TGF-β1诱导乳腺癌细胞发生EMT转化过程中能量代谢的研究
发布时间：2018-09-10 14:20
【摘要】：[目的]:本文通过外加TGF-β1,诱导乳腺癌细胞MCF-7从上皮细胞向间充质转化(epithelial-mesenchymal transition, EMT)。通过建立稳定的 EMT 细胞模型,探究MCF-7发生EMT转化过程中能量代谢方式的变化及其调控机制。[方法]:用含有TGF-β1 (2ng/ml)的培养基培养MCF-7细胞9天,之后换正常培养基培养9天。在这个过程中MCF-7细胞完成了从上皮细胞向间质细胞转化,再从间质细胞转化成上皮细胞的过程。实验中,我们用qPCR和western检测了 EMT上皮细胞标志蛋白E-耗黏蛋白(E-cadherin)、间质细胞标志蛋白波形蛋白(Vimentin)和转录因子Snail 1的表达,同时通过细胞侵袭实验检测了细胞侵袭能力的变化;用western检测了糖代谢相关蛋白PDH、LDH、NDUFB8、TFAM和COX1的表达变化;通过流式细胞术和qPCR检测了线粒体数量以及线粒体DNA拷贝数;用qPCR和western检测了 FASN、CPT1和CD36等与脂代谢相关基因或蛋白的表达;并检测了可能引起脂代谢变化的相关信号通路蛋白的表达变化;最后我们收集到20例乳腺癌临床样本,利用免疫组化方法检测了脂代谢相关蛋白在乳腺癌组织中的表达部位和表达水平的变化。通过开展上述研究,我们得到如下研究结果:[结果](1)成功构建了 TGF-β1诱导MCF-7从上皮细胞向间质细胞的转化,转化过程中观察到相应的细胞形态学的变化和粘附性降低,同时检测到E-cadherin表达降低,Vimentin和Snail1表达量升高。(2)MCF-7在发生EMT过程中细胞增殖能力降低,但ATP水平增高,细胞侵袭能力增加。(3)MCF-7在发生EMT过程中氧化磷酸化相关蛋白NDUFB8、TFAM和COX1表达显著增加,线粒体数量和线粒体DNA也明显增加。表明EMT转化过程中细胞线粒体氧化磷酸化活性增强。(4) EMT转化过程中FASN表达降低,CPT-1和CD36表达升高,细胞脂肪酸的合成途径降低脂肪酸β氧化增强。(5) TGF-β1处理MCF-7后检测到P-AMPK表达增高而ACC的表达降低,P-AMPK可能通过抑制ACC表达从而增强CPT-1的表达,增加细胞内脂肪酸氧化。(6)对20例乳腺癌临床肿瘤样本进行免疫组化分析发现在乳腺癌肿瘤组织中E-cadherin高表达的上皮细胞中FASN高表达,而在Vimentin高表达的间质细胞中CPT-1α高表达,表明在乳腺癌组织中存在上皮细胞和间质细胞的明显分区,在上皮细胞中脂肪酸合成加强利于脂肪储存,而间质细胞中加强脂肪酸β氧化,产生大量ATP用于细胞侵袭和转移。提示肿瘤组织的不同区域和不同细胞类型代谢方式不同。[结论]:TGF-β1诱导MCF-7发生EMT的过程中细胞增殖减慢,细胞侵袭能力增加,ATP水平增加,线粒体氧化磷酸化活性增强,脂肪酸β氧化增加,脂肪酸合成降低。同时乳腺癌肿瘤组织中也检测到了上皮细胞中脂肪酸合成加强,利于脂肪储存,而间质细胞中脂肪酸β氧化加强,产生大量ATP利于细胞侵袭和转移。提示肿瘤组织的不同区域和不同细胞类型代谢方式不同。本研究对肿瘤转移过程中的代谢特征有新的认识,为EMT转化过程中能量代谢与转移的关系研究奠定基础。
[Abstract]:[Objective] To induce the epithelial-mesenchymal transition (EMT) of breast cancer cell line MCF-7 by adding TGF-beta 1. To establish a stable EMT cell model and explore the changes of energy metabolism and its regulation mechanism during the EMT transformation of MCF-7. MCF-7 cells were cultured in medium for 9 days, then in normal medium for 9 days. During this process, MCF-7 cells completed the transformation from epithelial cells to mesenchymal cells, and then from mesenchymal cells to epithelial cells. The expression of Vimentin and Snail-1 was detected by cell invasion assay; the expression of PDH, LDH, NDUFB8, TFAM and COX1 were detected by Western blot; the number of mitochondria and the number of mitochondrial DNA copies were detected by flow cytometry and qPCR; and the number of mitochondrial DNA copies was detected by qPCR and qPCR. Western detected the expression of FASN, CPT1 and CD36 genes or proteins related to lipid metabolism; detected the expression of signal pathway proteins that may cause lipid metabolism changes; finally, we collected 20 breast cancer clinical samples and detected the expression of lipid metabolism related proteins in breast cancer tissues by immunohistochemical method. The results were as follows: (1) TGF-beta 1 was successfully constructed to induce the transformation of MCF-7 from epithelial cells to mesenchymal cells. The morphological changes and adhesion of MCF-7 cells were observed during the transformation, and the expression of E-cadherin, Vimentin and Snail 1 were decreased. (3) The expression of oxidative phosphorylation-related proteins NDUFB8, TFAM, COX1, mitochondrial DNA and mitochondrial DNA increased significantly during EMT. Phosphorylation activity was enhanced. (4) The expression of FASN decreased, the expression of CPT-1 and CD36 increased, and the synthesis pathway of cellular fatty acid decreased the oxidation of fatty acid beta. (5) After MCF-7 was treated with TGF-beta 1, the expression of P-AMPK increased and the expression of ACC decreased. P-AMPK may enhance the expression of CPT-1 and increase intracellular lipid by inhibiting the expression of ACC. Fatty acid oxidation. (6) Immunohistochemical analysis of 20 breast cancer samples showed that FASN was highly expressed in E-cadherin-overexpressed epithelial cells, while CPT-1a was highly expressed in Vimentin-overexpressed stromal cells, suggesting that there were distinct zones of epithelial cells and stromal cells in breast cancer tissues. Fatty acid synthesis in skin cells is beneficial to fat storage, whereas in stromal cells, fatty acid beta oxidation is enhanced, resulting in a large number of ATP for cell invasion and metastasis. In addition, ATP levels increased, mitochondrial oxidative phosphorylation activity increased, fatty acid beta oxidation increased, and fatty acid synthesis decreased. At the same time, fatty acid synthesis in epithelial cells was also detected in breast cancer tissues, which was conducive to fat storage, while fatty acid beta oxidation in stromal cells was enhanced, resulting in a large number of ATP conducive to cell invasion and metastasis. This study provides a new understanding of the metabolic characteristics of tumor metastasis and lays a foundation for the study of the relationship between energy metabolism and metastasis in the process of EMT transformation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2234698
[Abstract]:[Objective] To induce the epithelial-mesenchymal transition (EMT) of breast cancer cell line MCF-7 by adding TGF-beta 1. To establish a stable EMT cell model and explore the changes of energy metabolism and its regulation mechanism during the EMT transformation of MCF-7. MCF-7 cells were cultured in medium for 9 days, then in normal medium for 9 days. During this process, MCF-7 cells completed the transformation from epithelial cells to mesenchymal cells, and then from mesenchymal cells to epithelial cells. The expression of Vimentin and Snail-1 was detected by cell invasion assay; the expression of PDH, LDH, NDUFB8, TFAM and COX1 were detected by Western blot; the number of mitochondria and the number of mitochondrial DNA copies were detected by flow cytometry and qPCR; and the number of mitochondrial DNA copies was detected by qPCR and qPCR. Western detected the expression of FASN, CPT1 and CD36 genes or proteins related to lipid metabolism; detected the expression of signal pathway proteins that may cause lipid metabolism changes; finally, we collected 20 breast cancer clinical samples and detected the expression of lipid metabolism related proteins in breast cancer tissues by immunohistochemical method. The results were as follows: (1) TGF-beta 1 was successfully constructed to induce the transformation of MCF-7 from epithelial cells to mesenchymal cells. The morphological changes and adhesion of MCF-7 cells were observed during the transformation, and the expression of E-cadherin, Vimentin and Snail 1 were decreased. (3) The expression of oxidative phosphorylation-related proteins NDUFB8, TFAM, COX1, mitochondrial DNA and mitochondrial DNA increased significantly during EMT. Phosphorylation activity was enhanced. (4) The expression of FASN decreased, the expression of CPT-1 and CD36 increased, and the synthesis pathway of cellular fatty acid decreased the oxidation of fatty acid beta. (5) After MCF-7 was treated with TGF-beta 1, the expression of P-AMPK increased and the expression of ACC decreased. P-AMPK may enhance the expression of CPT-1 and increase intracellular lipid by inhibiting the expression of ACC. Fatty acid oxidation. (6) Immunohistochemical analysis of 20 breast cancer samples showed that FASN was highly expressed in E-cadherin-overexpressed epithelial cells, while CPT-1a was highly expressed in Vimentin-overexpressed stromal cells, suggesting that there were distinct zones of epithelial cells and stromal cells in breast cancer tissues. Fatty acid synthesis in skin cells is beneficial to fat storage, whereas in stromal cells, fatty acid beta oxidation is enhanced, resulting in a large number of ATP for cell invasion and metastasis. In addition, ATP levels increased, mitochondrial oxidative phosphorylation activity increased, fatty acid beta oxidation increased, and fatty acid synthesis decreased. At the same time, fatty acid synthesis in epithelial cells was also detected in breast cancer tissues, which was conducive to fat storage, while fatty acid beta oxidation in stromal cells was enhanced, resulting in a large number of ATP conducive to cell invasion and metastasis. This study provides a new understanding of the metabolic characteristics of tumor metastasis and lays a foundation for the study of the relationship between energy metabolism and metastasis in the process of EMT transformation.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
【参考文献】
相关期刊论文 前1条
1 Liem Minh Phan;Sai-Ching Jim Yeung;Mong-Hong Lee;;Cancer metabolic reprogramming: importance, main features, and potentials for precise targeted anti-cancer therapies[J];Cancer Biology & Medicine;2014年01期
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