Molecular Determinants of Vertebrate Vasculature and Development

脊椎动物脉管系统和发育的分子决定因素

• 批准号: RGPIN-2017-04787
• 负责人: Majumder, Mousumi
• 金额: $ 1.89万
• 依托单位: Brandon University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=710873
• 关键词: Molecular; Determinants; Vertebrate; Vasculature; Development

Background: Vertebrate vascular system is vital for blood circulation, gas ogenesis. Prostaglandin(PG) E2 promotes inflammation, can regulate stem cells and induce inflammation associated miRNAs by upregulating EP4, a receptor for PGE2. PGE2 mediated functions can be inhibited by blocking EP4 activity. The biology of PGE2 and miRNAs in vertebrate development and angiogenesis are not known. Long term goal: My research program will find novel molecular determinants responsible for embryonic growth and vasculogenesis in the vertebrate system. We shall determine the roles of PGE2 and miRNA in vertebrate development. I shall use Human Umbilical Vein Endothelial Cells (HUVEC) as in vitro model and Zebrafish as an in vivo model. Short-term objectives: Objective 1: To study angiogenic roles of PGE2 induced miRNAs in vitro. HUVEC cells will be treated with PGE2 to stimulate miRNA expression. miRNA will be overexpressed in this cells (HUVEC-miR) to test cell migration, proliferation, and tube formations, three functions require for angiogenesis. Objective 2: To investigate if EP4 activity regulates miRNA functions; we shall stimulate miRNA in HUVEC cells with EP4 agonist and inhibit miRNA functions in HUVEC-miR cells with EP4 antagonist. Functional dependence on EP4 mediated signaling (PI3K/AKT, ERK phosphorylation) will establish the link between miRNAs and EP4. Objective 3: (a) To study the role of the PGE2 in embryonic development and vasculogenesis in Zebrafish model; we shall inject PGE2 in fish embryo in a dose-dependent way and record developmental changes. We shall conduct microarrays with embryo extracts to identify miRNA changes. (b) To test miRNA functions, we shall knock down miRNAs in Zebrafish embryo and measure growth change. This pilot study will identify novel miRNAs regulated by PGE2 during vasculogenesis. Objective 4: To find gene expression changes due to PGE2 treatment in Zebrafish; we shall conduct microarrays in Vehicle and PGE2 treated fish embryo extracted mRNA to identify novel genes might involve in vertebrate development. Significance: With various approaches, this program will, therefore, add new determinants in the catalog of vertebrate development. Identification of the physiological roles of PGE2 and miRNA in vasculogenesis; EP4 signaling regulating miRNA functions, this program will, therefore, modify the course of currently used drugs targeting EP4.

背景：脊椎动物的血管系统对于血液循环、气体生成至关重要。前列腺素 (PG) E2 促进炎症，可以调节干细胞并通过上调 EP4（PGE2 受体）诱导炎症相关 miRNA。 PGE2 介导的功能可以通过阻断 EP4 活性来抑制。 PGE2 和 miRNA 在脊椎动物发育和血管生成中的生物学作用尚不清楚。 长期目标：我的研究计划将找到负责脊椎动物系统胚胎生长和血管生成的新型分子决定因素。我们将确定 PGE2 和 miRNA 在脊椎动物发育中的作用。我将使用人脐静脉内皮细胞（HUVEC）作为体外模型，使用斑马鱼作为体内模型。 短期目标： 目的1：研究PGE2诱导的miRNA的体外血管生成作用。 HUVEC 细胞将用 PGE2 处理以刺激 miRNA 表达。 miRNA 将在该细胞中过表达 (HUVEC-miR)，以测试细胞迁移、增殖和管形成，这是血管生成所需的三个功能。 目标2：探讨EP4活性是否调节miRNA功能；我们将用EP4激动剂刺激HUVEC细胞中的miRNA并用EP4拮抗剂抑制HUVEC-miR细胞中的miRNA功能。对 EP4 介导的信号传导（PI3K/AKT、ERK 磷酸化）的功能依赖性将建立 miRNA 和 EP4 之间的联系。 目标3：（a）研究PGE2在斑马鱼模型胚胎发育和血管生成中的作用；我们将以剂量依赖的方式将PGE2注射到鱼胚胎中并记录发育变化。我们将使用胚胎提取物进行微阵列分析，以鉴定 miRNA 的变化。 (b) 为了测试 miRNA 功能，我们将敲除斑马鱼胚胎中的 miRNA 并测量生长变化。这项试点研究将鉴定血管生成过程中受 PGE2 调节的新型 miRNA。 目标4：发现PGE2处理斑马鱼后基因表达的变化；我们将在载体和 PGE2 处理的鱼胚胎中提取 mRNA 进行微阵列，以鉴定可能参与脊椎动物发育的新基因。 意义：通过各种方法，该计划将因此在脊椎动物发育目录中添加新的决定因素。鉴定 PGE2 和 miRNA 在血管生成中的生理作用； EP4 信号传导调节 miRNA 功能，因此该程序将修改目前使用的针对 EP4 的药物的过程。