转录因子FOXA2调控乳腺癌上皮间质转化的机制研究

发布时间：2018-03-18 12:26

本文选题：转录因子FOXA2　切入点：上皮间质转化EMT　出处：《湖南大学》2015年博士论文　论文类型：学位论文

【摘要】：上皮-间质转化(EMT)过程是指上皮来源的细胞在诱导调控下,丧失了上皮表型的过程。经历了这个过程之后,细胞丧失自身极性、细胞间连接能力乃至上皮表型,但是获得了间质表型并增强了迁徙和侵袭能力。由此细胞可以离开上皮组织,侵袭进入细胞基质并通过循环系统向远端组织进行转移。EMT过程最早发现于胚胎发育和器官的分化过程中,起着重要的生理作用。随后,针对病理相关的EMT研究发现,EMT参与了癌症发生发展、组织器官纤维化病变等相关疾病过程。在这些过程中具有代表性的是,乳腺癌细胞的EMT过程将会导致明显的早起转移事件的发生,这种现象也是乳腺癌临床治疗过程中的一大难题。在相关乳腺癌EMT过程的研究中发现,基因表达调控在整个EMT过程中发挥着关键调控的作用。因此研究特定转录因子调节乳腺癌细胞的EMT分子机理,对阐明肿瘤转移机制和临床治疗中应对乳腺癌转移有着重要的意义。本文主要研究转录因子FOXA2在乳腺癌EMT过程中的功能机制。转录因子FOXA2是叉头框(Forkhead box)家族的成员,其生理功能在于胚胎发育以及多器官功能维持。同时,大量的肿瘤相关研究结果不仅表明FOXA2的表达情况与肿瘤发生以及恶性转化过程高度相关,而且证实FOXA2参与了多种肿瘤细胞的EMT调控过程。所以,我们认为转录因子FOXA2可能是乳腺癌细胞EMT过程中的关键调控因子。我们由乳腺癌临床样本的数据出发,运用乳腺癌E型细胞MCF-7和M型细胞MDA-MB-231作为EMT对应的细胞体系模型,研究乳腺癌细胞中FOXA2刺激E型基因和抑制M型基因的分子作用机制,并且在小鼠尾静脉注射转移模型中加以验证,得到以下结果:首先,我们在乳腺癌临床样本中检测了FOXA2以及E-cadherin、Vimentin等EMT标志基因的表达。同时考察了乳腺癌上皮型细胞MCF-7和间质型细胞MDA-MB-231中以上基因的m RNA和蛋白质表达水平,我们发现FOXA2和上皮表型以及EMT标志基因有着强烈的对应关系。另外,通过EGF诱导的MCF-10A这一EMT模型进一步确认了FOXA2随着EMT过程的发生而表达减弱的事实。然后,为了考察FOXA2对于乳腺癌上皮表型的维持作用,我们在乳腺癌上皮型细胞MCF-7中干扰了FOXA2的表达,发现细胞的迁移能力加强,在分子表达层面上皮型标志物表达下降而间质型标志物表达上升。相对应的,为了验证FOXA2对于乳腺癌间质型细胞迁移能力的抑制作用,我们通过慢病毒感染的方式筛选出稳定高表达FOXA2的在乳腺癌间质型细胞MDA-MB-231细胞株,发现该细胞株的迁移能力减弱,其中的上皮型标志物表达上升而间质型标志物表达下降。这说明FOXA2是维持乳腺癌细胞上皮特征的重要因子,也是抑制乳腺癌细胞发生EMT的关键因子。随后的Ch IP、EMSA、荧光素酶报告基因实验证实,FOXA2能够直接结合在下游靶基因E-cadherin和ZEB2的启动子上,并且分别对其进行正调控和负调控。也有文献指出,FOXA蛋白能够招募转录辅助抑制子TLE3联合作用。在我们的体系中,通过m RNA水平和蛋白水平的检测,我们发现TLE3的表达与上皮表型呈高度正相关。之后,通过Ch IP、Co-IP以及EMSA等实验,确认FOXA2通过招募TLE3到ZEB2的启动子上,进而抑制了ZEB2的表达。最后为了考察FOXA2在体内对于乳腺癌细胞转移的影响,我们通过尾静脉注射高表达FOXA2的MDA-MB-231细胞株的方式建立了裸鼠乳腺癌转移模型,确认FOXA2在体内可以抑制乳腺癌细胞向肺部的转移能力。
[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.

【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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