金属硫蛋白沉默对乳腺癌转移基因表达的影响
发布时间：2018-12-20 15:11
【摘要】：目的乳腺癌是较为普遍的一种恶性肿瘤,目前数十种与乳腺癌发病、发展及转移相关的基因已经被鉴定。本研究拟观察在乳腺癌MCF-7细胞中,当MT2A基因表达沉默时,乳腺癌细胞的繁殖及乳腺癌转移关联基因的表达变化。方法利用生物信息学分析金属硫蛋白MT2A的序列特点设计并合成干扰载体sh RNA-MT2A,转染乳腺癌MCF-7细胞获得MT2A基因沉默型细胞株。利用观察MT2A基因沉默型乳腺癌细胞的繁殖变化和黏附基质的能力变化,通过RT-PCR、western blot检测FGF-1、Twist、转铁蛋白、AOP-1和过氧化还原酶6等5个基因在转录和翻译水平上的变化。结果1设计并合成三条shRNA-MT2A干扰载体,通过实验最终确定一条干扰载体用于后续研究。2通过脂质体转染技术将sh RNA-MT2A转入乳腺癌MCF-7细胞株,得到MT2A基因沉默型细胞株,并通过荧光显微镜观察、半定量PCR技术和western blot技术对MT2A的表达情况作了鉴定。3在MT2A基因缺失时,癌细胞繁殖变化,发现乳腺癌细胞MCF-7繁殖速度降低,与基质的黏附能力增强。4实时定量PCR结果显示FGF-1和Twist蛋白在MT2A缺失的乳腺癌细胞中表达水平变化不大。5转铁蛋白在MT2A基因沉默的乳腺癌细胞中转铁蛋白的m RNA含量上升。6研究发现细胞内的MT2A沉默后,AOP-1和过氧化还原酶6的m RNA水平均上升,但是AOP-1基因表达上调水平高于过氧化还原酶6基因。7 western blot实验分析显示在MT2A基因沉默的乳腺癌细胞中FGF-1和Twist蛋白水平没有发生显著变化,而转铁蛋白、AOP-1和过氧化还原酶6三种蛋白在细胞中的含量出现上升。8构建MT2A与β-domain过表达细胞株,结果证实MT2A与β-domain的过表达不影响乳腺癌细胞的繁殖,而β-domain过表达促进细胞线粒体中细胞色素c氧化酶6B2亚基(CYP6B2)表达量上调。结论本研究证实MT2A基因沉默型乳腺癌细胞繁殖速度变慢、与基质的黏附能力增强;因为MT的主要功能是调节金属离子代谢和抗氧化,所以MT2A基因沉默影响到了铁离子调控蛋白转铁蛋白,也影响到了抗氧化蛋白AOP-1和过氧化还原酶6和细胞色素c氧化酶6B2亚基。
[Abstract]:Objective Breast cancer is a common malignant tumor. At present, dozens of genes related to the pathogenesis, development and metastasis of breast cancer have been identified. The aim of this study was to observe the proliferation of breast cancer cells and the expression of metastasis associated genes when the expression of MT2A gene was silenced in breast cancer MCF-7 cells. Methods using bioinformatics to analyze the sequence characteristics of metallothionein MT2A, interference vector sh RNA-MT2A, was synthesized and transfected into breast cancer MCF-7 cells to obtain MT2A gene silencing cell line. The reproductive changes and adhesion ability of MT2A gene silencing breast cancer cells were observed. FGF-1,Twist, transferrin was detected by RT-PCR,western blot. The transcriptional and transcriptional changes of AOP-1 and reductase 6. Results 1 three shRNA-MT2A interference vectors were designed and synthesized, and one interference vector was determined by experiment for further study. 2 sh RNA-MT2A was transfected into breast cancer MCF-7 cell line by liposome transfection technique. MT2A gene silencing cell lines were obtained, and the expression of MT2A was identified by fluorescence microscopy, semi-quantitative PCR and western blot techniques. 3 when the MT2A gene was absent, the proliferation of cancer cells changed. It was found that the MCF-7 reproduction rate of breast cancer cells decreased, Enhanced adhesion to matrix. 4 Real-time quantitative PCR results showed that the expression of FGF-1 and Twist proteins in MT2A deficient breast cancer cells showed little change. 5 transferrin in MT2A gene silenced breast cancer cell line transferrin The content of m RNA was increased. 6 the study found that after MT2A silencing, The levels of m RNA of both AOP-1 and reductase 6 increased. However, the up-regulation of AOP-1 gene expression was higher than that of reductase 6 gene. 7 western blot analysis showed that there was no significant change in FGF-1 and Twist protein levels in breast cancer cells with MT2A gene silencing. The content of AOP-1 and reductase 6 protein increased in the cells. 8 the overexpression of MT2A and 尾-domain showed that the overexpression of MT2A and 尾-domain did not affect the proliferation of breast cancer cells. Overexpression of 尾-domain increased the expression of cytochrome c oxidase 6B2 subunit (CYP6B2) in mitochondria. Conclusion this study confirmed that MT2A gene silencing breast cancer cells have slower reproductive rate and stronger adhesion to matrix. Because the main function of MT is to regulate metal ion metabolism and antioxidation, MT2A gene silencing affects ferrion-regulated protein transferrin. It also affected the antioxidant protein AOP-1, peroxidation reductase 6 and cytochrome c oxidase 6B2 subunit.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
本文编号：2388169
[Abstract]:Objective Breast cancer is a common malignant tumor. At present, dozens of genes related to the pathogenesis, development and metastasis of breast cancer have been identified. The aim of this study was to observe the proliferation of breast cancer cells and the expression of metastasis associated genes when the expression of MT2A gene was silenced in breast cancer MCF-7 cells. Methods using bioinformatics to analyze the sequence characteristics of metallothionein MT2A, interference vector sh RNA-MT2A, was synthesized and transfected into breast cancer MCF-7 cells to obtain MT2A gene silencing cell line. The reproductive changes and adhesion ability of MT2A gene silencing breast cancer cells were observed. FGF-1,Twist, transferrin was detected by RT-PCR,western blot. The transcriptional and transcriptional changes of AOP-1 and reductase 6. Results 1 three shRNA-MT2A interference vectors were designed and synthesized, and one interference vector was determined by experiment for further study. 2 sh RNA-MT2A was transfected into breast cancer MCF-7 cell line by liposome transfection technique. MT2A gene silencing cell lines were obtained, and the expression of MT2A was identified by fluorescence microscopy, semi-quantitative PCR and western blot techniques. 3 when the MT2A gene was absent, the proliferation of cancer cells changed. It was found that the MCF-7 reproduction rate of breast cancer cells decreased, Enhanced adhesion to matrix. 4 Real-time quantitative PCR results showed that the expression of FGF-1 and Twist proteins in MT2A deficient breast cancer cells showed little change. 5 transferrin in MT2A gene silenced breast cancer cell line transferrin The content of m RNA was increased. 6 the study found that after MT2A silencing, The levels of m RNA of both AOP-1 and reductase 6 increased. However, the up-regulation of AOP-1 gene expression was higher than that of reductase 6 gene. 7 western blot analysis showed that there was no significant change in FGF-1 and Twist protein levels in breast cancer cells with MT2A gene silencing. The content of AOP-1 and reductase 6 protein increased in the cells. 8 the overexpression of MT2A and 尾-domain showed that the overexpression of MT2A and 尾-domain did not affect the proliferation of breast cancer cells. Overexpression of 尾-domain increased the expression of cytochrome c oxidase 6B2 subunit (CYP6B2) in mitochondria. Conclusion this study confirmed that MT2A gene silencing breast cancer cells have slower reproductive rate and stronger adhesion to matrix. Because the main function of MT is to regulate metal ion metabolism and antioxidation, MT2A gene silencing affects ferrion-regulated protein transferrin. It also affected the antioxidant protein AOP-1, peroxidation reductase 6 and cytochrome c oxidase 6B2 subunit.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
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