本文選題：MiR-139-5p　切入點：RIP-seq技術(shù)　出處：《東北農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：每個miRNA都具有多個靶基因,因此對miRNA下游作用的靶基因進(jìn)行尋找及功能研究仍是臨床及科研的重要方向。而哺乳動物乳腺組織是動物體中少數(shù)的隨著年齡、發(fā)情周期和繁殖狀態(tài)而發(fā)生相應(yīng)周期性變化器官之一。因此對人乳腺中miR-139-5p靶基因的鑒定及驗證至關(guān)重要。研究發(fā)現(xiàn)miR-139與其靶基因共同作用可影響眾多生物學(xué)功能,如蛋白定位、細(xì)胞周期調(diào)控以及抑制腫瘤侵襲等。眾所周知,細(xì)胞周期的有序進(jìn)行是生物體生長、發(fā)育及繁殖的基礎(chǔ),而細(xì)胞周期加快或阻滯會影響細(xì)胞增殖及遷移能力,進(jìn)而至使腫瘤、癌癥以及基因突變的發(fā)生。為充分研究miR-139-5p的作用和功能,其靶基因的挖掘和鑒定及初步的功能驗證成為急需解決的問題。本研究將RNA結(jié)合蛋白免疫沉淀(RNA Binding Protein Immunoprecipitation,RIP)與新一代測序技術(shù)相結(jié)合,以MCF-10A細(xì)胞系為研究對象,鑒定與驗證miR-139-5p在乳腺上皮細(xì)胞中的靶基因。先運用針對目標(biāo)蛋白Ago2的抗體把相應(yīng)的RNA-蛋白復(fù)合物沉淀下來,然后經(jīng)過分離純化,對結(jié)合在復(fù)合物上的RNA進(jìn)行測序分析,找出成對樣品中的差異峰,進(jìn)而確認(rèn)miR-139-5p在MCF-10A中的靶基因,并對靶基因進(jìn)行KEGG和GO分析;最后運用q PCR等技術(shù)對miR-139-5p的靶基因及miR-139-5p部分功能進(jìn)行驗證。這些結(jié)果將為miR-139-5p在人乳腺中的功能研究提供新的參考。研究結(jié)果如下:(1)RIP-seq測序數(shù)據(jù)處理及生物信息學(xué)分析:RIP-seq測序后我們將由Negative Control組與miR-139-5p mimic組獲得的peak進(jìn)行比較,得到差異基因484個,這些差異基因就是miR-139-5p在MCF-10A細(xì)胞中的靶基因,這些靶基因可能是乳腺發(fā)育或乳腺進(jìn)化過程中重要的基因。利用KEGG對靶基因進(jìn)行通路分析,我們得到204種Pathway,其中顯著性通路有21條(P0.05),涉及到120個靶基因;在GO分析中發(fā)現(xiàn),顯著性GO條目448個(P0.05),主要涉及蛋白定位(protein localization)、細(xì)胞周期調(diào)控(regulation of cell cycle)、細(xì)胞器組成(organelle part)、結(jié)合RNA(RNA binding)和結(jié)合核酸(nucleic acid binding)等;(2)miR-139-5p靶基因的驗證選取的18個靶基因在mRNA水平被miR-139-5p顯著下調(diào),表明其表達(dá)受miR-139-5p調(diào)節(jié)。選擇細(xì)胞周期信號通路對miR-139-5p的靶基因進(jìn)行功能驗證,表明miR-139-5p能顯著抑制G1/S細(xì)胞周期轉(zhuǎn)變,進(jìn)而抑制細(xì)胞增殖,這種調(diào)節(jié)是信號通路中多個靶基因共同作用的結(jié)果。
[Abstract]:Each miRNA has multiple target genes, so it is still an important direction of clinical and scientific research to search for the downstream target genes of miRNA, and mammal mammary tissue is a small number of animals with age. Therefore, it is very important for the identification and verification of miR-139-5p target gene in human mammary gland. It has been found that the interaction of miR-139 and its target gene can affect many biological functions, such as protein localization. Cell cycle regulation and inhibition of tumor invasion. It is well known that the orderly development of cell cycle is the basis of organism growth, development and reproduction, and cell cycle acceleration or arrest will affect cell proliferation and migration ability, and thus make tumor, Cancer and gene mutation. To fully study the role and function of miR-139-5p, In this study, RNA binding protein Binding Binding Protein immunoprecipitation technique was combined with a new generation of sequencing technology to study MCF-10A cell line. The target genes of miR-139-5p in mammary epithelial cells were identified and verified. The corresponding RNA-protein complexes were precipitated with antibodies against the target protein Ago2, then purified and sequenced. The differential peaks in paired samples were found, and the target genes of miR-139-5p in MCF-10A were confirmed, and the target genes were analyzed by KEGG and go. Finally, Q PCR and other techniques were used to verify the target gene and miR-139-5p function of miR-139-5p. These results will provide a new reference for the study of the function of miR-139-5p in human mammary gland. The results are as follows: RIP-seq sequencing data processing and bioinformatics. We'll compare the peak from the Negative Control group with the peak from the miR-139-5p mimic group after analyzing the RIP-seq sequencing. A total of 484 differentially expressed genes were obtained, which are the target genes of miR-139-5p in MCF-10A cells. These target genes may be important genes in breast development or breast evolution. KEGG is used to analyze the pathway of target genes. We got 204 kinds of Pathways, of which there were 21 P0.05 genes involved in 120 target genes, which were found in go analysis. Significant go entries included 448 P0.05s, mainly involving protein localization, cell cycle regulation of cell cycle, organelle binding (RNA(RNA binding) and nucleic acid binding. The 18 target genes selected were significantly down-regulated by miR-139-5p at the mRNA level. The results indicated that the expression of miR-139-5p was regulated by miR-139-5p. The function of target gene of miR-139-5p was verified by selecting cell cycle signaling pathway, which indicated that miR-139-5p could significantly inhibit the cell cycle transition of G1 / S cells and then inhibit the proliferation of G1 / S cells. This regulation is the result of the interaction of multiple target genes in the signaling pathway.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

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