本文選題：轉(zhuǎn)錄因子 + 數(shù)據(jù)庫(kù)　； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：轉(zhuǎn)錄因子(transcription factor,TF)是一類通過結(jié)合特定DNA序列而選擇性的激活或抑制基因表達(dá)的蛋白質(zhì),在轉(zhuǎn)錄調(diào)控過程發(fā)揮重要功能,鑒定和注釋是其生物學(xué)功能研究的基礎(chǔ)。TF和miRNA分別作為轉(zhuǎn)錄水平和轉(zhuǎn)錄后水平的關(guān)鍵調(diào)控因子,可以形成前饋環(huán)或反饋環(huán)協(xié)同調(diào)控靶基因表達(dá),該種調(diào)控方式已被成功的應(yīng)用到乳腺癌和神經(jīng)腦膠質(zhì)瘤等的研究中。我們合作方之前的研究發(fā)現(xiàn),慢性髓系細(xì)胞白血病(Chronic myeloid leukemia,CML) K562細(xì)胞系釋放的微泡,在持續(xù)加樣14天后正常單個(gè)核細(xì)胞群體中出現(xiàn)少量類似急性白血病的癌細(xì)胞,21天后絕大部分單個(gè)核細(xì)胞轉(zhuǎn)變成急性白血病表型的癌細(xì)胞。這個(gè)系統(tǒng)可作為穩(wěn)定產(chǎn)生癌變細(xì)胞和研究CML急變的模型,但轉(zhuǎn)化過程中的分子機(jī)理還不清楚。本研究,首先系統(tǒng)地預(yù)測(cè)了已測(cè)序動(dòng)物物種的TF并進(jìn)行詳細(xì)的功能注釋和數(shù)據(jù)庫(kù)構(gòu)建,通過構(gòu)建TF-miRNA共調(diào)控網(wǎng)絡(luò),分析了 K562微泡誘導(dǎo)正常單個(gè)核細(xì)胞癌變模型中基因的失調(diào)機(jī)制,并對(duì)關(guān)鍵TF和miRNA的作用機(jī)理進(jìn)行闡述,取得以下重要結(jié)果。第一,通過文獻(xiàn)閱讀和數(shù)據(jù)庫(kù)檢索,收集了完整的動(dòng)物轉(zhuǎn)錄因子家族并為每個(gè)家族構(gòu)建隱馬爾科夫模型(Hidden Markov Model,HMM),利用HMM對(duì)所有已測(cè)序動(dòng)物物種的TF進(jìn)行了全面地預(yù)測(cè)和注釋,并構(gòu)建成數(shù)據(jù)庫(kù)AnimalTFDB。首先,該數(shù)據(jù)庫(kù)共包含65個(gè)動(dòng)物物種,70個(gè)TF家族,72336個(gè)TF基因、21053個(gè)轉(zhuǎn)錄輔因子和6502個(gè)染色質(zhì)重塑因子,是目前最完整的動(dòng)物轉(zhuǎn)錄因子數(shù)據(jù)庫(kù)。其次,我們分別在基因和家族層面對(duì)三類調(diào)控因子進(jìn)行注釋和分析,提供了大量有用的信息。在基因?qū)用?包括基因的基本信息、功能結(jié)構(gòu)域、GO注釋、KEGG和BioCarta注釋、同源基因、基因表達(dá)量、基因表型、蛋白相互作用、TF結(jié)合位點(diǎn)和靶基因信息等。在TF家族層面,我們?yōu)槊總�(gè)TF家族提供了結(jié)構(gòu)和功能描述,對(duì)同一家族的DNA結(jié)合結(jié)構(gòu)域進(jìn)行多序列比對(duì)和進(jìn)化樹構(gòu)建。此外,該數(shù)據(jù)庫(kù)還提供了在線的TF預(yù)測(cè)平臺(tái)和BLAST搜索工具,方便研究者對(duì)新測(cè)序物種進(jìn)行轉(zhuǎn)錄因子預(yù)測(cè)。第二,為了研究K562微泡誘導(dǎo)正常單個(gè)核細(xì)胞癌變過程中的作用機(jī)制,我們對(duì)K562分泌的微泡、正常單個(gè)核細(xì)胞(MNC)、轉(zhuǎn)化一周(1W)、兩周(2W)和三周(3W)的細(xì)胞進(jìn)行轉(zhuǎn)錄組和小RNA測(cè)序,并將MNC-1W、1W-2W、2W-3W作為細(xì)胞轉(zhuǎn)化的三個(gè)階段。第一步,利用多種分析方法研究了不同轉(zhuǎn)化階段細(xì)胞間的差異。通過差異表達(dá)分析,得到了 3717個(gè)差異基因和143個(gè)差異miRNA。GO富集分析發(fā)現(xiàn),三個(gè)轉(zhuǎn)化過程中多數(shù)差異基因富集在免疫相關(guān)的信號(hào)通路,另外在2W-3W過程中大量細(xì)胞增殖和凋亡相關(guān)基因出現(xiàn)差異表達(dá)�；蚓垲惙治霭l(fā)現(xiàn),在3W樣本中上調(diào)的基因多參與細(xì)胞增殖、DNA損傷修復(fù)、氧化還原反應(yīng)和新陳代謝等過程,下調(diào)的基因多與細(xì)胞凋亡、免疫應(yīng)答和信號(hào)傳遞有關(guān);miRNA聚類結(jié)果顯示,在3W樣本中上調(diào)的miRNA多為促癌miRNA,比如miR-17-92和miR-183/96/182,下調(diào)的miRNA多為抑癌miRNA,比如let-7家族、miR-181家族和miR-15a/16-5p等。第二步,利用TF-miRNA共調(diào)控網(wǎng)絡(luò)分析,發(fā)現(xiàn)大量差異TF和miRNA與上述基因的失調(diào)有關(guān),尤其是微泡中高表達(dá)的TF (如YBX1、MYC、STAT5A、GATA2)和miRNA(如 miR-146b-5p、miR-17-92、miR-92b-3p),這些調(diào)控因子與 BCR-ABL1 一起上調(diào)細(xì)胞增殖、DNA損傷修復(fù)、能量代謝和PI3K/AKT通路基因的表達(dá),下調(diào)細(xì)胞凋亡、免疫系統(tǒng)和腫瘤抑制基因的表達(dá)。而且,這些高表達(dá)調(diào)控因子還可激活促癌TF和miRNA,抑制抑癌TF和miRNA的表達(dá)。我們篩選了 miR-146b-5p進(jìn)行實(shí)驗(yàn)驗(yàn)證,熒光素酶報(bào)告實(shí)驗(yàn)和細(xì)胞內(nèi)轉(zhuǎn)染實(shí)驗(yàn)顯示,miR-146b-5p通過抑制NUMB和NOTCH2的表達(dá),加速正常單個(gè)核細(xì)胞到白血病細(xì)胞的轉(zhuǎn)化,在誘導(dǎo)過程起到重要作用。本研究系統(tǒng)的預(yù)測(cè)了動(dòng)物轉(zhuǎn)錄因子并提供豐富的注釋信息,為動(dòng)物轉(zhuǎn)錄因子的功能和比較基因組學(xué)研究提供了可靠的數(shù)據(jù)資源。通過對(duì)K562微泡誘導(dǎo)正常單個(gè)核細(xì)胞癌變模型的差異比較和調(diào)控網(wǎng)絡(luò)分析,發(fā)現(xiàn)了促進(jìn)細(xì)胞轉(zhuǎn)化的關(guān)鍵TF和miRNA,并通過實(shí)驗(yàn)證實(shí)了 miR-146b-5p上調(diào)可以加速正常單個(gè)核細(xì)胞癌變,為CML的發(fā)生發(fā)展和治療提供了新的研究思路和分子靶標(biāo)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q78;R733.7

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本文編號(hào)：1768412