细胞内核仁素在VEGF介导的血管新生中的作用及其机制

It has been reported that nucleolin expressed on the endothelial cell surface is involved in regulation of angiogenesis, however, nucleolin，distributing mainly within the cells, is a multifunctional RNA binding protein which regulates target genes expression at post-transcriptional level. What kind of roles of intracellular nucleolin plays in VEGF-mediated angiogenesis and how it works, these two questions remain unclear. In previous works, we found that (i) nucleolin expression was up-regulated in VEGF-treated endothelial cells and localized mainly in non-membrane protein fraction; (ii) by using bioinformatics searching engine, 13 genes including COX2, MM9, eNOS etc were found that their mRNA sequences contain the specific nucleolin binding element for interacting with nucleolin protein; (iii) down-regulation of nucleolin inhibited, while enhanced expression of nucleolin increased VEGF-mediated tube formation and expression of several angiogenesis-related genes, such as COX-2, eNOS, MMP9,endoglin, integrin αV etc;(iv) Nucleolin can bind to the mRNA of MMP9, endoglin and integrin αV by using co-immunoprecipitation analysis on RNA-protein interaction. In present, based on these findings, we hypothesize that intracellular nucleolin regulates positively VEGF-mediated angiogenesis by specifically interacting with several angiogenesis-related genes mRNA and controlling their stability. Our project was designed based on our previous works. To clarify the novel mechanisms by which intracellular nucleolin regulates angiogenesis, and provide new clues for prevention and treatment of ischemic heart disease, we will, from aspects of protein-RNA interaction and mRNA stability (regulation of genes expression at post-transcriptional level), perform in-depth study on the roles of nucleolin in VEGF-mediated angiogenesis and investigate how nucleolin control the mRNA stability of angiogenesis-associated genes by using cell and transgenic mouse models.

细胞内重要的RNA结合蛋白- - 核仁素在血管新生中发挥何种作用及如何发挥作用，目前尚不清楚。申请者前期发现：①VEGF可诱导核仁素表达上调，且主要分布在细胞内,而不是移位至胞膜；②生物信息学分析发现MMP9，整合素αV等13个血管新生相关基因的mRNA中含有核仁素结合元件。③核仁素低表达明显抑制VEGF介导的内皮细胞管型形成和血管新生相关基因表达，过表达核仁素增强VEGF的促血管新生作用及上述相关基因表达；④免疫沉淀分析初步显示核仁素可与MMP9等3个基因的mRNA结合。据此，申请者推测细胞内核仁素通过与某些血管新生相关基因mRNA结合，影响其稳定性，而正性调节VEGF介导的血管新生。本项目拟进一步从蛋白质-mRNA相互作用角度,以细胞和核仁素转基因动物为模型，探讨细胞内核仁素对VEGF介导的血管新生的影响及其机制，以阐明核仁素在血管新生中的作用新机制，为缺血性心脏病的防治提供新的实验线索。

核仁素是一个主要位于细胞核仁中的磷酸化蛋白，可穿梭于胞核胞浆甚至细胞膜表面。核仁素是一个多功能蛋白参与rDNA转录、rRNA合成、运输、核糖体装配，细胞增殖、凋亡等过程，细胞膜表面核仁素以受体形式介导多种因子的促/抑血管新生作用，然而细胞内核仁素在血管新生过程有何影响，如何影响，并不清楚。在本项目资助下，围绕核仁素具有RNA结合并在转录后调节基因表达的特性，从整体细胞水平，采用转基因工程动物以及分子细胞生物学技术，深入探讨了上述两个问题。主要发现如下：1、证实了核仁素的促血管新生作用及其在转录后调控血管新生相关基因表达的分子机制，并找到了核仁素调控的mRNA靶VEGF和MMP9。同时建立了心肌缺血时血管新生过程中核仁素介导心肌细胞与内皮细胞之间的交互作用理论。2、证实了羟基红花黄色素A单体（HSYA）的促血管新生和改善心功能的作用及其分子机制：HSYA上调核仁素表达促进血管新生，并通过核仁素稳定血管新生相关基因MMP9和VEGFa mRNA，促进其蛋白表达，从而促进血管新生。3、证实了HSYA心肌细胞保护作用及其分子机制，阐明了HSYA通过上调心肌细胞HSP70表达，促进HSP70与核仁素相互作用，稳定核仁素蛋白水平，从而通过核仁素上调BCL-2/BAX比值，而抑制线粒体凋亡信号通路的活化，发挥抗心肌细胞凋亡作用。上述研究充分显示核仁素在缺血心脏血管新生中的多方位调节作用及其机制，亦为以核仁素为靶点的血管新生调控药物的研发提供了理论依据。