Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells  

作　　者：Harun M Said Buelent Polat Susanne Stein Mathias Guckenberger Carsten Hagemann Adrian Staab Astrid Katzer Jelena Anacker Michael Flentje Dirk Vordermark 

机构地区：[1]Department of Radiation Oncology, University of Wuerzburg, 97080 Würzburg, Germany [2]Department of Hematology and Oncology, Johannes Gutenberg University, Ⅲ Medical School, 55101 Mainz, Germany [3]Department of Neurosurgery, Tumorbiology Laboratory, University of Wuerzburg, 97080 Würzburg, Germany [4]Department of Radiation Oncology, Paul Scherer Institute, CH-5100 Villingen, Switzerland [5]Department of Gynaecology and Obstetrics,University of Wuerzburg, 97080 Würzburg, Germany [6]Department of Radiation Oncology, University-Halle-Wittenberg, 06110 Halle, Germany

出　　处：《World Journal of Clinical Oncology》2012年第7期104-110,共7页世界临床肿瘤学杂志（英文版）

基　　金：Supported by Deutsche Forschungsgemeinschaft DFG,VO 871/2-3,to Vordermark D;the IZKF Würzburg,B25,to Hagemann C

摘　　要：AIM: To study short ds RNA oligonucleotides(si RNA)as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1(NDRG1) gene induced under different physiological conditions(Normoxia and hypoxia) modulating NDRG1 transcription, m RNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The p SUPERNDRG1 vectors were designed, two sequences were selected from the human NDRG1 c DNA(5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor(HIF)-1α m RNA sequences in Gen Bank. NDRG1 m RNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia(P < 0.05 was considered significant).RESULTS: si RNA- and iodoacetate(IAA)-mediated downregulation of NDRG1 m RNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it canrepresent a potential target for tumor treatment in human glioblastoma. The si RNA method can represent an elegant altAIM: To study short ds RNA oligonucleotides(si RNA)as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1(NDRG1) gene induced under different physiological conditions(Normoxia and hypoxia) modulating NDRG1 transcription, m RNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The p SUPERNDRG1 vectors were designed, two sequences were selected from the human NDRG1 c DNA(5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor(HIF)-1α m RNA sequences in Gen Bank. NDRG1 m RNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia(P < 0.05 was considered significant).RESULTS: si RNA- and iodoacetate(IAA)-mediated downregulation of NDRG1 m RNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it canrepresent a potential target for tumor treatment in human glioblastoma. The si RNA method can represent an elegant alt

关 键 词：N-MYC DOWN regulated gene 1 Short DSRNA OLIGONUCLEOTIDES HUMAN CANCER diseases Brain CANCER Radiotherapy 

分 类 号：R739.41[医药卫生—肿瘤]