The Effects Of Beclin 1 And Relative Genes On Autophagy Induced By Irradiation

Autophagy is a process which can autolysis by lysosome, is the main regulatory mechanism of decomposing long-life proteins, and is the only way to degrade organelles. It has been demonstrated that the tumor death is associated with autophagy instead of apoptosis about the tumor therapy. The effect of Beclin 1 gene on autophagy is to control the cell growth and the mechanisms of suppressing tumors. It has been reported that Beclin 1 gene is very important in autophagy, embryonic development and maintaining the homoiostasis in vivo. And it is very important for tumor because of autophagy by mutant and modulation inbalance. The effect of p53 gene on autophagy is still confussing. It was reported that p53 gene can induce autophagy, but can also suppress autophagy. The relationship between Beclin 1 gene and p53 gene and the effect of them on autophagy provide a new way for tumor therapy.In this study, we demonstrated the effect of Beclin 1 and p53 gene on human breast cancer cell (MCF7) by irradiation via analytical cytology and molecular biology. The result will be helpful to approach the cytology and biology foundation of Beclin 1 gene on cell effect induced by irradiation.1 The effect of irradiation on LC31.1 The structure of MCF7PQN-GFP-LC3The cell model was made up by calcium phosphate coprecipitation method. Immunocytochemistry was applied to detect the result. Results showed that it was successful to the structure of MCF7PQN-GFP-LC3.1.2 The effect of irradiation on LC3Immunocytochemistry was applied to detect and Laser confocal microscopy was applied to observe the changes of autophagy associated protein LC3 in MCF7PQN-GFP-LC3 at 24 h and 48 h after IR with 8 Gy and 12 Gy separated. Results showed that there were spotted autophagy associated proteins LC3 in MCF7 cells.2 Structure of the cell model and the detection of normal biological indicator2.1 Expression of Beclin 1 gene in MCF7Beclin Ri modelThe RNAi and the vector of Super-Beclin 1-RNAi were applied to interact the expression of Beclin 1 in MCF7 cells to establish the MCF7Beclin Ri model.2.2 Detection of cell multiplication timeThe MCF7 cells were cultured in six pore plate with the concentration of 103 per well.Cell counting was done after 48 h, 72 h, 96 h, 120 h and the growing curve was painted.The cell multiplication time of MCF7 is 12.5 h. The MCF7Beclin Ri and MCF7Psuper Retro cells were cultured separately in six pore plate with the concentration of 104 per well.Cell counting was done after 48 h,72 h,96 h,120 h,144 h and the growing curve was painted.The cell multiplication time of MCF7Beclin Ri and MCF7Psuper Retro cells is 20.968 h and 12.71 h.2.3 The change of survival rate after irradiationnThe MTT was applied to detect the change of survival rate. Results showed that the survival rate was lower than that of the control. The survival rate of MCF7 cell was lowest at the dose of 12 Gy; The survival rate of MCF7Beclin Ri cell was lowest at the dose of 10 Gy; The survival rate of MCF7Psuper Retro cell was lowest at the dose of 12 Gy.3 Dose and time effect changes of Beclin 1, p53 and p21 proteins after IR3.1 Dose effect changes of Beclin 1 proteins in cells 16h after IR with different doses of X-rays.Western Blot was applied to detect the changes of Beclin 1 total proteins at 16h after IR with doses of 2-8Gy. Results showed that Beclin 1 total protein in MCF7, MCF7Beclin Ri and MCF7Psuper Retro cells increased as compared with control.3.2 Time effect changes of Beclin 1 proteins in cells 16 h after IR with different doses of X-rays.Western Blot was applied to detect the changes of Beclin 1 protein at 4 h-32 h after IR with 4 Gy. Results showed that Beclin 1 protein increased at 4 h-16 h in MCF7, MCF7Beclin Ri and MCF7Psuper Retro cells compared with control. 3.3 Dose effect changes of p53 proteins in cells 16h after IR with different doses of X-rays.Western Blot was applied to detect the changes of p53 total proteins at 16 h after IR with doses of 2-8 Gy. Results showed that p53 total protein in MCF7, MCF7Beclin Ri and MCF7Psuper Retro cells increased as compared with control.3.4 Time effect changes of p53 proteins in cells 16 h after IR with different doses of X-rays.Western Blot was applied to detect the changes of p53 protein at 4 h-32 h after IR with 4 Gy. Results showed that p53 total protein increased at 4 h-16 h in MCF7 cells, at 4 h, 16 h in MCF7Beclin Ri cells and at 4 h,16 h in MCF7Psuper Retro cells as compared with control.3.5 Dose effect changes of p21 proteins in cells 16 h after IR with different doses of X-rays.Western Blot was applied to detect the changes of p21 total proteins at 16 h after IR with doses of 2-8 Gy. Results showed that p21 total protein increased in MCF7 cells with doses of 2-8 Gy , in MCF7Beclin Ri cells with doses of 4 Gy,8 Gy and in MCF7Psuper Retro cells with doses of 4-8 Gy as compared with control.3.6 Time effect changes of p21 proteins in cells 16h after IR with different doses of X-raysWestern Blot was applied to detect the changes of p21 protein at 4 h-32 h after IR with 4 Gy. Results showed that p21 total protein increased at 4 h-16 h in MCF7 cells, at 4 h and 16 h in MCF7Beclin Ri cells and at 16 h in MCF7Psuper Retro cells as compared with control.This study was focus on the effect and mechanism of Beclin 1 gene on autophagy in MCF7 cells induced by irradiation and will provide the theory evidence for the gene-irradiation combined treatment of breast cancer.