CYP3A4基因启动子受AFB1调控位点的鉴定

Identification of the AFB1 response element of the human CYP3A4 promoter

目的构建不同长度人CYP3A4基因启动子荧光素酶报告质粒并予鉴定,检测L02细胞中各荧光素酶报告质粒相对荧光素酶活性,并分析AFB1处理对其影响,找出AFB1调控人CYP3A4基因可能的启动子区域。方法采用PCR技术扩增不同长度人CYP3A4基因启动子片段,将其插入荧光素酶报告质粒p GL3-basic中荧光素酶编码序列之前,构建含不同长度CYP3A4启动子的报告质粒p GL3-basic-CYP3A4-1~7,将报告质粒转染L02细胞,检测其荧光素酶活性来表示CYP3A4对应启动子片段的转录活性。检测各报告质粒在AFB1处理下与DMSO对照相比荧光素酶活性上升的比例,找出AFB1上调CYP3A4启动子转录活性的作用区域。结果报告质粒测序结果证实,上述荧光素酶报告质粒均构建成功。将报告质粒转染L02细胞,AFB1和DMSO处理后检测结果表明p GL3-basic-CYP3A4-1~6和p GL3-basic-CYP3A4-7相对荧光素酶活性受AFB1上升比率差异有统计学意义（P〈0.05）,p GL3-basic-CYP3A4-1至p GL3-basic-CYP3A4-6这6个报告质粒相对荧光素酶活性受AFB1上升比率差异无统计学意义（P〉0.05）。结论成功构建了人CYP3A4基因不同长度启动子片段的荧光素酶报告质粒,并初步确定AFB1可以通过调控CYP3A4启动子-200~0 nt的结合位点来上调CYP3A4的转录活性。

Objective To construct luciferase reporter plasmids carrying different length of human CYP3A4 promoter, and detect their activity in L02 cells in response to Aflatoxin B1 for the prediction of AFB1 response element. Methods Different length of CYP3A4 promoter sequences were amplified by PCR and cloned into luciferase reporter plasmid p GL3-basic; after sequencing these reporter plasmids were named p GL3-basic-CYP3A4-1 to 7, and transfected into L02 cells and detect the luciferase activities in response to AFB1 or DMSO. Results The CYP3A4 promoter sequences in p GL3-basic-CYP3A4-1 to 7 plasmids were verified for the by DNA sequencing. The luciferase activities up-regulated by AFB1 in plasmids p GL3-basic-CYP3A4-1 to 6 were higher than that in plasmid p GL3-basic-CYP3A4-7,but showed no significant differences among p GL3-basic-CYP3A4-1 to 6. The AFB1 response element may be CYP3A4（-200 ~0 nt）.Conclusion Different lengths of human CYP3A4 promoter reporter plasmids were successfully constructed. The possible AFB1 response element of the human CYP3A4 promoter is from-200 to 0 nt of the sequence.