Fra-1在乳腺癌细胞中高表达原因及其启动子高转录活性的分子机制研究  

Causes of Fra-1 high expression in breast cancer cells and molecular mechanism of Fra-1 high transcription activity

作　　者：周秋媛 李虹 王红莉 李伟 徐艳华 

机构地区：[1]湖北省恩施州中心医院病理科,湖北恩施445000

出　　处：《国际检验医学杂志》2017年第22期3113-3115,3119,共4页International Journal of Laboratory Medicine

摘　　要：目的探讨Fra-1在乳腺癌细胞中高表达原因,以及Fra-1启动子高转录活性的分子机制。方法选取2株人乳腺癌细胞株MDA231和MCF-7、1株人脐静脉内皮细胞株ECV304进行研究。采用实时荧光定量逆转录-聚合酶链反应法和蛋白质印迹法分别检测Fra-1mRNA和蛋白的表达情况;构建Fra-1双荧光报告载体,检测Fra-1启动子转录活性;构建Fra-1启动子短截报告载体及相关位点的突变型报告载体,检测短截报告载体及突变型载体转录活性;通过凝胶迁移阻滞试验观察生物素标记的特异性探针与活化因子蛋白-1(AP1)结合情况。结果乳腺癌细胞株MDA231和MCF-7中,Fra-1的mRNA和蛋白的相对表达量及其启动子全长报告载体转录活性均高于人脐静脉内皮细胞株ECV304,差异有统计学意义(P<0.05);在构建的一系列Fra-1启动子短截报告载体中,只有pGL3B-256活性明显下降,差异有统计学意义(P<0.05);AP1突变型报告载体pGL3B-M2、特异性蛋白1及AP1双突变报告载体pGL3B-M3活性明显低于野生型报告载体pGL3B-M1,差异有统计学意义(P<0.05);凝胶迁移阻滞试验观察到生物素标记的特异性探针与AP1结合发生了明显的阻滞效应。结论在体外培养的乳腺癌细胞株MDA231和MCF-7中,反式作用因子AP1通过激活Fra-1基因启动子上调其表达。Objective To explore the causes of Fra-1 high expression in breast cancer cells and molecular mechanism of Fra-1 high transcription activity.Methods Two human breast cancer cell lines MDA231 and MCF-7 and 1 human umbilical vein endothelial cell ECV304 were selected as the research objects.Real time fluorescent quantitative reverse transcription-polymerase chain reaction and Western blotting were used to detect the Fra-1 mRNA and protein expression;the Fra-1 dual fluorescence reporter vector was constructed,and the Fra-1 promotor transcription activity was detected;the Fra-1 promotor short-cut reporter vector and mutation reporter vector at related loci were constructed,and the transcription activity of short-cut reporter vector and mutation reporter vector was detected;the binding situation of specific probe marked by biotin with activator protein-1（AP1）was observed by using the gel migration block test.Results The relative expression of mRNA and protein and promotor whole length reporter vector transcription activity of Fra-1 in human breast cancer cell lines MDA231 and MCF-7 cells were higher than that in human umbilical vein endothelial cell MCV304,the difference was statistically significant（P0.05）;in constructed series Fra-1 promotor shortcut reporter vectors,only the activity of pGL3 B-256 was obviously decreased,the difference was statistically significant（P0.05）;the activity of AP1 mutation reporter vector pGL3 B-M2 and SP1 and AP1 dual mutation reporter vector pGL3 B-M3 was significantly lower than that of wild repoter vector pGL3 B-M1,the difference was statistically significant（P0.05）;the gel migration block test found that the binding of specific probe marked by biotin with AP1 had obvious blocking effect.Conclusion In in vitro cultured breast cancer cell lines MDA231 and MCF-7,the trans-acting factor AP1 up-regulates its expression by activating Fra-1 gene promoter.

关 键 词：启动子 乳腺癌 反式作用因子 

分 类 号：R737.9[医药卫生—肿瘤]