动物转录因子数据库构建及K562微泡致癌机理的调控网络研究

发布时间：2018-04-18 12:39

  本文选题：转录因子 + 数据库　； 参考：《华中科技大学》2015年博士论文

【摘要】：转录因子(transcription factor,TF)是一类通过结合特定DNA序列而选择性的激活或抑制基因表达的蛋白质,在转录调控过程发挥重要功能,鉴定和注释是其生物学功能研究的基础。TF和miRNA分别作为转录水平和转录后水平的关键调控因子,可以形成前馈环或反馈环协同调控靶基因表达,该种调控方式已被成功的应用到乳腺癌和神经脑胶质瘤等的研究中。我们合作方之前的研究发现,慢性髓系细胞白血病(Chronic myeloid leukemia,CML) K562细胞系释放的微泡,在持续加样14天后正常单个核细胞群体中出现少量类似急性白血病的癌细胞,21天后绝大部分单个核细胞转变成急性白血病表型的癌细胞。这个系统可作为稳定产生癌变细胞和研究CML急变的模型,但转化过程中的分子机理还不清楚。本研究,首先系统地预测了已测序动物物种的TF并进行详细的功能注释和数据库构建,通过构建TF-miRNA共调控网络,分析了 K562微泡诱导正常单个核细胞癌变模型中基因的失调机制,并对关键TF和miRNA的作用机理进行阐述,取得以下重要结果。第一,通过文献阅读和数据库检索,收集了完整的动物转录因子家族并为每个家族构建隐马尔科夫模型(Hidden Markov Model,HMM),利用HMM对所有已测序动物物种的TF进行了全面地预测和注释,并构建成数据库AnimalTFDB。首先,该数据库共包含65个动物物种,70个TF家族,72336个TF基因、21053个转录辅因子和6502个染色质重塑因子,是目前最完整的动物转录因子数据库。其次,我们分别在基因和家族层面对三类调控因子进行注释和分析,提供了大量有用的信息。在基因层面,包括基因的基本信息、功能结构域、GO注释、KEGG和BioCarta注释、同源基因、基因表达量、基因表型、蛋白相互作用、TF结合位点和靶基因信息等。在TF家族层面,我们为每个TF家族提供了结构和功能描述,对同一家族的DNA结合结构域进行多序列比对和进化树构建。此外,该数据库还提供了在线的TF预测平台和BLAST搜索工具,方便研究者对新测序物种进行转录因子预测。第二,为了研究K562微泡诱导正常单个核细胞癌变过程中的作用机制,我们对K562分泌的微泡、正常单个核细胞(MNC)、转化一周(1W)、两周(2W)和三周(3W)的细胞进行转录组和小RNA测序,并将MNC-1W、1W-2W、2W-3W作为细胞转化的三个阶段。第一步,利用多种分析方法研究了不同转化阶段细胞间的差异。通过差异表达分析,得到了 3717个差异基因和143个差异miRNA。GO富集分析发现,三个转化过程中多数差异基因富集在免疫相关的信号通路,另外在2W-3W过程中大量细胞增殖和凋亡相关基因出现差异表达。基因聚类分析发现,在3W样本中上调的基因多参与细胞增殖、DNA损伤修复、氧化还原反应和新陈代谢等过程,下调的基因多与细胞凋亡、免疫应答和信号传递有关;miRNA聚类结果显示,在3W样本中上调的miRNA多为促癌miRNA,比如miR-17-92和miR-183/96/182,下调的miRNA多为抑癌miRNA,比如let-7家族、miR-181家族和miR-15a/16-5p等。第二步,利用TF-miRNA共调控网络分析,发现大量差异TF和miRNA与上述基因的失调有关,尤其是微泡中高表达的TF (如YBX1、MYC、STAT5A、GATA2)和miRNA(如 miR-146b-5p、miR-17-92、miR-92b-3p),这些调控因子与 BCR-ABL1 一起上调细胞增殖、DNA损伤修复、能量代谢和PI3K/AKT通路基因的表达,下调细胞凋亡、免疫系统和肿瘤抑制基因的表达。而且,这些高表达调控因子还可激活促癌TF和miRNA,抑制抑癌TF和miRNA的表达。我们筛选了 miR-146b-5p进行实验验证,荧光素酶报告实验和细胞内转染实验显示,miR-146b-5p通过抑制NUMB和NOTCH2的表达,加速正常单个核细胞到白血病细胞的转化,在诱导过程起到重要作用。本研究系统的预测了动物转录因子并提供丰富的注释信息,为动物转录因子的功能和比较基因组学研究提供了可靠的数据资源。通过对K562微泡诱导正常单个核细胞癌变模型的差异比较和调控网络分析,发现了促进细胞转化的关键TF和miRNA,并通过实验证实了 miR-146b-5p上调可以加速正常单个核细胞癌变,为CML的发生发展和治疗提供了新的研究思路和分子靶标。
[Abstract]:Transcription factors (transcription factor TF) is a kind of specific DNA sequences by combining selective activation or inhibition of gene expression of proteins, play an important role in the process of transcription, identification and annotation is the study of its biological function based.TF and miRNA as a key regulator of transcription level and post transcriptional level, can be formed the feedforward loop or feedback loop co regulation of target gene expression, study the regulation methods have been successfully applied to breast cancer and brain tumor. So our research partners had found that chronic myeloid leukemia (Chronic myeloid, leukemia, CML) microbubbles K562 cell line release, appear a few similar acute leukemia cancer cells in the sample after 14 days of continuous normal mononuclear cells group, 21 days after the change of the vast majority of mononuclear cells of acute myeloid leukemia phenotype of cancer cells. This A system can be used to produce stable malignant cells and study of CML blast model, but the molecular mechanism in the process of transformation is not clear. This study first systematically predicted sequenced animal species TF and detailed functional annotation and database construction, CO regulatory network through the construction of TF-miRNA, analyzes the mechanism of dysfunction of K562 microbubbles induced model of normal mononuclear cells in cancer gene, the mechanism of the key TF and miRNA are described, the following important results. First, by reading literature and database retrieval, collection of animal transcription factor family complete and construct a hidden Markov model for each family (Hidden Markov, Model, HMM) based on HMM all animal species have been sequenced TF comprehensively prediction and annotation, and to build a database of AnimalTFDB. first, the database contains a total of 65 animal species, 70 TF family, 72 336 TF genes, 21053 transcription cofactors and chromatin remodeling factor 6502, is currently the most complete animal transcription factor database. Secondly, we are facing three kinds of genes and family level regulator annotation and analysis, provides a lot of useful information. At the level of genes, including the basic information of genes. The functional domain, GO notes, KEGG and BioCarta annotations, homologous gene, gene expression, gene expression, protein interaction, TF binding site and target gene information. In the TF family level, we provide the structure and function of TF for each family, the same family of DNA binding domain of multiple sequence alignment and phylogenetic tree construction. In addition, the database also provides online prediction of TF platform and BLAST search tools, convenient for researchers to predict transcription factors in newly sequenced species. Second, in order to study the K562 microbubbles induced normal Mechanism of nuclear cell carcinogenesis, we microbubble on K562 secretion, normal mononuclear cells (MNC), into a week (1W), two weeks (2W) and three weeks (3W) cell transcriptome and small RNA sequencing, and MNC-1W, 1W-2W, three stage 2W-3W as the cell transformation. The first step, using a variety of analytical methods to study the differences between different stages of cell transformation. Through the analysis of differential expression, obtained 3717 genes and 143 differentially miRNA.GO enrichment analysis showed that most of the differences of the three gene conversion process is enriched in immune related signaling pathway in 2W-3W process cell proliferation and apoptosis related gene differential expression. Gene cluster analysis found up-regulated in 3W in a sample of genes involved in cell proliferation, DNA repair, and the process of redox reaction The new supersedes the old., cut the base due to multiple cell apoptosis and immune response A related transmission and signal; miRNA clustering results showed that the increase in the 3W sample miRNA for cancer miRNA, such as miR-17-92 and miR-183/96/182, downregulation of miRNA as tumor suppressor miRNA, such as let-7 family, miR-181 family and miR-15a/16-5p. The second step, using TF-miRNA control network analysis, found large differences in TF disorders and miRNA and the gene, especially the micro bubble in the high expression of TF (such as YBX1, MYC, STAT5A, GATA2) and miRNA (such as miR-146b-5p, miR-17-92, miR-92b-3p), these factors together with BCR-ABL1 increased cell proliferation, DNA repair, energy metabolism and expression of genes of the PI3K/AKT pathway, down regulated cells apoptosis, immune system and tumor suppressor gene expression. Moreover, these high expression can also activate cancer promoting TF and miRNA, inhibit the expression of tumor suppressor TF and miRNA. We screened miR-146b-5p experiments, fluorescence Show the luciferase reporting test and transfection experiments, miR-146b-5p by inhibiting expression of NUMB and NOTCH2, to accelerate the transformation of normal mononuclear cells to leukemia cells, to play an important role in the induction process. This study predicts animal transcription factors and provide rich annotation information, and provide reliable data for functions and resources comparative genomic studies of animal transcription factor. The differences of K562 microbubbles induced model of normal mononuclear cells and cancerous regulatory network analysis, found the key to promote cell transformation of TF and miRNA, and through the experiment confirmed the upregulation of miR-146b-5p can accelerate the normal mononuclear cells canceration, provided research ideas and new molecules the target for the development and treatment of CML.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q78;R733.7

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