MiR-1 is a Critical Regulator of VEGF-Induced Angiogenesis

MiR-1 是 VEGF 诱导的血管生成的关键调节因子

• 批准号: 7777216
• 负责人: Seyedtaghi Takyar
• 金额: $ 11.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777216
• 关键词: Angiogenic Factor; Biological; Blood Circulation; Blood Vessels; Cell Culture Techniques; Cell Differentiation process; Cell Proliferation; Cells; Chronic; Data; Development; Disease; Down-Regulation; ENG gene; Embryonic Development; Endothelial Cells; Functional RNA; Future; Gene Expression; Growth; Growth Factor; Healed; Hemorrhage; In Vitro; Invaded; Lung; Maintenance; Malignant Neoplasms; Measures; Messenger RNA; MicroRNAs; Mus; Neoplasm Metastasis; Neoplasms; PECAM1 gene; PTPRC gene; Pathologic Processes; Pathway interactions; Permeability; Phosphorylation; Play; Precipitation; Process; Property; RNA-Induced Silencing Complex; Recruitment Activity; Reverse Transcriptase Polymerase Chain Reaction; Role; Route; Signal Pathway; Signal Transduction; Small RNA; Sorting - Cell Movement; Staining method; Stains; Supplementation; Technology; Testing; Tissues; Trachea; Transgenic Mice; Transgenic Model; Transgenic Organisms; Translations; Tube; VEGF165; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor B; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factors; Wound Healing; Wounds and Injuries; angiogenesis; carcinogenesis; healing; in vivo; injury and repair; migration; neovascularization; novel; public health relevance; research study; response; stem; tumor; vascular bed

DESCRIPTION (provided by applicant): Project Summary VEGF (Vascular Endothelial Growth Factor) is one of the main angiogenic factors involved in the development and maintenance of blood vessels and is shown to play a central role in various pathological processes such as wound healing, carcinogenesis and metastasis. MicroRNAs are a recently recognized group of non-coding small RNA molecules that regulate gene expression by inhibiting the translation of mRNAs or facilitating their degradation. We characterized the lung microRNA profile of the CC10-rtTA-VEGF transgenic mice in microarray experiments and confirmed our findings by stem-loop RT-PCR. The level of miR-1 was quantified in (CD45-, CD31+) and (CD45-, CD105+) cells separated from total lung by FACS sorting. We measured the level of miR- 1 in primary mouse lung endothelial cells in vitro after stimulation with VEGF. The effect of miR-1 supplementation on VEGF-induced proliferation and cord formation was studied in cell culture. The in vivo effect of miR-1 on angiogenesis after intranasal delivery was characterized by staining of the trachea from VEGF transgenic mice with CD31. We found that the levels of miR-1 were consistently diminished in the total lung RNAs from CC10-rtTa- VEGF transgenic mice, endothelial cells of these mice separated by FACS sorting, and mouse primary lung endothelial cells stimulated by VEGF in vitro, each compared to appropriate controls. MiR-1 supplementation downregulated VEGF-induced endothelial cell proliferation and cord formation in vitro and intranasal delivery of miR-1 inhibited angiogenesis in the bronchial circulation in vivo. Hypothesis: MiR-1 is a critical regulator of VEGF-induced endothelial cell responses To test the validity of this hypothesis we propose to: Aim #1: Characterize the spectrum of VEGF-induced endothelial cell responses regulated by miR-1 in vitro. Aim #2: Characterize the effect of miR-1 over-expression on VEGF responses in vivo (by A. direct delivery and B. transgenic modeling). Future Directions: Define the mechanism of miR-1 effects (by A. characterizing the effect of miR-1 on signaling and, B: identifying mRNAs recruited to RNA Induced Silencing complex (RISC). PUBLIC HEALTH RELEVANCE: Project Narrative "Angiogenesis" means growth of blood vessels anywhere in the body, and this process is crucial for normal development and healing of wounds and injuries. However, if this process becomes excessive it can cause chronic bleeding and a non-healing wound. Tumors also need blood vessels to grow and invade the surrounding tissues, so they secrete substances (growth factors) that promote the growth of vasculature around them. We have found that one of the small cellular RNAs produced within the vascular cells can inhibit the excessive vessel growth in response to the above growth factors and can be used to modulate the tendency of blood vessels to grow excessively in cancer and chronic wounds.

描述（由申请人提供）：项目概述VEGF（血管内皮生长因子）是参与血管发育和维持的主要血管生成因子之一，并显示在各种病理过程中发挥核心作用，如伤口愈合、致癌和转移。MicroRNA是近年来发现的一类非编码小RNA分子，通过抑制mRNA的翻译或促进其降解来调节基因表达。 我们在微阵列实验中表征了CC 10-rtTA-VEGF转基因小鼠的肺microRNA谱，并通过茎环RT-PCR证实了我们的发现。在通过FACS分选从总肺分离的（CD 45-，CD 31+）和（CD 45-，CD 105+）细胞中定量miR-1的水平。我们在体外检测了VEGF刺激后原代小鼠肺内皮细胞中miR- 1的水平。在细胞培养中研究了miR-1补充对VEGF诱导的增殖和索形成的影响。通过用CD 31对VEGF转基因小鼠的气管进行染色来表征鼻内递送后miR-1对血管生成的体内作用。 我们发现，与适当的对照相比，来自CC 10-rtTa-VEGF转基因小鼠、通过FACS分选分离的这些小鼠的内皮细胞和体外由VEGF刺激的小鼠原代肺内皮细胞的总肺RNA中的miR-1水平一致地降低。miR-1补充下调VEGF诱导的内皮细胞增殖和体外索形成，并且miR-1的鼻内递送抑制体内支气管循环中的血管生成。假设：miR-1是VEGF诱导的内皮细胞反应的关键调节剂为了测试这一假设的有效性，我们提出：目的#1：表征由miR-1在体外调节的VEGF诱导的内皮细胞反应的谱。目的#2：表征miR-1过表达对体内VEGF应答的影响（通过A.直接交付和B.转基因建模）。未来方向：定义miR-1效应的机制（A.表征miR-1对信号传导的作用，以及，B：鉴定募集到RNA诱导沉默复合物（RISC）的mRNA。 公共卫生关系：“血管生成”是指血管在身体任何地方的生长，这一过程对伤口和损伤的正常发育和愈合至关重要。然而，如果这个过程变得过度，它可能会导致慢性出血和不愈合的伤口。肿瘤还需要血管生长并侵入周围组织，因此它们分泌促进周围血管生长的物质（生长因子）。我们已经发现，在血管细胞内产生的小细胞RNA之一可以抑制响应于上述生长因子的过度血管生长，并且可以用于调节血管在癌症和慢性伤口中过度生长的趋势。