转录因子E2F4对哮喘相关基因Six1的转录调控

The regulation of the transcription factor E2F4 to human asthma-related gene Six1

目的:构建哮喘相关同源异型盒基因1(sine oculis homeobox homolog 1,Six1)启动子片段的荧光素酶报告质粒,以预测功能性转录因子结合位点,并研究E2F转录因子4(E2F transcription factor 4,E2F4)对哮喘相关基因Six1的影响及其机制。方法:将Six1基因启动子片段(-351~+100 nt)插入荧光素酶报告基因载体pGL3-basic中,获得Six1基因启动子荧光素酶报告质粒pSix1-451。通过双荧光素酶报告基因系统测定Six1基因启动子片段在HEK-293和BEAS-2B细胞中的活性,并使用生物学手段预测其潜在的转录因子结合位点。将pSix1-451与E2F4小干扰或过表达质粒共转染至HEK-293和BEAS-2B细胞后,检测其荧光素酶活性以确定E2F4对Six1基因的启动作用。通过荧光定量PCR和蛋白免疫印迹实验观察敲低和过表达E2F4后对Six1基因表达量的影响。结果:成功构建有活性的Six1启动子片段荧光素酶报告质粒,且在此片段内含有E2F4等转录因子结合位点。转录因子E2F4在启动子、mRNA及蛋白水平对Six1存在正向调控。结论:转录因子E2F4对哮喘相关基因Six1存在正向转录调控。

Objective:To construct a luciferase reporter plasmid for human asthma-related sine oculis homeobox homolog 1(Six1)promoter fragment,predict the potential transcription factor binding sites,and to study the effect of E2F transcription factor 4(E2F4)on Six1. Methods:Six1 promoter fragment(-351~+100 nt)was synthesized and inserted into the luciferase reporter gene vector pGL3-basic,the activity of recombinant plasmid p Six1-451 in HEK-293 and BEAS-2B cells was measured by dual luciferase reporter gene assay,and the potential transcriptional binding sites were predicted by bioinformatics methods. Then pSix1-451 and siE2F4 or E2F4 overexpression plasmids were co-transfected into HEK-293 and BEAS-2B cells,the luciferase activity was measured to determine the role of E2F4 in Six1 gene transcription. The effects of knockdown and overexpression of E2F4 on Six1 gene expression were measured by qRT-PCR and Western blotting experiments. Results:The luciferase reporter plasmid of human Six1 promoter fragment was successfully constructed,and its activity was verified. The fragment contained transcription factor binding sites,such as E2F4. The regulatory effect of E2F4 on Six1 was verified. Conclusion:The transcription factor E2F4 positively promotes the expression of human asthma-related gene Six1.