摘要
目的:通过构建LKB1基因获得性肺癌细胞模型,了解转录因子Sp1对LKB1-血管内皮生长因子(VEGF)通路的调控作用。方法:应用巢式PCR(nPCR)方法扩增LKB1基因编码区,构建LKB1真核表达载体;W estern印迹检测LKB1在A549细胞中的表达;siRNA技术干扰Sp1的表达;RT-PCR联合ELISA方法检测VEGF的变化;SEAP检测系统检测Sp1的转录活性。结果:成功构建LKB1基因稳定表达细胞模型;LKB1蛋白下调VEGF表达和Sp1转录活性;siRNA介导的Sp1沉默伴随VEGF表达下调。结论:转录因子Sp1参与LKB1基因对VEGF的表达调控。
Objective：To construct the stable cell model of ectopic LKB1 expression and to study the regulation role of Sp1 in LKB1-VEGF signal pathway in lung cancer cells. Methods： DNA fragment encoding LKB1 protein was amplified by nested-PCR（nPCR） from human fetal brain cDNA library and sub-clonod into the eukaryotic expression vector pcDNA3.1. Western blot was used to study the expression status of LKB1 protein in AS49 cells. Transcription factor Sp1 was silenced by small interference RNA（siRNA）. RT-PCR and ELISA were applied to examine the expression status of VEGF. SEAP assay was used to estimate the transcription activity of Sp1. Resstlts：Stable cell line of A.549 which expresses ectopic LKB1 was constructed. LKB1 remarkably suppressed VEGF expression and the transcription activity of Sp1. SiRNA targeting Sp1 effectively decreased the expression of Sp1, and the silencing of Sp1 resulted in remarkable down-regnlation of VEGF gene. Conclusion： Transcription factor Sp1 plays an important role in expression of VEGF regulated by LKB1.
出处
《军事医学科学院院刊》
CSCD
北大核心
2007年第1期11-15,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金资助项目(30270590)
