The Roles Of LOX And P53 In Radioresistance Of Tumor Cells

The application of radiotherapy plays an irreplaceable role in cancer treatment.DNA damage repair is considered to be an important factor affecting radiosensitivity.It is believed that LOX is highly expressed in some malignant tumors and is involved in regulating the occurrence,deterioration,adhesion and invasion of tumors.LOX with catalytic activity not only locates outside the cell,but also returns to the nucleus to participate in signal transduction and gene transcription regulation.P53 is located at the center of stress signals,such as ionizing radiation,proto-oncogene activation,hypoxia,and so on.As a transcription factor,p53 promotes the transcription of downstream target genes to achieve its function.Therefore,exploring the roles and molecular mechanisms of LOX and p53 in radiation resistance of cancer cells can provide new therapeutic ideas for radiotherapy.Purposes:1.To elucidate the mechanism and signal pathway of LOX and p53 regulating radiation sensitivity of cancer cells.2.To elucidate the relationship between LOX and p53 in a series of biological function changes induced by ionizing radiation and their regulatory role.Methods:1.Real-time fluorescence quantitative PCR was used to detect LOX gene expression in two colon cancer cell lines,lung cancer cell lines,osteosarcoma cell lines and liver cancer cell lines at 0 h,3 h,6 h,9 h,12 h and 24 h after irradiation with 4 Gy X-ray.2.Construction and validation of recombinant pEGFP-LOX full and pEGFP-LOX mature.3.The transfection efficiency of LOX siRNA plasmid was verified by real-time fluorescence quantitative PCR and Western Blot.4.The transfection efficiency of LOX in H460 and HCT116 p53 wt cells was verified by real-time fluorescence quantitative PCR.5.After transfection of p53 siRNA,the expression of LOX gene and protein in radiation-activated HCCT116 p53 wt cell line was detected by RT-PCR and Western Blot.Transfection of p53 plasmid into H1299 cell line and detection of LOX protein expression level after irradiation.6.The effect of silencing LOX on the colony forming ability of irradiated HCCT116 p53 wt and HCT116 p53-/-cell lines was detected by clonogenic assay.7.Flow cytometry was used to analyze the early apoptotic rate of HCT116 p53 wt and HCT116 p53-/-cell lines induced by ionizing radiation after LOX silencing.8.After LOX silencing,the DNA double strand break repair ability of HCT116 p53 wt cells induced by ionizing radiation was detected by gamma H2 AX immunofluorescence staining.9.The effect of overexpression of LOX on the survival rate of irradiated H460 and HCT116 p53 wt cell lines was investigated by counting living cells.10.Flow cytometry was used to detect apoptotic levels of H 460 and HCT 116 p53 wt cells induced by ionizing radiation after overexpression of LOX.11.Flow cytometry was used to detect G2/M arrest in H 460 and HCT 116 p53 wt cells induced by ionizing radiation after overexpression of LOX.12.The effects of ionizing radiation on the expression of Rad51,Ku70 and Bax in LOX full-HCT116 p53 wt cell line.Results:Ⅰ.Changes of LOX transcription levels in different p53 phenotype tumor cells before and after radiationThe expression of LOX in p53 wild-type cells(HCT116 p53 wt,H460,U2 OS,HepG2)was significantly increased at 3 h,6 h,9 h,12 h,and 24 h after 4 Gy X-ray treatment(p < 0.001),but There was no significant change in p53-deficient cells(HCT116 p53-/-,H1299,SAOS2,Hep3B).Ⅱ.P53 regulates LOX transcription and protein levels induced by radiationP53 siRNA can inhibit LOX transcription and translation of HCT116 p53 wt cells after irradiation,while transfection of p53 plasmid can up-regulate LOX protein expression in H1299 cells after irradiation.Ⅲ.Silencing LOX Enhances Radiosensitivity of p53 Wild-type Cell LinesTransfection of LOX siRNA silenced LOX mRNA and protein expression in HCT116 p53 wt cells.Silencing LOX significantly reduced the number of clones of HCT116 p53 wt cells(p < 0.05),but there was no significant change in HCT116 p53-/-cells.After treatment with 4Gy,LOX siRNA increased the early apoptotic rate of HCT116 p53 wt cells(p <0.05),but had no significant effect on HCT116 p53-/-cells.Prior to irradiation,regardless of whether or not LOX siRNA was transfected,there was no significant effect on the early apoptotic rate of HCT116 p53 wt cells.X-ray treatment of 4Gy of HCT116 p53 wt cells was performed.The percentage of γ-H2 AX positive cells was up-regulated 6 h after irradiation.The percentage of γ-H2AX-positive cells in the cells silenced by LOX was up-regulated 24 h after irradiation(p <0.05).Ⅳ.LOX reduces radiation sensitivity of p53 wild-type cellsThe recombinant plasmid pEGFP-LOX was constructed using HCT116 cDNA as a template.LOX mRNA levels were significantly increased in H460 and HCT116 p53 wt cells transfected with LOX full and LOX mature plasmids(p <0.05);after treatment with 8 Gy,transfected LOX full and LOX mature plasmids significantly upregulated LOX mRNA in cells(p <0.05).Before irradiation,LOX full and LOX mature had no significant effect on the proliferation of H460 and HCT116 p53 wt cells.After 8 Gy of X-ray treatment,LOX full and LOX mature significantly inhibited cell proliferation(p < 0.05).Before irradiation,LOX full and LOX mature had no significant effect on the G2/M phase of H460 and HCT116 p53 wt cells.Ionizing radiation increased the G2/M phase of the cells,while transfection of LOX full and LOX mature significantly inhibited G2/M blockade of H460 and HCT116 p53 wt cells(p <0.05).Before irradiation,LOX full and LOX mature had no significant effect on the apoptosis rate of H460 and HCT116 p53 wt cells.Ionizing radiation increased the apoptosis of H460 and HCT116 p53 wt,while transfection of LOX full and LOX mature significantly inhibited the apoptosis rate of H460 and HCT116 p53wt(p <0.05).Before irradiation,regardless of whether or not the LOX full plasmid was transfected,the expression of Cdc2 and CyclinB1 protein in HCT116 p53 wt cells did not change significantly.After 8 Gy X-ray treatment,overexpression of LOX significantly up-regulated the Cdc2 protein expression of HCT116 p53 wt cells and down-regulated the expression of CyclinB1 protein(p <0.001).Transfection of LOX full plasmid significantly up-regulated the expression of anti-apoptotic protein Bcl-xl in HCT116 p53 wt cells(p <0.001),and down-regulated the expression of Bax protein.Prior to irradiation,no change in translational levels of Rad51 and Ku70 in HCT116 p53 wt cell lines was induced regardless of whether or not LOX full was transfected.After administration of 8 Gy dose,the expression of Rad51 and Ku70 protein in HCT116 p53 wt cells transfected with LOX full was up-regulated(p <0.05).Conclusions:1.The expression of LOX in p53 wild-type cancer cells was induced by ionizing radiation,while the p53-deficient cells were not affected.2.Silencing LOX significantly inhibited the clonality of HCT116 p53 wt cells after irradiation,thereby enhancing radiosensitivity,but had no significant effect on HCT116 p53-/-cell radiation sensitivity.3.Silencing LOX can promote early apoptosis of HCT116 p53 wt cells after irradiation,but does not affect the early apoptosis of HCT116 p53-/-cells,and inhibit DNA damage repair of HCT116 p53 wt cells.4.LOX overexpression significantly promotes the growth of HCT116 and H460 cells after irradiation.5.LOX overexpression can up-regulate Bcl-xl,down-regulate the expression of Bax,inhibit the apoptosis of HCT116 p53 wt and H460 cells induced by ionizing radiation,up-regulate Cdc2,down-regulate the expression of CyclinB1,and thus inhibit the G2/M phase block of HCT116 p53 wt and H460 cells induced by ionizing radiation.6.LOX overexpression can up-regulate the expression of Rad51 and Ku70 in HCT116 p53 wt cells after irradiation,which may promote damage repair through this pathway.