本文選題：ANG　切入點：microRNA　出處：《浙江大學》2010年博士論文

【摘要】：血管生成素(Angiogenin, ANG;亦稱RNase A family 5)可以特異性地定位于血管內(nèi)皮細胞及一些腫瘤細胞的細胞核,在血管新生和腫瘤發(fā)生中起重要作用,但其具體的作用機制尚未完全明了。microRNA是一類非編碼小RNA,通過在轉(zhuǎn)錄后水平對基因表達的調(diào)控而影響生物學過程,包括血管生成及腫瘤形成等,但目前并不了解其與ANG的關(guān)系。為此,本論文從直接受ANG調(diào)控的microRNAs和直接靶向調(diào)控ANG的microRNAs兩個方面研究了ANG特異的microRNAs及其生物學功能。 為探索受ANG影響和調(diào)控的microRNAs,本論文首先利用microRNA芯片篩選的方法,在血管內(nèi)皮細胞中鑒定受ANG刺激后表達發(fā)生改變的microRNAs。通過篩選,共得到了26個在ANG刺激前后發(fā)生差異表達的microRNAs,包括17個上調(diào)和9個下調(diào)的microRNAs.生物信息學分析顯示,這些microRNAs參與多種生物學過程,包括腫瘤發(fā)生和轉(zhuǎn)移、血管生成、神經(jīng)發(fā)育、細胞凋亡等。利用數(shù)據(jù)庫MicroCosm,初步預(yù)測了這些microRNAs的靶基因。另一方面,本論文利用ChIP-on-Chip方法探索了ANG與microRNAs啟動子區(qū)的結(jié)合情況,發(fā)現(xiàn)有121個microRNAs啟動子區(qū)可能與ANG結(jié)合,其中有9個啟動子控制的microRNAs與上述利用microRNA芯片篩選得到的受ANG直接調(diào)控的microRNAs相同,包括8個上調(diào)和1個下調(diào)的microRNAs。在此基礎(chǔ)上,利用ChIP-QPCR的方法對所得到的部分microRNA啟動子區(qū)做進一步驗證,發(fā)現(xiàn)miR-149、miR-17、miR-378和miR-641等確實受ANG的調(diào)控,說明ANG具有轉(zhuǎn)錄因子的活性,可以通過結(jié)合microRNAs的啟動子區(qū)而對其表達水平進行調(diào)控。 ANG是一個在血管內(nèi)皮細胞和腫瘤細胞中高表達的蛋白質(zhì),本身也可能受到microRNAs的靶向調(diào)控。為此,本論文首先通過生物信息學方法預(yù)測得到8個可能靶向結(jié)合到ANG基因3'UTR的microRNAs,然后分析了它們對ANG mRNA和蛋白質(zhì)表達水平的影響,發(fā)現(xiàn)miR-1208、miR-196b、miR-296、miR-409、miR-570和miR-641共六個microRNAs確實可以調(diào)節(jié)ANG的表達,并對靶細胞的增殖、遷移、粘附和管腔形成均有一定影響。在此基礎(chǔ)上,本論文著重對miR-409進行了較深入的研究,發(fā)現(xiàn)miR-409不僅可以抑制內(nèi)皮細胞的管腔形成和細胞增殖,也可以抑制腫瘤細胞HT1080的增殖及血管擬態(tài)；小鼠移植瘤實驗也證明了miR-409可以抑制腫瘤的生長、血管生成和轉(zhuǎn)移。進一步,本論文在結(jié)腸癌病人的組織中發(fā)現(xiàn)miR-409的表達較相應(yīng)癌旁組織的表達下降,并且miR-409的表達與腫瘤的轉(zhuǎn)移密切相關(guān)。以上結(jié)果說明ANG本身可以受多種microRNAs的調(diào)控,其中miR-409可以直接靶向下調(diào)ANG的表達,影響ANG在血管生成及腫瘤發(fā)生、發(fā)展中的作用。 綜合上述兩部分的研究結(jié)果,說明ANG不僅可以調(diào)控microRNAs的表達,而且其本身也同時被多種microRNAs所調(diào)控。通過這些microRNAs組成的調(diào)控網(wǎng)絡(luò),一方面決定了細胞中ANG的表達水平,另一方面也放大了ANG對細胞行為的影響,從而實現(xiàn)其對血管生成和腫瘤發(fā)生的促進作用。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R363

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