领域的前沿。将为学员提供良好的培训环境。 该研究的成功将为 DC 发育中的新分子调节剂以及 circAEBP2 对 DC 的影响提供见解。这项研究将增进我们对 circRNA、转录后调控和 DC 的了解。