The role of circular RNA AEBP2 in dendritic cells

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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.