Study On The Mechanism Of Transcriptional Regulation Of IEX-1 Gene Induced By As₂O₃ In Human Osteosarcoma MG-63

Objective As₂O₃ is a chinese traditional medicine.In our previous study,we have found that 1.0μmol/L As₂O₃ could induce MG-63 cell apoptosis in vitro.And then we identified that IEX-1 gene expression was down regulated after the addition of 1.0μmol/L As₂O₃ using cDNA Array, RT-PCR,Northern Blot.IEX-1,a recently discovered early response gene, regulates cell growth and apoptosis.We want to found the exact transcriptional mechanism of IEX-1 in the present study.Methods Promoters of IEX-1 gene were obtained by PCR from gDNA extracted from peripheral blood and inserted in to luciferase reporter plasmids pGL3-Basic;Double stranded oligonucleotide containing p53 binding site were end-labeled with[γ-³²p]ATP using T4 polynucleotide kinase and purified with the Microspin G-25 columns. The expression level of p53 and IEX-1 protein were detected after incubation of 1.0μmol/L As₂O₃ using western blotting.Results Transcriptional factor p53 was up-regulated after the addition of 1.0μmol/L As₂O₃,and the increased p53 protein bind the promoter region of IEX-1 gene,which repress the transcription and result in down-regulation of IEX-1 gene expression.Conclusion We further confirm IEX-1 gene is associated with osteosarcoma and find the exact transcriptional mechanism of IEX-1 after the addition of As₂O₃.