血管生成素特异microRNAs的鉴定与功能分析

发布时间：2018-04-03 02:23

本文选题：ANG　切入点：microRNA　出处：《浙江大学》2010年博士论文

【摘要】：血管生成素(Angiogenin, ANG;亦称RNase A family 5)可以特异性地定位于血管内皮细胞及一些肿瘤细胞的细胞核,在血管新生和肿瘤发生中起重要作用,但其具体的作用机制尚未完全明了。microRNA是一类非编码小RNA,通过在转录后水平对基因表达的调控而影响生物学过程,包括血管生成及肿瘤形成等,但目前并不了解其与ANG的关系。为此,本论文从直接受ANG调控的microRNAs和直接靶向调控ANG的microRNAs两个方面研究了ANG特异的microRNAs及其生物学功能。为探索受ANG影响和调控的microRNAs,本论文首先利用microRNA芯片筛选的方法,在血管内皮细胞中鉴定受ANG刺激后表达发生改变的microRNAs。通过筛选,共得到了26个在ANG刺激前后发生差异表达的microRNAs,包括17个上调和9个下调的microRNAs.生物信息学分析显示,这些microRNAs参与多种生物学过程,包括肿瘤发生和转移、血管生成、神经发育、细胞凋亡等。利用数据库MicroCosm,初步预测了这些microRNAs的靶基因。另一方面,本论文利用ChIP-on-Chip方法探索了ANG与microRNAs启动子区的结合情况,发现有121个microRNAs启动子区可能与ANG结合,其中有9个启动子控制的microRNAs与上述利用microRNA芯片筛选得到的受ANG直接调控的microRNAs相同,包括8个上调和1个下调的microRNAs。在此基础上,利用ChIP-QPCR的方法对所得到的部分microRNA启动子区做进一步验证,发现miR-149、miR-17、miR-378和miR-641等确实受ANG的调控,说明ANG具有转录因子的活性,可以通过结合microRNAs的启动子区而对其表达水平进行调控。 ANG是一个在血管内皮细胞和肿瘤细胞中高表达的蛋白质,本身也可能受到microRNAs的靶向调控。为此,本论文首先通过生物信息学方法预测得到8个可能靶向结合到ANG基因3'UTR的microRNAs,然后分析了它们对ANG mRNA和蛋白质表达水平的影响,发现miR-1208、miR-196b、miR-296、miR-409、miR-570和miR-641共六个microRNAs确实可以调节ANG的表达,并对靶细胞的增殖、迁移、粘附和管腔形成均有一定影响。在此基础上,本论文着重对miR-409进行了较深入的研究,发现miR-409不仅可以抑制内皮细胞的管腔形成和细胞增殖,也可以抑制肿瘤细胞HT1080的增殖及血管拟态；小鼠移植瘤实验也证明了miR-409可以抑制肿瘤的生长、血管生成和转移。进一步,本论文在结肠癌病人的组织中发现miR-409的表达较相应癌旁组织的表达下降,并且miR-409的表达与肿瘤的转移密切相关。以上结果说明ANG本身可以受多种microRNAs的调控,其中miR-409可以直接靶向下调ANG的表达,影响ANG在血管生成及肿瘤发生、发展中的作用。综合上述两部分的研究结果,说明ANG不仅可以调控microRNAs的表达,而且其本身也同时被多种microRNAs所调控。通过这些microRNAs组成的调控网络,一方面决定了细胞中ANG的表达水平,另一方面也放大了ANG对细胞行为的影响,从而实现其对血管生成和肿瘤发生的促进作用。
[Abstract]:Angiogenin A family 5 (RNase A family 5) can specifically locate the nuclei of vascular endothelial cells and some tumor cells, and play an important role in angiogenesis and tumorigenesis.However, its specific mechanism is not fully understood. MicroRNA is a class of non-coding small RNAs that affect biological processes, including angiogenesis and tumor formation, through the regulation of gene expression at post-transcriptional level, but the relationship between microRNA and ANG is not understood.Therefore, the specific microRNAs and its biological functions of ANG were studied from two aspects: microRNAs regulated directly by ANG and microRNAs which regulated ANG directly.In order to explore the microRNAs affected and regulated by ANG, the microRNAs stimulated by ANG were identified in vascular endothelial cells by microRNA microarray screening method.A total of 26 microRNAs differentially expressed before and after ANG stimulation were obtained, including 17 up-regulated and 9 down-regulated microRNAs.Bioinformatics analysis shows that these microRNAs are involved in many biological processes, including tumorigenesis and metastasis, angiogenesis, neurogenesis, apoptosis and so on.Using the database MicroCosm, the target genes of these microRNAs were preliminarily predicted.On the other hand, we use ChIP-on-Chip method to explore the binding between ANG and microRNAs promoter, and we find that 121 microRNAs promoters may bind to ANG.Nine of the promoter-controlled microRNAs were the same as the microRNAs directly regulated by ANG, including 8 up-regulated and 1 down-regulated microRNAs.On this basis, the ChIP-QPCR method was used to further verify some of the microRNA promoter regions. It was found that miR-149 miR-17 miR-378 and miR-641 were indeed regulated by ANG, which indicated that ANG had the activity of transcription factors.The expression level of microRNAs can be regulated by binding to its promoter region.ANG is a highly expressed protein in vascular endothelial cells and tumor cells and may be regulated by microRNAs.In this paper, eight microRNASs that might be targeted to ANG gene 3'UTR were predicted by bioinformatics, and their effects on ANG mRNA and protein expression were analyzed.It was found that miR-1208 miR-296 miR-296 miR-409 miR-570 and miR-641 could regulate the expression of ANG, and had some effects on the proliferation, migration, adhesion and lumen formation of target cells.On this basis, the miR-409 was studied in depth. It was found that miR-409 could not only inhibit the formation and proliferation of endothelial cells, but also inhibit the proliferation and vascular mimicry of HT1080.Mouse tumor transplantation test also demonstrated that miR-409 can inhibit tumor growth, angiogenesis and metastasis.Furthermore, we found that the expression of miR-409 in colon cancer tissues was lower than that in corresponding adjacent tissues, and the expression of miR-409 was closely related to tumor metastasis.These results suggest that ANG itself can be regulated by a variety of microRNAs, in which miR-409 can directly target down-regulate the expression of ANG, and affect the role of ANG in angiogenesis, tumorigenesis and development.The results show that ANG can not only regulate the expression of microRNAs, but also be regulated by many kinds of microRNAs.Through these regulatory networks of microRNAs, the expression level of ANG in cells is determined on the one hand, and the effect of ANG on cell behavior is magnified on the other hand, so that it can promote angiogenesis and tumorigenesis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

【共引文献】