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

World Journal of Clinical Oncology 2012年第7期3卷 104-110页

作者： Harun M Said Buelent Polat Susanne Stein mathias guckenberger Carsten Hagemann Adrian Staab Astrid Katzer Jelena Anacker Michael Flentje Dirk Vordermark Department of Radiation Oncology University of Wuerzburg 97080 Würzburg Germany Department of Hematology and Oncology Johannes Gutenberg University Ⅲ Medical School 55101 Mainz Germany Department of Neurosurgery Tumorbiology Laboratory University of Wuerzburg 97080 Würzburg Germany Department of Radiation Oncology Paul Scherer Institute CH-5100 Villingen Switzerland Department of Gynaecology and Obstetrics University of Wuerzburg 97080 Würzburg Germany Department of Radiation Oncology University-Halle-Wittenberg 06110 Halle Germany

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. T

关键词： N-Myc down regulated gene 1 Short dsRNA oligonucleotides Human cancer diseases Brain cancer Radiotherapy

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