Study On The Role Of LyGDI In Apoptosis Sensitivity Of Cancer Cell Induced By Radiation And Regulation Of P53

Objective:To construct eukaryotic expressing vector of lyGDI andΔN19lyGDI with fluorescence tag and To establish stable cell lines transfected with lyGDI andΔN19lyGDI genes,in order to investigate effects and mechanisms of the overexpression of LyGDI and D19LyGDI on the radiation-sensitivity of cells;To study effects of the recovery of P53 on LyGDI in cells.Methods:1.Design the forward and reverse primers of lyGDI andΔN19lyGDI.Gene fragments of lyGDI andΔN19lyGDI were amplified by PCR,then eukaryotic expression vectors of pEGFP-DF-lyGDI and pEGFP-DF-ΔN19lyGDI were constructed respectively. After verification by enzymecutting,PCR and sequencing;The expression of fusion proteins were detected by immunoblotting and the localization of fusion proteins in cells were observed by immunohistochemistry after the genes were transfected into MCF-7 cells mediated by lipofection.2.Vector of pcDNA3.1-p53 was transfected into L929 cell lines mediated by lipofection.Then,stable cell lines were verified by RT-PCR and immunoblotting was finished.The change of cell proliferation ability was detected by growth curve.After cells were irradiated by X-ray,colony formation testing was performed to detect the change of radiation-sensitivity and flow cytometry was used to check the change of the apoptosis rate of irradiated cells,immunoblotting was done to detect the expression of related proteins.3.By using the L929-lyGDI/ΔN19lyGDI and A549-lyGDI cell lines,which were successfully constructed by stable transfection,the effect of exogenous lyGDI on the rate of apoptosis induced by radiation in two cell lines were detected.In order to study the effect of LyGDI on the radiation-sensitivity of the cells,the related proteins expression were detected by immunoblotting. Results:The genes order were completely correct,which were proved by sequencing. The interest proteins expression was detected by immunoblotting and immunohistochemistry. The recovery of P53 protein in L929 cells inhibitted the ability of cell proliferation, promoted the cell apoptosis induced by radiation,and induced the truncation of LyGDI;the rates of apoptosis induced by radiation were increased in both L929-lyGDI and L929-ΔN19lyGDI cells,and the fusion proteins were disrupted into corresponding LyGDI andΔN19LyGDI proteins after cells were irradiated;the rates of apoptosis induced by radiation were increased in A549-lyGDI cells,the radiation-sensitivity was up-regulated,the truncation of LyGDI protein intoΔN19LyGDI protein was observed in 4 to 24 hours after cells irradiated by 8Gyγ-ray.Conclusion:1.Eukaryotic expression vectors of pEGFP-DF-lyGDI and pEGFP-DF-ΔN19lyGDI with fluorescence tag were constructed successfully;2.The recovery of P53 expression in L929 cells inhibitted the ability of cell proliferation,enhanced the radiation-sensitivity and induced the cleavage of LyGDI;3.The overexpression of LyGDI promoted the radiation-sensitivity in L929 and A549 cell lines.