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乳腺癌细胞特异性MGA启动子的扩增及功能分析

李金桥, 梁培龙, 吴峰, 杨超, 张洪玉, 魏力军, 戴钟铨, 李莹辉

李金桥, 梁培龙, 吴峰, 杨超, 张洪玉, 魏力军, 戴钟铨, 李莹辉. 乳腺癌细胞特异性MGA启动子的扩增及功能分析[J]. 肿瘤防治研究, 2015, 42(12): 1177-1182. DOI: 10.3971/j.issn.1000-8578.2015.12.002
引用本文: 李金桥, 梁培龙, 吴峰, 杨超, 张洪玉, 魏力军, 戴钟铨, 李莹辉. 乳腺癌细胞特异性MGA启动子的扩增及功能分析[J]. 肿瘤防治研究, 2015, 42(12): 1177-1182. DOI: 10.3971/j.issn.1000-8578.2015.12.002
LI Jinqiao, LIANG Peilong, WU Feng, YANG Chao, ZHANG Hongyu, WEI Lijun, DAI Zhongquan, LI Yinghui. Amplification and Function of Specific Mammaglobin A Promoter in Breast Cancer Cells[J]. Cancer Research on Prevention and Treatment, 2015, 42(12): 1177-1182. DOI: 10.3971/j.issn.1000-8578.2015.12.002
Citation: LI Jinqiao, LIANG Peilong, WU Feng, YANG Chao, ZHANG Hongyu, WEI Lijun, DAI Zhongquan, LI Yinghui. Amplification and Function of Specific Mammaglobin A Promoter in Breast Cancer Cells[J]. Cancer Research on Prevention and Treatment, 2015, 42(12): 1177-1182. DOI: 10.3971/j.issn.1000-8578.2015.12.002

乳腺癌细胞特异性MGA启动子的扩增及功能分析

基金项目: 国家重点基础研究发展计划(973)项目(2011CB707704)
详细信息
    作者简介:

    李金桥(1990-),男,硕士在读,主要从事肿瘤发生与防治的细胞分子生物学研究

    通讯作者:

    戴钟铨,E-mail:daizhq77@163.com

    李莹辉,E-mail:yinghuidd@vip.sina.com

  • 中图分类号: R737.9

Amplification and Function of Specific Mammaglobin A Promoter in Breast Cancer Cells

  • 摘要: 目的 探讨人乳腺球蛋白(mammaglobin A, MGA)基因启动子的乳腺肿瘤细胞特异性和调控元件,并验证MGA启动子载体系统在活体肿瘤组织中的表达活性。方法 PCR扩增5.6 kb大小的人MGA启动子片段,并在此基础上构建四个含有萤火虫荧光素酶报告基因的重组载体;瞬时转染到乳腺癌细胞系T47D和人胚肾细胞系HEK293T后,利用双荧光素酶报告基因体系,检测各个MGA启动子活性;稳定转染MGA重组载体到乳腺癌细胞系T47D(T47DEM),进行裸鼠皮下造瘤,活体成像检测报告基因活性。结果 (1)双增强子的启动子活性低于单增强子启动子(降低82%);(2)定点删除YY1结合元件后,启动子活性下降76.2%,乳腺肿瘤细胞特异性下降38.4%;(3)定点删除Nkx2.5后,启动子活性没有明显变化,但特异性降低31.9%;(4)细胞株T47DEM在体形成肿瘤后,表现出明显的报告基因活性。结论 研究了两个增强子叠加和两个转录因子结合位点(YY1和Nkx2.5)对MGA启动子活性和特异性的影响;构建的细胞株(T47DEM)可以用于治疗乳腺癌药物的在体筛选。

     

    Abstract: Objective To investigate the specificity and regulatory element of human mammaglobin A (MGA) promoter, and validate its expression activity in living breast tumor tissues. Methods The 5.6kb MGA promoter was amplified by PCR from human genome. Then, four recombinant plasmids with different fragment or element mutation of MGA promoter driving firefly luciferase reporter were constructed. After transient transfection into breast cancer cell line T47D and human embryonic kidney cell line HEK293T, dual luciferase reporter gene system was used to determine the activity of these MGA promoter fragments. T47D cells stably transfected with MGA recombinant vector(T47DEM) was implanted subcutaneously in nude mice, and the subcutaneous breast tumor tissue were imaged by in vivo imaging system(IVIS). Results (1) Superposition of two enhancers markedly reduced the promoter activity by 82%; (2) Site-specific deletion of YY1 binding element decreased both activity (76.2%) and specificity (38.4%) of MGA promoter; (3) The deletion of Nkx2.5 binding site had no effect on the activity of MGA promoter but significantly reduced the specificity (31.9%); (4) In vivo image of the subcutaneous breast tumor tissue developed form (T47DEM) showed obviously the firefly luciferase report gene activity. Conclusion The effect of two transcription factor binding sites(YY1 and Nkx2.5) and superimposing two enhancers to MGA promoter are investigated and the constructed T47DEM cell line could be used for the in vivo drug screening of breast cancer.

     

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出版历程
  • 收稿日期:  2015-01-04
  • 修回日期:  2015-03-17
  • 刊出日期:  2015-12-24

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