Snail诱导的FTL在乳腺癌发生发展中的作用

发布时间：2018-04-02 19:24

本文选题：FTL　切入点：Snail　出处：《大连医科大学》2017年硕士论文

【摘要】：目的:乳腺癌在全世界女性癌症发病率居首位,并且易转移复发,所以也是死亡率很高的疾病。近年来许多研究表明,铁代谢相关基因和肿瘤的发生发展有密切关系,可作为癌症预后指标。铁代谢基因铁储蛋白的亚基FTL,虽然在许多癌症中高表达,包括乳腺癌、结直肠癌、肝癌和胰腺癌等,但其在乳腺癌中的作用研究较少,对肿瘤影响的机制不是很清楚。Snail主要又是介导乳腺癌发生转移的重要蛋白,而且近些年的研究发现Snail对肿瘤的代谢调控也很重要。我们之前研究发现,Snail在乳腺正常细胞和乳腺癌细胞MCF-7中都能上调FTL的表达,说明Snail可能通过介导FTL的表达对乳腺癌的发生发展产生作用。因此,我们探究了FTL和Snail在乳腺癌发生发展中的作用,及FTL对肿瘤细胞的迁移、生长的促进作用。方法:为研究Snail和FTL在乳腺癌发生发展的作用,snail是如何调控FTL的表达,我们首先在多种肿瘤细胞中分别构建了Snail过表达及敲低的稳转细胞株,并用实时荧光定量PCR技术检测Snail对FTL表达的影响。为了进一步验证Snail对FTL的调控作用,我们构建了FTL启动子的荧光素酶报告基因质粒。在HEK293T细胞中,共转染Snail与荧光素酶报告基因质粒,并检测Snail对FTL启动子活性的影响。下一步我们进行了FTL的功能研究。我们首先在正常乳腺细胞系MCF-10A、人乳腺癌细胞MCF-7和MDA-MB-231中构建了FTL过表达和敲低的稳转细胞系,并在过表达snail的MCF-10A、MCF-7和MDA-MB-231细胞中构建了FTL过表达和敲低的稳转细胞系。CCK8、Transwell和流式细胞检测分别检测细胞的生长,迁移能力和细胞周期变化。结果:在正常乳腺细胞系MCF-10A、人乳腺癌细胞MCF-7细胞中,snail可以促进FTL的表达,敲低snail能减少FTL的表达,但在人乳腺癌细胞MDAMB-231中FTL的表达没有发现受snail的影响。荧光素酶基因实验显示,snail可能并不直接作为转录激活因子调控FTL的表达,而是通过其他方式调控FTL的表达。在过表达FTL的细胞中,细胞生长和迁移能力增强;相应的,在敲低FTL时,细胞的生长和迁移能力下降,细胞周期被阻滞在G2/M期。结论:1.我们的研究表明snail可促进FTL的表达,但不是通过其转录调控直接结合于FTL的启动子,可能通过其他调控途径间接促进FTL表达。2.FTL可促进乳腺癌的生长和迁移。
[Abstract]:Objective: breast cancer rate ranks first in the world of female cancer incidence, and easy to recurrence and metastasis, so the disease is very high mortality rate. Recently many studies have shown that there is a close relationship between the occurrence and development of iron metabolism related genes and cancer, cancer can be used as a prognostic indicator. Subunit FTL gene of iron metabolism of iron storage protein, though in many cancers, high expression, including breast cancer, colorectal cancer, liver cancer and pancreatic cancer, but its role in breast cancer research on the mechanism of tumor effect is not very clear.Snail is an important protein mediated breast cancer metastasis, and recent studies revealed that the metabolic regulation of tumor Snail it is very important. Our previous study found that Snail can increase the expression of FTL in normal breast cells and breast cancer cells MCF-7, Snail expression may be mediated through the production of FTL and development of breast cancer The role of students. Therefore, we study the FTL and Snail in the progression of breast cancer, and FTL on the migration of tumor cells and growth promoting effects. Methods: To study the Snail and FTL in the occurrence and development of breast cancer, Snail expression is how to control the FTL, we first in a variety of tumor cells respectively. Construction of Snail overexpression and knockdown of stabletransfection cell lines, and affect the detection of Snail real-time fluorescence quantitative PCR technology on the expression of FTL. In order to further verify the regulation effect of Snail on FTL, we constructed the luciferase reporter plasmid FTL promoter. In HEK293T cells, CO transfected with the luciferase reporter gene Snail effect of plasmid Snail and to detect the activity of the FTL promoter. Next we study the functions of FTL. We first MCF-10A in normal breast cell lines, MCF-7 and MDA-MB-231 in human breast cancer cells to construct FTL Expression and knockdown of stabletransfection cell lines, and the expression of snail in MCF-10A, MCF-7 and MDA-MB-231 cells to construct FTL overexpression and knockdown of stabletransfection cell lines.CCK8, Transwell and flow cytometry were used to detect cell growth, migration and cell cycle changes. Results: in normal breast cell line MCF-10A, human breast cancer MCF-7 cells, snail can promote the expression of FTL, knockdown of snail can reduce the expression of FTL, but the expression in human breast cancer cell MDAMB-231 FTL found no influence of snail. Luciferase gene display experiment, snail can not directly as the expression of activating transcription factor regulation of FTL, but through the expression regulation of FTL in other ways. Over expression of FTL cells, increase the growth and migration of cells; accordingly, knocking at low FTL, decreased growth and cell migration, cell cycle arrest in G2/M phase. Conclusion: 1., our research shows that snail can promote the expression of FTL, but not directly through its transcriptional regulation, and directly bind to the promoter of FTL. It may indirectly promote FTL expression by other regulatory pathways, and.2.FTL can promote the growth and migration of breast cancer.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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