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EDN1基因5′上游AP-1顺式调控元件在同型半胱氨酸诱导HUVECs EDN1基因转录中的作用

Effects of AP-1 element on endothelin-1 gene transcription in homocysteine-induced HUVECs

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【作者】 段金虹徐海珊戴顺龄王小明吴云清张彦东程锦轩孙仁宇李良陈槐卿

【Author】 DUAN Jin-hong1,XU Hai-shan1,DAI Shun-ling1,WANG Xiao-ming1,WU Yun-qing1,ZHANG Yan-dong1,CHENG Jin-xuan1,SUN Ren-yu1,LI Liang2,CHEN Huai-qing2(1 Institute of Basic Medical Sciences,Peking Union Medical College and Chinese Academy of Medical Sciences,Beijing 100005,China;2 Institute of Biomedical Engineering,West China Center of Medical Sciences,Sichuan University,Chengdu 610041,China)

【机构】 中国医学科学院基础医学研究所 中国协和医科大学基础医学院中国医学科学院基础医学研究所中国协和医科大学基础医学院四川大学华西医学中心生物医学工程研究所北京100005四川成都610041

【摘要】 目的:探讨EDN1基因5′上游AP-1顺式调控元件在同型半胱氨酸(Hcy)诱导HUVECs EDN1基因转录中的作用。方法:用荧光探针DCF检测HUVECs内活性氧的含量;用RT-PCR法检测EDN1mRNA的表达,用Western blotting法测定c-jun/AP-1蛋白的表达;同时用双抗夹心法测定HUVECs上清液中EDN1的分泌;利用重组质粒的瞬时转染技术观察了HUVECs的AP-1报告基因的活性。结果:Hcy能明显增加HUVECs ROS的生成,促进EDN1的分泌和提高EDN1mRNA的表达;瞬时转染分析实验结果显示Hcy能明显诱导质粒pGL3-EDN1-AP-1(-115/+135)荧光素酶的表达,但对质粒pGL3-EDN1-AP-1(-115/+135)的突变体pGL3-EDN1-AP-1MU(-115/+135)荧光素酶的基础表达及Hcy的诱导作用均明显降低。结论:推测Hcy可能改变HUVECs内的氧化还原状态,促进ROS的释放,激活核转录因子AP-1,通过EDN1基因中AP-1顺式作用元件调控该基因转录。

【Abstract】 AIM:The objective of this study was to observe the effects of AP-1 element on endothelin-1(EDN1)gene transcription in homocysteine(Hcy)-induced human umbilical vein endothelial cells(HUVECs).METHODS:The redox level in Hcy-induced HUVECs was determined by using the fluorescent product DCF.The influences of Hcy on expressions of EDN1 mRNA and protein of c-jun/AP-1 in HUVECs were measured by the methods of RT-PCR and Western blotting,respectively.The EDN1 secretion level of HUVECs induced by Hcy was determined by enzymatic immunoassay.The transient transfection assay was performed and luciferase activity of transcriptional factor AP-1 reporter gene induced by Hcy was detected.RESULTS:The Hcy significantly increased EDN1 secretion,EDN1 mRNA expression and oxidative stress in HUVECs.Hcy remarkably induced the expression of pGL3-EDN1-AP-1(115/+135)luciferase reporter gene plasmid.However,basic expression of mutation of pGL3-EDN1-AP-1(-115/+135)luciferase reporter gene plasmid was downregulated markedly in HUVECs induced by Hcy.CONCLUSION:Redox-active and release of ROS are changed by Hcy.Activated AP-1 element plays an important role in EDN1gene transcription in HUVECs induced by Hcy.

【基金】 国家自然科学基金资助项目(No.30170376)
  • 【文献出处】 中国病理生理杂志 ,Chinese Journal of Pathophysiology , 编辑部邮箱 ,2007年09期
  • 【分类号】R543
  • 【被引频次】7
  • 【下载频次】121
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