【摘要】：目的乳腺癌是較為普遍的一種惡性腫瘤,目前數(shù)十種與乳腺癌發(fā)病、發(fā)展及轉(zhuǎn)移相關(guān)的基因已經(jīng)被鑒定。本研究擬觀察在乳腺癌MCF-7細(xì)胞中,當(dāng)MT2A基因表達(dá)沉默時,乳腺癌細(xì)胞的繁殖及乳腺癌轉(zhuǎn)移關(guān)聯(lián)基因的表達(dá)變化。方法利用生物信息學(xué)分析金屬硫蛋白MT2A的序列特點(diǎn)設(shè)計并合成干擾載體sh RNA-MT2A,轉(zhuǎn)染乳腺癌MCF-7細(xì)胞獲得MT2A基因沉默型細(xì)胞株。利用觀察MT2A基因沉默型乳腺癌細(xì)胞的繁殖變化和黏附基質(zhì)的能力變化,通過RT-PCR、western blot檢測FGF-1、Twist、轉(zhuǎn)鐵蛋白、AOP-1和過氧化還原酶6等5個基因在轉(zhuǎn)錄和翻譯水平上的變化。結(jié)果1設(shè)計并合成三條shRNA-MT2A干擾載體,通過實(shí)驗(yàn)最終確定一條干擾載體用于后續(xù)研究。2通過脂質(zhì)體轉(zhuǎn)染技術(shù)將sh RNA-MT2A轉(zhuǎn)入乳腺癌MCF-7細(xì)胞株,得到MT2A基因沉默型細(xì)胞株,并通過熒光顯微鏡觀察、半定量PCR技術(shù)和western blot技術(shù)對MT2A的表達(dá)情況作了鑒定。3在MT2A基因缺失時,癌細(xì)胞繁殖變化,發(fā)現(xiàn)乳腺癌細(xì)胞MCF-7繁殖速度降低,與基質(zhì)的黏附能力增強(qiáng)。4實(shí)時定量PCR結(jié)果顯示FGF-1和Twist蛋白在MT2A缺失的乳腺癌細(xì)胞中表達(dá)水平變化不大。5轉(zhuǎn)鐵蛋白在MT2A基因沉默的乳腺癌細(xì)胞中轉(zhuǎn)鐵蛋白的m RNA含量上升。6研究發(fā)現(xiàn)細(xì)胞內(nèi)的MT2A沉默后,AOP-1和過氧化還原酶6的m RNA水平均上升,但是AOP-1基因表達(dá)上調(diào)水平高于過氧化還原酶6基因。7 western blot實(shí)驗(yàn)分析顯示在MT2A基因沉默的乳腺癌細(xì)胞中FGF-1和Twist蛋白水平?jīng)]有發(fā)生顯著變化,而轉(zhuǎn)鐵蛋白、AOP-1和過氧化還原酶6三種蛋白在細(xì)胞中的含量出現(xiàn)上升。8構(gòu)建MT2A與β-domain過表達(dá)細(xì)胞株,結(jié)果證實(shí)MT2A與β-domain的過表達(dá)不影響乳腺癌細(xì)胞的繁殖,而β-domain過表達(dá)促進(jìn)細(xì)胞線粒體中細(xì)胞色素c氧化酶6B2亞基(CYP6B2)表達(dá)量上調(diào)。結(jié)論本研究證實(shí)MT2A基因沉默型乳腺癌細(xì)胞繁殖速度變慢、與基質(zhì)的黏附能力增強(qiáng);因?yàn)镸T的主要功能是調(diào)節(jié)金屬離子代謝和抗氧化,所以MT2A基因沉默影響到了鐵離子調(diào)控蛋白轉(zhuǎn)鐵蛋白,也影響到了抗氧化蛋白AOP-1和過氧化還原酶6和細(xì)胞色素c氧化酶6B2亞基。
[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.
【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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本文編號：2388169