血管内皮生长因子对胃癌BGC-823细胞多药耐药相关蛋白1启动子活性的影响

Effect of vascular endothelial growth factor on the activity of multidrug resistance-associated protein 1 promoter in gastric cancer cell line BGC-823

目的:检测血管内皮生长因子(VEGF)对多药耐药相关蛋白1(MRP1)基因启动子转录活性的影响并初步探讨其作用机制。方法:合成MRP1启动子片段,克隆到荧光素酶报告基因载体PGL3-Basic中;用脂质体2000将重组的报告基因载体与内参质粒β-半乳糖苷酶(β-gal)瞬时共同转染人胃癌BGC-823细胞,检测VEGF作用后MRP1启动子活性的改变;继之,采用PI3K/AKT信号通路抑制剂LY294002预处理转染的细胞,再检测VEGF对MRP1启动子转录活性的影响。结果:酶切鉴定和DNA序列分析表明成功地构建了重组PGL3-Basic-MRP1载体;荧光素酶活性分析表明重组PGL3-Basic-MRP1在BGC-823细胞中具有启动子活性(144±3.0),与空载体PGL3-Basic(60±4.910)相比,转录活性增高2.4倍,差异有统计学意义(P<0.01);VEGF能以剂量依赖的方式上调MRP1启动子区的转录活性,与无VEGF作用组(171±6.083)相比,最大活性(924±19.975)增高5.4倍(P<0.05);LY294002能够显著抑制VEGF对MRP1启动子区的转录激活,当LY294002浓度为50μmol/ml时,MRP1启动子活性(392±25.541)与无LY294002作用组(1001±11.533)相比下降2.5倍(P<0.05)。结论:VEGF对MRP1启动子活性具有上调作用,PI3K/AKT信号通路在这一过程中发挥了重要作用。

Objective：To assess the efficacy and mechanism of vascular endothelial growth factor（VEGF） on the transcriptional activity of the the multidrug resistance-associated protein 1（MRP1） promoter.Methods：The promoter of MRP1 was synthesized and cloned into the luciferase reporter gene vector PGL3-Basic.The recombinant plasmid was transiently co-transfected into BGC-823 cells with control vector β-gal by lipofectamine 2000 reagent,and then detected the alteration of the MRP1 promoter activity after being treated with VEGF.Subsequently,pretreated co-transfected cells with PI3K/AKT signaling pathway inhibitor LY294002,and then evaluated the effect of VEGF on the MRP1 promoter activity.Results：The consequence of restriction enzyme digestion and nucleotide sequence analysis confirmed that the recombinant plasmid was successfully constructed.The analysis of the luciferase reporter gene activity demonstrated that the recombinant plasmid displayed marked activity（144±3.0） in BGC-823 cells,about 2.4-fold than that seen with control vector PGL3-Basic（60±4.910,P0.01）.Importantly,this activity was up-regulated by VEGF in a dose-dependent manner,and the most pronounced activity（924±19.975） was at least 5.4-fold above that when cells cultured without VEGF（171±6.083,P0.05）;Furthermore,LY294002 significantly supressed the effect of VEGF on the MRP1 promoter activity.Compared with cells cultured in the Absence of LY294002（1 001±11.533）,the activity of the MRP1 promoter was reduced 2.5-fold when cells were exposured to 50 μmol/ml LY294002 （392±25.541） （P0.05）.Conclusion：VEGF could augment the activity of the MRP1 promoter,and PI3K/AKT signaling pathway was proposed to be a critical factor underlying this mechanism of action.