miR-146a和miR-10a/b在斑马鱼胚胎血管发育中的功能研究

During vertebrate embryogenesis, the stereotyped pattern of the circulatory system is initially established by conserved genetic pathways. MicroRNAs (miRNAs) are single-stranded small none-coding RNAs, generally 19-25 nucleotides in length, that act as repressors of gene expression. It is evidenced that miRNAs regulate vascular development, but systematic analysis of miRNAs profile in endothelial cells (ECs) is thus far lacking. Lately, we took advantage of deep sequencing analysis to examine the miRNAs expression in arterial and venous ECs derived from human, chicken embryo, and zebrafish embryo. Around 40 common miRNAs were found to be conservatively presented in ECs of all the three species. Among the common group, the miR-146a and miR-10a/b are abundantly expressed. Furthermore, each expression level of the three miRNAs in zebrafish ECs is higher than that of the miR-126/miR-126b, which is recognized as endothelial specific miRNA. Bioinformatical analysis reveals that miR-146a and miR-10a/b potentially regulate several genes, involved in blood vessel development, including gng2, krit1, fli1b and mib, etc. In addition, our preliminary data show one of the miRNAs regulated the tip cell formation in branching intersegmental vessels. Up to date, there is no report to show the experimental evidence regarding the three miRNAs specifically involved in genetic pathways that control blood vessel development and zebrafish embryonic development. In current project, for the first time we suppose to investigate the roles of miR-146a and miR-10a/b in regulating zebrafish embryonic blood vessel formation. The outcome of this project will help understanding the full repertoire of action of miRNAs in vasculogenesis and angiogenesis and allow us to open a new avenue for target-designing drugs to control the formation of blood vessels in a variety of pathological conditions caused by insufficient or excessive blood supply.

MicroRNA(miRNA)是内源性的单链非编码小RNA，通过与靶基因mRNA的3'UTR结合，抑制其表达。miRNA被证实：通过调节基因表达来调控内皮细胞行为和血管发育。但是目前系统分析血管内皮细胞miRNA 表达谱的研究还很欠缺。我们利用流式细胞仪(FACs)从22小时Tg(fli1:EGFP)斑马鱼中分选出内皮细胞，另获得鸡和人内皮细胞。深度测序(deep sequencing)分析这些内皮细胞的miRNA表达，发现约40个在这些物种均有表达，在进化上保守。其中miR-146a和miR-10a/b不仅表达于斑马鱼、鸡和人类内皮细胞，而且在斑马鱼内皮细胞中的表达水平高于内皮细胞特异高表达的miR-126s，生物信息学分析显示它们可能调控一系列血管发育相关基因。此外预实验提示其中一个miRNA下调影响tip cell的形成。因此本项目拟研究它们在斑马鱼胚胎血管形成中的功能。

MicroRNA(miRNA)是内源性的单链非编码小 RNA，通过与靶基因 mRNA 的 3’UTR 结合，抑制其表达。miRNA 被证实通过调节基因表达来调控内皮细胞行为和血管发育。但是目前系统分析血管内皮细胞 miRNA 表达谱的研究还很欠缺。我们利用流式细胞仪(FACs) 从受精后22 小时 Tg(fli1:EGFP)斑马鱼中分选出内皮细胞，此外我们通过手术另获得鸡胚的内皮细胞并且购买了人脐带血管的动脉和静脉内皮细胞。深度测序(deep sequencing)分析这些内皮细胞 miRNA的表达，发现约40个miRNA在这三个物种的血管内皮细胞均有表达，提示其在进化上保守。其中 miR-146a 和 miR-10a/b 不仅表达于斑马鱼、鸡和人类内皮细胞，而且在斑马鱼内皮细胞中的表达水平高于内皮细胞特异高表达的 miR-126s，生物信息学分析显示它们可能调控一系列血管发育相关基因。本项目研究中我们在已取得数据的基础上，进一步确定 miR-146a 和 miR-10a/b 在斑马鱼血管内皮 细胞的时空表达谱。建立了miR-146a 和 miR-10a/b 下调的斑马鱼胚胎模型。此外实验结果显示其中 miR-10 a/b下调影响内皮tip cell的形成。因此在之后的工作中我们主要聚焦在miR-10 a/b的功能研究上。然后进一步的确定了miR-10a/b 下调后导致斑马鱼胚胎血管系统发育异常的相关表型并且对表型进行细胞生物学机制阐释。最终我们找到了一个miR-10a/b调控血管新生的一个新的靶基因mib1。除此之外，我们还在人的内皮细胞模型上验证了本结论，结果显示：miR-10a/b调控血管内皮细胞的行为在进化上保守。本研究结果会进一步帮助理解 miRNA 如何调控血管的新生，为之后靶向设计药物来控制血管的形成提供理论依据。