本文選題：轉(zhuǎn)錄因子FOXA2　切入點(diǎn)：上皮間質(zhì)轉(zhuǎn)化EMT　出處：《湖南大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：上皮-間質(zhì)轉(zhuǎn)化(EMT)過程是指上皮來源的細(xì)胞在誘導(dǎo)調(diào)控下,喪失了上皮表型的過程。經(jīng)歷了這個(gè)過程之后,細(xì)胞喪失自身極性、細(xì)胞間連接能力乃至上皮表型,但是獲得了間質(zhì)表型并增強(qiáng)了遷徙和侵襲能力。由此細(xì)胞可以離開上皮組織,侵襲進(jìn)入細(xì)胞基質(zhì)并通過循環(huán)系統(tǒng)向遠(yuǎn)端組織進(jìn)行轉(zhuǎn)移。EMT過程最早發(fā)現(xiàn)于胚胎發(fā)育和器官的分化過程中,起著重要的生理作用。隨后,針對病理相關(guān)的EMT研究發(fā)現(xiàn),EMT參與了癌癥發(fā)生發(fā)展、組織器官纖維化病變等相關(guān)疾病過程。在這些過程中具有代表性的是,乳腺癌細(xì)胞的EMT過程將會導(dǎo)致明顯的早起轉(zhuǎn)移事件的發(fā)生,這種現(xiàn)象也是乳腺癌臨床治療過程中的一大難題。在相關(guān)乳腺癌EMT過程的研究中發(fā)現(xiàn),基因表達(dá)調(diào)控在整個(gè)EMT過程中發(fā)揮著關(guān)鍵調(diào)控的作用。因此研究特定轉(zhuǎn)錄因子調(diào)節(jié)乳腺癌細(xì)胞的EMT分子機(jī)理,對闡明腫瘤轉(zhuǎn)移機(jī)制和臨床治療中應(yīng)對乳腺癌轉(zhuǎn)移有著重要的意義。本文主要研究轉(zhuǎn)錄因子FOXA2在乳腺癌EMT過程中的功能機(jī)制。轉(zhuǎn)錄因子FOXA2是叉頭框(Forkhead box)家族的成員,其生理功能在于胚胎發(fā)育以及多器官功能維持。同時(shí),大量的腫瘤相關(guān)研究結(jié)果不僅表明FOXA2的表達(dá)情況與腫瘤發(fā)生以及惡性轉(zhuǎn)化過程高度相關(guān),而且證實(shí)FOXA2參與了多種腫瘤細(xì)胞的EMT調(diào)控過程。所以,我們認(rèn)為轉(zhuǎn)錄因子FOXA2可能是乳腺癌細(xì)胞EMT過程中的關(guān)鍵調(diào)控因子。我們由乳腺癌臨床樣本的數(shù)據(jù)出發(fā),運(yùn)用乳腺癌E型細(xì)胞MCF-7和M型細(xì)胞MDA-MB-231作為EMT對應(yīng)的細(xì)胞體系模型,研究乳腺癌細(xì)胞中FOXA2刺激E型基因和抑制M型基因的分子作用機(jī)制,并且在小鼠尾靜脈注射轉(zhuǎn)移模型中加以驗(yàn)證,得到以下結(jié)果:首先,我們在乳腺癌臨床樣本中檢測了FOXA2以及E-cadherin、Vimentin等EMT標(biāo)志基因的表達(dá)。同時(shí)考察了乳腺癌上皮型細(xì)胞MCF-7和間質(zhì)型細(xì)胞MDA-MB-231中以上基因的m RNA和蛋白質(zhì)表達(dá)水平,我們發(fā)現(xiàn)FOXA2和上皮表型以及EMT標(biāo)志基因有著強(qiáng)烈的對應(yīng)關(guān)系。另外,通過EGF誘導(dǎo)的MCF-10A這一EMT模型進(jìn)一步確認(rèn)了FOXA2隨著EMT過程的發(fā)生而表達(dá)減弱的事實(shí)。然后,為了考察FOXA2對于乳腺癌上皮表型的維持作用,我們在乳腺癌上皮型細(xì)胞MCF-7中干擾了FOXA2的表達(dá),發(fā)現(xiàn)細(xì)胞的遷移能力加強(qiáng),在分子表達(dá)層面上皮型標(biāo)志物表達(dá)下降而間質(zhì)型標(biāo)志物表達(dá)上升。相對應(yīng)的,為了驗(yàn)證FOXA2對于乳腺癌間質(zhì)型細(xì)胞遷移能力的抑制作用,我們通過慢病毒感染的方式篩選出穩(wěn)定高表達(dá)FOXA2的在乳腺癌間質(zhì)型細(xì)胞MDA-MB-231細(xì)胞株,發(fā)現(xiàn)該細(xì)胞株的遷移能力減弱,其中的上皮型標(biāo)志物表達(dá)上升而間質(zhì)型標(biāo)志物表達(dá)下降。這說明FOXA2是維持乳腺癌細(xì)胞上皮特征的重要因子,也是抑制乳腺癌細(xì)胞發(fā)生EMT的關(guān)鍵因子。隨后的Ch IP、EMSA、熒光素酶報(bào)告基因?qū)嶒?yàn)證實(shí),FOXA2能夠直接結(jié)合在下游靶基因E-cadherin和ZEB2的啟動(dòng)子上,并且分別對其進(jìn)行正調(diào)控和負(fù)調(diào)控。也有文獻(xiàn)指出,FOXA蛋白能夠招募轉(zhuǎn)錄輔助抑制子TLE3聯(lián)合作用。在我們的體系中,通過m RNA水平和蛋白水平的檢測,我們發(fā)現(xiàn)TLE3的表達(dá)與上皮表型呈高度正相關(guān)。之后,通過Ch IP、Co-IP以及EMSA等實(shí)驗(yàn),確認(rèn)FOXA2通過招募TLE3到ZEB2的啟動(dòng)子上,進(jìn)而抑制了ZEB2的表達(dá)。最后為了考察FOXA2在體內(nèi)對于乳腺癌細(xì)胞轉(zhuǎn)移的影響,我們通過尾靜脈注射高表達(dá)FOXA2的MDA-MB-231細(xì)胞株的方式建立了裸鼠乳腺癌轉(zhuǎn)移模型,確認(rèn)FOXA2在體內(nèi)可以抑制乳腺癌細(xì)胞向肺部的轉(zhuǎn)移能力。
[Abstract]:Epithelial mesenchymal transition (EMT) is the process of epithelial cells in the induction of control, loss of epithelial phenotype. After this process, the cells lose their polarity, cell junction ability and epithelial phenotype, but the mesenchymal phenotype and enhances the migration and invasion of the cells. You can leave the epithelial tissue, differentiation and invasion into the extracellular matrix to the distal tissue through the circulatory system to transfer.EMT process was first found in embryonic development and organ, and plays an important role. Then, according to EMT research related to pathology found that EMT involved in cancer development, the process of diseases related to organ fibrosis lesions etc. in the process of representative, will be EMT of breast cancer cells leads to obvious early metastasis event, this phenomenon is the process of clinical treatment of breast cancer A major problem in the study of breast cancer. Found in the EMT process, the regulation of gene expression plays a key regulatory role in the whole EMT process. Therefore research on molecular mechanism of EMT specific transcription factors regulate breast cancer cells, is of great significance to clarify the mechanism of tumor metastasis to breast cancer metastasis and clinical treatment the function mechanism of FOXA2. This paper mainly studies the transcription factor EMT in breast cancer. In the process of transcription factor forkhead box FOXA2 (Forkhead box) family members, its physiological function is to maintain the embryonic development and organ function. At the same time, a large number of tumor related research results not only show that the expression of FOXA2 and tumor occurrence and malignant transformation the process is highly relevant, and confirmed that FOXA2 is involved in EMT regulation of a variety of tumor cells. Therefore, we propose that the transcription factor FOXA2 may be in the process of breast cancer cell EMT The key regulatory factor. We by clinical samples of breast cancer data of breast cancer cells by type E MCF-7 and type M MDA-MB-231 cells as the corresponding EMT cell system model of breast cancer cells stimulated with FOXA2 E gene and molecular mechanism of inhibitory effects of M gene, and injected into tail vein of mice in model transfer to verify, get the following results: first, we detected FOXA2 and E-cadherin in clinical samples of breast cancer, Vimentin and EMT marker gene expression. At the same time, the effects of M RNA and protein expression levels in breast cancer epithelial cells MCF-7 and interstitial cell MDA-MB-231 in these genes, we found that FOXA2 and epithelial phenotype and EMT marker genes have a relationship strong. In addition, induced by EGF MCF-10A of the EMT model further confirmed the FOXA2 with the EMT process and the reduced expression of the fact. Then, in order to investigate the role of FOXA2 in maintaining mammary epithelial phenotype, we interfere with the expression of FOXA2 in breast cancer epithelial cell MCF-7, found to strengthen the ability of cell migration, expression levels of epithelial markers decreased expression of mesenchymal marker expression rise in molecular. Correspondingly, in order to verify for FOXA2 inhibition of breast cancer interstitial cell migration, we infected by lentivirus screened out the stable high expression of FOXA2 in breast cancer interstitial cells MDA-MB-231 cell line, found the migration ability of the cells decreased, the epithelial marker expression increased mesenchymal marker expression decreased. This FOXA2 is an important factor in maintaining breast cancer cell epithelial features, is also a key factor to suppress breast cancer cells EMT. Then the Ch IP EMSA, luciferase reporter experiments showed that FOXA2 Can be directly combined with E-cadherin gene and ZEB2 promoter, respectively, and the positive and negative regulation on it. It has been pointed out that FOXA protein can inhibit the recruitment of transcriptional aided joint action TLE3. In our system, through the detection of M RNA and protein level, we found that the expression of TLE3 and the epithelial phenotype is highly relevant. Then, by Ch IP, Co-IP and EMSA experiment, confirm the FOXA2 by recruiting TLE3 to the promoter of ZEB2, thereby inhibiting the expression of ZEB2. Finally, in order to investigate the effect of FOXA2 in vivo for metastatic breast cancer cells, we by intravenous injection of MDA-MB-231 high expression cell line FOXA2 the way to establish breast cancer metastasis model in nude mice, confirming that FOXA2 can inhibit breast cancer cell metastasis to the lung in vivo.

【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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