抑瘤基因NGX6启动子的克隆与功能鉴定

Cloning and Identification of Promoter of Suppressed-tumor Gene NGX6

NGX6是一个结直肠癌候选抑瘤基因,其转录调控机制不明.采用生物信息学技术预测其启动子区,并构建NGX6启动子荧光素酶报告基因重组体pGL3/Enhancer/1126.荧光素酶活性检测结果表明该区域具有强启动子活性.应用PromoterInspector program,FistEF,CpGplot和MatInspector Professional软件分析发现,NGX6基因转录调控区为一个不含TATA盒,而含有CAAT盒的GC富集区.凝胶迁移阻滞实验确定NGX6基因启动子区域具有Sp1特异性结合位点,Sp1特异性阻断剂光神霉素(mithramycin A)能明显抑制NGX6启动子的活性和NGX6基因的表达;封闭内源性Sp1能下调NGX6基因mRNA表达水平.

Transcriptional regulation mechanisms have not been clearly illuminated for NGX6 gene, which is a candidate of tumor suppressor gene in colorectal cancer, pGL3/Enhancer/1126 vector, a recombinant reporter gene vectors of the transcription regulatory region of NGX6 gene, was constructed based on bioinformatic techniques and identified by luciferase assay system. No canonical TATA boxes, but several CAAT and GC boxes were observed in the transcription regulatory region by the online analysis programs Promoterlnspector program, FistEF, CpGplot and Matlnspector Professional. Transcriptional factor Sp 1 was validated to bind to NGX6 promoter by electrophoretic mobility shift assay （EMSA）. Inhibition of the Sp 1 binding to NGX6 promoter by mithramycin A significantly reduced the promoter activity. The endogenous expression of NGX6 in mRNA level was down-regulated by mithramycin A and blocking with Spl.