The role of circular RNA AEBP2 in dendritic cells

环状RNA(CircRNAs)是一类新的内源性表达的非编码RNA，由后向剪接产生并形成共价闭合环。越来越多的证据表明，CircRNAs调控基因表达，并在许多生理过程中发挥重要作用。然而，目前尚不清楚CircRNA是否参与了树突状细胞(DC)的发育，树突状细胞是免疫系统的中心。 DC是一种独特的专职抗原提呈细胞，能够连接天然免疫系统和获得性免疫系统。尽管在了解DC的发展和功能方面取得了进展，但参与DC开发的新的分子调控因子仍有待于揭开面纱，以全面了解DC的发展和功能。我们最近的CircRNA微量分析结果表明，CircRNA可能是一种新的有趣的DC调节因子，因为我们的初步数据表明，CircRNAs在DC中大量表达，并且成熟DC(MDCs)和未成熟DC(ImDC)的CircRNAs表达谱存在显著差异。CircAEBP2(CircAEBP2)是这些改变的CircRNAs之一，在MDCS中显著上调。此外，用转化生长因子β治疗可诱导imDC并下调大约AEBP2的表达。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，我们假设大约AEBP2是DC发育和分化为成熟和免疫原性DC所必需的，通过与RelB和STAT1的相互作用。有两个特定的目标来检验我们的假设： 目的：探讨CircAEBP2在树突状细胞中的作用。我们将体外培养骨髓来源的DC，将含有CircAEBP2 siRNA的DC转染到下调的CircAEBP2或与CircAEBP2的表达载体共同上调CircAEBP2，然后评估DC在混合淋巴细胞反应(MLR)中的成熟状态和功能(激活和极化T细胞，诱导调节性T细胞和耗尽的T细胞)。 目的：探讨CircAEBP2调控树突状细胞的分子机制。我们将对CircAEBP2过表达的树突状细胞进行一系列RIP检测，以使用CircAEBP探针下调CircAEBP2结合蛋白，并使用抗RelB和STAT1蛋白的抗体下调CircRNAs，然后通过Western blotting和qRT-PCR来确定CircAEBP2与这些蛋白的相互作用。 本研究的目的是探索CircRNA的新作用，发现决定DC发育、分化和功能的新的分子调控因子，这是基本的科学问题。这项拟议的研究处于CircRNA和免疫学领域的前沿。它将为学员提供良好的培训环境。 这项研究的成功将为DC的发展提供新的分子调控因子，以及CircAEBP2对DC的影响。这项研究将促进我们对CircRNA、转录后调控和DC的了解。

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.