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

项目概述：血管内皮细胞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，在伤口愈合、癌变和转移等多种病理过程中发挥核心作用。MicroRNAs是最近发现的一组非编码小RNA分子，通过抑制mRNAs的翻译或促进其降解来调节基因的表达。我们在基因芯片实验中对CC10-RTTA-VEGF转基因小鼠的肺microRNA图谱进行了表征，并通过茎环RT-PCR证实了我们的发现。用流式细胞仪对分离的(CD45-，CD31+)和(CD45-，CD105+)细胞进行miR-1定量。我们测定了体外培养的小鼠肺内皮细胞经血管内皮生长因子刺激后miR-1的表达水平。在细胞培养中研究miR-1对血管内皮生长因子诱导的细胞增殖和脐带形成的影响。通过用CD31对血管内皮生长因子转基因小鼠的气管进行染色，研究miR-1对鼻腔给药后血管生成的体内影响。我们发现，在CC10-RTTA-VEGF转基因小鼠的总肺RNA中，miR-1的水平持续降低，这些小鼠的内皮细胞经FACS分选分离，以及在体外经VEGF刺激的小鼠原代肺内皮细胞中，每一个都与适当的对照相比都是降低的。在体外，补充miR-1可抑制血管内皮生长因子诱导的内皮细胞的增殖和脐带的形成，而在体内，经鼻给药的miR-1可抑制支气管循环中的血管生成。假设：MIR-1是血管内皮生长因子诱导内皮细胞反应的关键调节因子。为了验证这一假说的有效性，我们提出：目的1：在体外研究由miR-1调控的血管内皮生长因子诱导的内皮细胞反应谱。目的#2：通过A.直接转染法和B.转基因模型研究miR-1过表达对体内血管内皮细胞生长因子反应的影响。未来方向：确定miR-1效应的机制(通过A.表征miR-1对信号的影响；以及B：识别招募到RNA诱导沉默复合体(RISC)的mRNAs。

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).