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目的本文旨在对NGAL基因启动子区及其附近是否存在TPA反应元件进行研究。方法(1)采用PCR法从食管癌细胞中克隆NGAL基因5′侧翼区416～+84片段,然后分别插入质粒pGLB和pGLE,构建pGLB416和pGLE416荧光素酶报告基因表达载体;(2)将pGLB416和pGLE416分别同pRLTK共转染食管癌细胞EC109;(3)用TPA刺激转染的EC109,检测细胞相对荧光素酶活力,通过相对荧光素酶活力变化判定NGAL基因5′侧翼416～+84区是否存在TPA反应元件,同时进行生物信息学分析。结果无论是pGLB416还是pGLE416,与pRLTK共转染食管癌细胞EC109后,用TPA刺激,与其相应的空载体相比,转染细胞荧光素酶活力均极显著升高(t检验,P<0.01),约分别升高46.82和46.41倍;而TPA刺激组与没有TPA刺激组相比,pGLB416和pGLE416转染EC109细胞荧光素酶活力分别显著升高了5.17倍和7.37倍(t检验,P<0.01)。生物信息学分析显示,NGAL基因5′侧翼416～+84区段至少存在4个潜在的TPA反应元件。结论食管癌细胞NGAL基因5′侧翼416～+84区段存在着TPA反应元件。
Objective This study aimed to investigate the presence or absence of TPA response elements in the NGAL gene promoter region. Methods (1) PCR was used to clone segments 416 ~ + 84 of 5 ’flanking region of NGAL gene from esophageal cancer cells, and then inserted into pGLB and pGLE respectively to construct pGLB416 and pGLE416 luciferase reporter gene expression vectors; (2) pGLB416 And pGLE416 were co-transfected with pRLTK esophageal cancer cells EC109; (3) TPA stimulated transfected EC109 cells were detected relative luciferase activity by comparing the relative luciferase activity of NGAL gene 5 ’flanking 416 ~ +84 TPA response elements exist, while bioinformatics analysis. Results Both pGLB416 and pGLE416 transfected esophageal cancer cells EC109 and pRLTK stimulation, TPA stimulation, compared with the corresponding empty vector, transfected cells luciferase activity were significantly increased (t test, P <0.01) , Respectively, increased by 46.82 and 46.41 respectively. Compared with TPA-stimulated group, the luciferase activity of pGLB416 and pGLE416 transfected EC109 cells increased by 5.17 and 7.37 times (t <0.01) ). Bioinformatics analysis showed that there are at least 4 potential TPA response elements in 416 ~ + 84 of the 5 ’flanking region of NGAL gene. Conclusion TPA response elements exist in 416 ~ + 84 of 5 ’flank of NGAL gene in esophageal cancer cells.