title: [Construction of SIRT1 promoter expression vector and its activity analysis as well as influence of AML1-ETO on transcriptional regulation of SIRT1 gene].
abstract: 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 SIRT1 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 XhoI/HindIII-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 XhoI/HindIII 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.
  
  
  CONCLUSIONS
  The SIRT1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene 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 carried out via promoting its promoter activity.
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Qian
authors: Q
authors: Wang
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China. E-mail: lipingruirui@163.com.
authors: Li-Ping
authors: LP
authors: Dou
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Yong-Hui
authors: YH
authors: Li
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Li-Li
authors: LL
authors: Wang
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Lei
authors: L
authors: Zhou
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Dan-Dan
authors: DD
authors: Li
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors: Li-Jun
authors: LJ
authors: Wang
authors: Department of hematology, Chinese PLA General Hospital, Beijing 100853, China. E-mail: chunhuiliyu@yahoo.com.
authors: Li
authors: L
authors: Yu
mesh_terms: D002460:Cell Line; D050676:Core Binding Factor Alpha 2 Subunit; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007938:Leukemia; D008156:Luciferases; D015514:Oncogene Proteins, Fusion; D011401:Promoter Regions, Genetic; D000075142:RUNX1 Translocation Partner 1 Protein; D056564:Sirtuin 1; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D015514:Oncogene Proteins, Fusion; D000075142:RUNX1 Translocation Partner 1 Protein; D008156:Luciferases; C447939:SIRT1 protein, human; D056564:Sirtuin 1