摘要
目的:本研究旨在分析AML1-ETO阳性白血病中SIRT1基因的表达及调控机理,寻找SIRT1核心启动子区域。方法:利用实时定量PCR方法在AML1-ETO阳性白血病细胞株和白血病临床样本中研究SIRT1基因的表达情况;构建SIRT1基因启动子-荧光素酶报告载体,分析其在293T细胞中的活性。采用PCR方法从含有SIRT1基因转录起始位点5'区的基因片段中扩增SIRT1基因核心启动子序列,插入经XhoⅠ和HindⅢ双酶切的荧光素酶报告载体p GL3-Basic,采用阳离子脂质体Super Fect包裹荧光素酶报告重组子转染293T细胞,应用双荧光素酶检测试剂盒测定荧光素酶活性。采用双荧光素酶报告基因检测方法,研究AML1-ETO对SIRT1启动子转录活性的调节作用。结果:AML1-ETO的表达与SIRT1的表达呈正相关;成功构建了6种SIRT1基因序列的荧光素酶报告重组子,并通过了酶切以及基因测序方法的鉴定;荧光素酶报告重组子在293T细胞中的荧光素酶活性明显高于阴性对照,明确了SIRT1基因核心启动子位置;研究结果证明融合蛋白AML1-ETO可以正向调控SIRT1启动子活性,SIRT1对AML1-ETO阳性白血病发生发展的影响。研究表明:SIRT1可能是AML1-ETO的靶基因之一。结论:成功构建了SIRT1基因启动子-荧光素酶报告载体,找到了SIRT1基因核心启动子区,且核心启动子在293T细胞中有较高的活性。AML1-ETO通过促进SIRT1启动子的活性对其进行转录调控。
Objective ： This study was aimed to analyze the expression and regulation mechanism of SIRT1 in AML1 - ETO positive leukemia to find the core promoter. Methods ：The real-time RT-PCR was used to detect the expression of SIRT1 in AML1-ETO positive leukemia cell line and clinical samples of leukemia patients, a S1RT1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the 293T cell line. A series of possible core promoter fragments of the SIRT1 5 ＇-untranslated region were amplified by PCR, the PCR products were cloned into Xho I/Hind^I-digested pGL3-Basic reporter vector, the poly-cationic compound SuperFect reporter vector complexes were transfected into 293T cells. The dual-luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity. Results： The six kinds of promoter fragment of SIRT1 gene were successfully constructed and cloned into the pGL3-Basic reporter vector, which was authenticated by Xho I/Hind 111 co-digestion and DNA sequencing. The luciferase activity of the promoter construct was significantly higher than that of the pGL3-Basic promoter in 293T cells. The luciferase report gene assay was also used to detect the regulation of AML1-ETO on the transcription activity of SIRT1 promoter. The results showed that the expression level of SIRT1 increased with the increase mens of AML1-ETO, the promoter of SIRT1 could be bound by AML1-ETO. Conclusion：The SIRT1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transferene in 293T cells. The SIRT1 core promoter possesses higher activity in 293T cells and can promote significantly expression of luciferase reporter gene in 293T cells. The transcription regulation of AML1-ETO on SIRT1 is carded out via promoting its promoter activity.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2015年第1期12-18,共7页
Journal of Experimental Hematology
基金
国家自然科学基金资助项目(30800482
81270610
81170518
30971297)
北京市科技新星计划项目(2011114)
北京市自然科学基金(7132217)
军队高新技术项目(2010gxjs091)
作者简介
窦立萍，副主任医师，副教授．E-mail：lipingruirui@163．com
共同通讯作者：于力，主任医师，教授，博士生导师．E-mail：chunhuiliyu@yahoo．com；
