The Expression Of DEK Proto-oncogene In Astrocyte Tumors And Its Effect On Proliferation As Well As Apoptosis Of The U251 Cells

Objective:At present,new methods for treating astrocyte tumors are becoming more and more crucial.Following with the development of molecular biology,molecular genetics,molecular immunology and transgene technology,many genes have been identified to be firmly connected with the genesis,development and prognosis of astrocyte tumors.Therefore,treatment of astrocyte tumors with gene therapy may be a promising method in clinic.DEK proto-oncogene was originally discovered in subtype of acute myeloid leukeakia.In addition,researches demonstrate that DEK proto-oncogene is highly expressed in liver cancer,throat squamosum carcinoma,cancer of the cervix,nasopharyngeal carcinoma and malignant melanoma,and is relevant to the genesis,development of tumors as well as the drug resistance of malignant melanoma.However,the expression level of DEK gene in astrocyte tumors tissue and the correlation between DEK gene and the genesis,development of astrocyte tumors.is still unclear.This study was performed to detect the expression level of DEK gene in astrocyte tumors,to determine the relationship between its expression level and the grade of astrocyte tumors,and to investigate the role of DEK in the genesis as well as the development of astrocyte tumors.Methods1.RT-PCR assay was used to measure the mRNA levels of DEK gene in normal brain samples and astrocyte tumors samples of different grade.2.Western blot assay was performed to measure the protein levels of DEK gene in normal brain tissue and astrocyte tumors tissue of different grade.3.Immunohistochemistry was used to detect the distribution and expression of DEK in normal brain tissue and astrocyte tumors tissue of different grade.4.RT-PCR assay was used to detect the mRNA expression of DEK gene in U251 cells after gene silencing by Small interefering RNA(siRNA).5.Western blot assay was performed to measure the protein levels of DEK gene in U251 cells after gene silencing by siRNA.6.Detecting the effect of DEK gene silencing by siRNA on the growth of U251 cells.7.Detecting the effects of DEK gene silencing by siRNA on the apoptopsis and generation cycle of U251 cells.8.Western blot assay was used to investigate the protein expression of p53,p21,Bcl-2 and c-myc in U251 cells after DEK gene silencing by siRNA.9.Detecting the effect of DEK gene silencing by siRNA on the activity of Caspase-3 in U251 cells.Results1.In normal brain tissue,both mRNA and protein expression levels of DEK were extremely low.However,DEK was commonly expressed in astrocyte tumors tissue of different grade.In addition,there were significant differences in the mRNA and protein expression levels of DEK between low grade and high grade astrocyte tumors.2.DEK was localized in the cell nucleus.Compared with that of normal brain tissue,the immunostaining of DEK in astrocyte tumors tissue of different grade was significantly increased.Additionally,there were significant differences in the immunostaining levels of DEK between low grade and high grade astrocyte tumors.3.The mRNA and protein expression levels of DEK in U251 cells after DEK gene silencing by siRNA were significantly decreased when compared with that of control or non-transfected group.By contrast,there was no significant difference between control and non-transfected groups.4.The growth rate of U251 cells began to significantly decrease at 24 h after DEK gene silencing by siRNA and reached the lowest point at 72 h.There was no significant difference between control and non-transfected groups.5.Compared with that of control or non-transfected group,the apoptosis rate in U251 cells after DEK gene silencing by siRNA was significantly increased.On the contrary,the proliferation index in U251 cells after DEK gene silencing by siRNA was significantly decreased.6.Compared with that of control or non-transfected group,the protein expression of p53 and p21 inU251 cells after DEK gene silencing by siRNA were significantly increased,meanwhile,the protein expression of Bcl-2 and c-myc were significantly decreased.There was no significant difference between control and non-transfected groups.7.The activity of Caspase-3 in U251 cells after DEK gene silencing by siRNA was significantly elevated as compared to control or non-transfected group.Conclusions1.mRNA and protein levels of DEK in astrocyte tumors tissue were both significantly upregulated along with the increased grade of astrocyte tumors.2.Targeting DEK siRNA can effectively exert its gene silencing potency in U251 cells.3.In U251 cells after DEK gene silencing by siRNA,the growth rate and was decreased,the proliferation was inhibited,and the apoptosis rate was increased,which suggest that DEK gene is relevant to the growth and proliferation of U251 cells in vitro.4.In U251 cells after DEK gene silencing by siRNA,the protein expression of p53 and p21 as well as the activity of caspase-3 was increased,the protein expression of Bcl-2 was decreased,suggesting that the p53-dependent apoptosis approach may play an important role in DEK proto-oncogene-induced formation and development of astrocyte tumors.5.The transcriptional activation of p53 and p21 induced by downregulation of DEK gene may negatively regulate cell cycle which can inhibit the generation cycle of U251 cells.6.In U251 cells after DEK gene silencing by siRNA,the protein expression of c-myc was significantly decreased,suggesting that DEK gene may participate in cell transformation and tumorigenesis through activating c-myc oncogene.