miR-139-5p在乳腺上皮细胞MCF-10A中靶基因的鉴定及功能验证

发布时间：2018-03-29 14:14

本文选题：MiR-139-5p　切入点：RIP-seq技术　出处：《东北农业大学》2017年硕士论文

【摘要】：每个miRNA都具有多个靶基因,因此对miRNA下游作用的靶基因进行寻找及功能研究仍是临床及科研的重要方向。而哺乳动物乳腺组织是动物体中少数的随着年龄、发情周期和繁殖状态而发生相应周期性变化器官之一。因此对人乳腺中miR-139-5p靶基因的鉴定及验证至关重要。研究发现miR-139与其靶基因共同作用可影响众多生物学功能,如蛋白定位、细胞周期调控以及抑制肿瘤侵袭等。众所周知,细胞周期的有序进行是生物体生长、发育及繁殖的基础,而细胞周期加快或阻滞会影响细胞增殖及迁移能力,进而至使肿瘤、癌症以及基因突变的发生。为充分研究miR-139-5p的作用和功能,其靶基因的挖掘和鉴定及初步的功能验证成为急需解决的问题。本研究将RNA结合蛋白免疫沉淀(RNA Binding Protein Immunoprecipitation,RIP)与新一代测序技术相结合,以MCF-10A细胞系为研究对象,鉴定与验证miR-139-5p在乳腺上皮细胞中的靶基因。先运用针对目标蛋白Ago2的抗体把相应的RNA-蛋白复合物沉淀下来,然后经过分离纯化,对结合在复合物上的RNA进行测序分析,找出成对样品中的差异峰,进而确认miR-139-5p在MCF-10A中的靶基因,并对靶基因进行KEGG和GO分析;最后运用q PCR等技术对miR-139-5p的靶基因及miR-139-5p部分功能进行验证。这些结果将为miR-139-5p在人乳腺中的功能研究提供新的参考。研究结果如下:(1)RIP-seq测序数据处理及生物信息学分析:RIP-seq测序后我们将由Negative Control组与miR-139-5p mimic组获得的peak进行比较,得到差异基因484个,这些差异基因就是miR-139-5p在MCF-10A细胞中的靶基因,这些靶基因可能是乳腺发育或乳腺进化过程中重要的基因。利用KEGG对靶基因进行通路分析,我们得到204种Pathway,其中显著性通路有21条(P0.05),涉及到120个靶基因;在GO分析中发现,显著性GO条目448个(P0.05),主要涉及蛋白定位(protein localization)、细胞周期调控(regulation of cell cycle)、细胞器组成(organelle part)、结合RNA(RNA binding)和结合核酸(nucleic acid binding)等;(2)miR-139-5p靶基因的验证选取的18个靶基因在mRNA水平被miR-139-5p显著下调,表明其表达受miR-139-5p调节。选择细胞周期信号通路对miR-139-5p的靶基因进行功能验证,表明miR-139-5p能显著抑制G1/S细胞周期转变,进而抑制细胞增殖,这种调节是信号通路中多个靶基因共同作用的结果。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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