胃癌Pdx1启动子载体构建及其受DNA甲基化的调节

Construction of a reporter vector regulated by the Pdx1 promoter and evaluation of the effect of DNA methylation on Pdx1 promoter activity in gastric cancer cells

目的:构建胰十二指肠同源盒基因1(pancreatic duodenum homeobox 1,Pdx1)启动子调控的荧光素酶基因表达载体,筛选Pdx1启动子,探讨Pdx1启动子活性受DNA甲基化的影响.方法:针对Pdx1基因启动子区域进行PCR扩增,经限制性酶切法将扩增产物克隆至荧光素酶基因报告载体中,构建pGL3-Pdx1重组质粒.荧光素酶检测法检测胃癌细胞中Pdx1各段报告基因的启动子活性,并用DNA甲基化酶SssI处理胃癌细胞,比较各报告基因的启动子活性变化.结果:经过酶切和PCR法鉴定成功构建了9个携带Pdx1启动子的重组荧光素酶基因报告载体;荧光素酶检测法显示与pGL3-basic比较,含有F383的3个报告基因F383、F720和F1039有强启动子活性,组间比较无显著性差异;与对照组比较,SssI甲基化酶处理的F383、F720和F1039启动子活动明显降低(P<0.05).结论:成功获得由Pdx1启动子调控的荧光素酶基因表达载体,并筛选出具有强启动子活性的3个报告基因(F383、F720和F1039),且其启动子活性受DNA甲基化影响.其中F383可能是Pdx1启动子核心,这些结果为研究胃癌中Pdx1基因沉默的表观遗传学机制建立了基础.

AIM：To construct a reporter vector regulated by the Pdx1 promoter to determine the effect of DNA methylation on the promoter activity of Pdx1 gene. METHODS：PCR amplification was performed to obtain nine potential Pdx1 promoter fragments,which were then cloned into the pGL3-basic vector to obtain recombinant pGL3-Pdx1 constructs.Promoter activity of different Pdx1 fragments in gastric cancer cells was detected by luciferase assay to identify the potential promoter area.The activity of the Pdx1 promoter with or without SssI methylase treatment was also evaluated by luciferase assay.RESULTS：Luciferase assay showed that three fragments（F383,F720 and F1039） ,all of which contained the F383 sequence,had stronger promoter activity than pGL3-basic control.The promoter activity of these three fragments decreased significantly after SssI methylase treatment（all P0.05） .CONCLUSION：A luciferase reporter gene sys-tem containing the Pdx1 promoter was successfully constructed.F383 is the potential core area of the Pdx1 promoter.These results provide a basis for studying the epigenetic mechanism of Pdx1 gene silencing in gastric cancer.