摘要
目的克隆并构建葡萄糖转运蛋白1(GLUT1)启动子的荧光素酶报告基因载体并检测其活性。方法以ZR75-1细胞提取的基因组为模板进行PCR扩增,将获得的GLUT1启动子片段酶切后连接至荧光素酶报告载体pGL4-basic中,重组质粒pGL4-basic-GLUT1经测序正确后与空载体分别转染293T细胞,并检测启动子活性;再将pGL4-basic-GLUT1同myc-HIF1α或对照质粒pXJ-40-myc转染ZR75-1细胞,分别在高糖和低糖条件下培养,检测启动子活性,探讨低氧诱导因子1α(HIF1α)对GLUT1启动子活性的影响。结果DNA测序显示,扩增片段与GLUT1启动子片段的序列一致。荧光素酶活性实验显示,转染pGL4-basic-GLUT1后,GLUT1启动子较对照组有较高活性(P<0.001);低糖条件下,pGL4-basic-GLUT1与myc-HIF1α共转染,GLUT1启动子活性更强(P=0.042)。结论成功构建了GLUT1启动子报告基因载体,明确了HIF1α在高糖和低糖条件下对GLUT1启动子活性的增强作用,为深入研究GLUT1在肿瘤细胞代谢中的作用奠定了基础。
Objective To clone and construct a GLUT1 promoter luciferase reporter gene vector and to investigate its transcriptional activity.Methods A GLUT1 promoter was amplified from ZR75-1 genome by PCR and cloned into the luciferase reporter gene vector pGL4-basic after digestion of restriction enzyme.The resulting plasmid was determined by DNA sequencing before the recombinant plasmid pGL4-basic-GLUT1 and empty vector pGL4-basic were transfected into293 T cell line respectively and the promoter activity was analyzed.PGL4-basic-GLUT1 was co-transfected with myc-HIF1αor control plasmid pXJ-40-myc into ZR75-1 cells which were cultured at high and low glucose levels,and the promoter activity was analyzed to explore the influence of HIF1αon GLUT1 promoter transcription activity.Results DNA sequencing showed that the sequence of the amplified fragment was consistent with the GLUT1 promoter region.Luciferase reporter assays showed that following transfection of pGL4-basic-GLUT 1,the GLUT1 promoter had higher activity than control(P<0.001).Furthermore,under low glucose conditions,co-transfection of pGL4-basic-GLUT1 with myc-HIF1αcould lead to stronger activity of the GLUT1 promoter(P=0.042).Conclusion A GLUT1 promoter reporter gene is cloned successfully,which confirms the enhancement of GLUT1 promoter activity by HIF1αat high and low glucose levels,and is expected to contribute to further study on the role of GLUT1 in tumor cell metabolism.
作者
任鑫鑫
罗菲
罗沙柳
刘坤
马超
梁迎春
李玲
蔺静
REN Xin⁃xin;LUO Fei;LUO Sha⁃liu;LIU Kun;MA Chao;LIANG Ying⁃chun;LI Ling;LIN Jing(Shanxi Medical University,Taiyuan 030000,China;The Fourth Medical Center,General Hospital of PLA,Beijing 100048,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Medical College,Guizhou University,Guiyang 550025,China;Scientific Research and Training Section,General Hospital of Central Theater Command of PLA,Wuhan 430072,China;Institute of Cancer Stem Cells,Dalian Medical University,Dalian,Liaoning 116044,China)
出处
《军事医学》
CAS
北大核心
2020年第7期510-514,共5页
Military Medical Sciences
基金
国家自然科学基金(81372596,81902838)
北京市自然科学基金(7204284)
