Changes And Mechanisms Of Genes Expression In Irradiation-induced Autophagy In Human Breast Cancer Cells

Autophagic cell death (Autophagic cell death, hereinafter referred to as autophagy), also known as typeâ…¡programmed cell death, is an alternative to apoptosis programmed cell death. Autophagy is a cellular stress or injury in the state, will be the formation of degradation products wrapped autophagosome (autophagosomes) by lysosomal degradation, to maintain the cell's own metabolic needs and organelles update. Has been reported in human breast cancer cell line MCF-7 in the ionizing radiation can cause the emergence of bubbles of autophagy, suggesting that radiation can induce autophagy. Ionizing radiation biological effects on cells, mainly through specific cell factor to regulate gene expression according to the corresponding order to regulate gene expression after irradiation of cells from the corresponding internal and external changes in signal transduction.DRAM and TIGAR genes are two important autophagy-related genes, but the radiation-induced breast cancer autophagic cell death and TIGAR whether genes involved in DRAM, has not been reported. In this study, breast cancer cells to establish the model of radiation-induced autophagy in order to investigate the autophagy genes in the DRAM and TIGAR changes and other autophagy-related genes in the mechanism, the search for new cancer treatment programs provide a theoretical basis.The present study, cell biology and molecular biology methods, the main research of the X-ray irradiation on human breast cancer cell line MCF-7 autophagy-related genes in the impact and role in establishing silencing and DRAM over-expression model to investigate autophagy changes in the pathway and the possible mechanism of action, the search for new cancer treatment programs provide a theoretical basis.1 An ionizing radiation-induced MCF-7 autophagyIonizing radiation for the detection of cells in the autophagy marker LC3 after expression, vaccination MCF7-PQN-GFP-LC3 cells in the petri dish containing a glass coverslip, respectively, to give OGy,4 and 8Gy dose irradiation, with a fluorescence microscope found in irradiated cells after expression of green fluorescent protein; detected by western-bloting LC3 LC3 protein expression after irradiation, issued on the rise. In order to detect ionizing radiation can induce cancer cell MCF7 death, we used colony formation assay validation and found that MCF7 cells were irradiated 4Gy and 8Gy after irradiation, the formation of colonies were 18 and 4, significantly less than the control group (45); the same time with MDC staining induced by ionizing radiation in MCF7 cells the number of autophagic vacuoles, found by 4Gy irradiation, the percentage of autophagic vacuoles increased significantly in the autophagy inhibitor before irradiation with 3-MA treatment of cells, the percentage of autophagic vesicles significantly decreased compared with irradiation, before irradiation with the inhibitor Z-VAD on apoptosis of cells after treatment, the percentage of autophagic vacuoles, compared with sham irradiated group was significantly increased, indicating that the inhibition of the MCF7 apoptosis pathway can promote the occurrence of autophagy.2 Ionizing radiation can induce human breast cancer MCF7 cells and autophagy-related protein pathwayIonizing radiation can induce autophagy may be radiation-induced cell damage. To investigate the ionizing radiation-induced autophagy mechanism, we MCF7 cells and autophagy-related protein pathway in-depth study and discussion. In tumor cells, the traditional cell signaling pathways are:(1) PI3K-â… /AKT/mTOR pathway, (2) PI3K-â…¢/Beclinl pathway, (3) P53 pathway. We were on the selected genes of the pathway for the study, we detected by Western bloting method by OGy,4Gy and 8Gy after irradiation the level of their expression of macrophage-related changes. MCF7 cells were irradiated 4Gy and 8gGy, PI3K-â… /AKT/mTOR AKT pathway on the expression level decreased significantly, which in 4Gy 16h after exposure to a minimum value (compared with sham control group decreased by about 40%.) PI3K-â…¢/Beclinl Beclinl pathway protein levels on irradiated in 4Gy significant upward trend, the highest in the 32h value (compared with sham irradiated group increased by about 40%); BCL2 protein levels tended to increase, which in 4Gy 16h after irradiation, reached the highest value (compared with sham irradiated group increased 40%), the 8Gy irradiation, BCL2 protein levels tended to increase in 2-32h duration of each group were higher than those in sham control group, in which the highest in the 4h (compared with sham irradiated group increased approximately 50%). Although autophagy induced by ionizing radiation the specific mechanism is unclear, but these studies have shown that ionizing radiation may be through PI3K-â… /AKT/mTOR, PI3K-â…¢/Beclinl, P53 interaction of signaling pathways such as MCF7 cells induced autophagy. 3.MCF-7 cells after irradiation of autophagy genes in autophagy inhibitor 3-MA and the apoptosis inhibitor Z-VAD treatment changesThe use of Realtime RT-PCR method to detect autophagy gene mRNA, dose-effect study shows that, MCF7 cells after irradiation by 4Gy, autophagy autophagy-related genes changed significantly, while use autophagy inhibitor 3-MA treatment or apoptosis inhibitor Z-VAD treatment after the irradiation group, LC3mRNA expression level was significantly lower compared with the irradiated group, but still higher than the sham control group and autophagy inhibitor 3-MA treatment group; MCF7 cell extracts from 4Gy 24h after irradiation, with the western-Detection of autophagy-related protein bloting found that irradiation significantly related to changes in autophagy protein, was added before irradiation autophagy inhibitor 3-MA treatment, LC3 protein levels decreased compared with the control group, about 20% lower than the radiation group; Before radiation, apoptosis inhibitor Z-VAD added after treatment, LC3 protein expression level of 3-MA group close to about 20% lower than the radiation group. These studies have shown that ionizing radiation may be through PI3K-â… /AKT/mTOR, PI3K-â…¢/Beclinl, P53 and other signaling pathways induced by interaction of autophagy MCF7 cells, and prompted apoptosis in MCF7 cells, autophagy may exist on the positive regulatory role.4 MCF-7 cells after exposure to DRAM and TIGAR changes in gene expressionIonizing radiation to detect autophagy marker gene expression changes, using real-time quantitative PCR, DRAM and TIGAR gene dosage-effect relationship between the outcome. DRAM gene mRNA expression levels in 4Gy 4h after irradiation increased by 7 times (compared with the control group OGy) in 8Gy peaked 4h after irradiation, compared with the control group, increased by 11 times, in 8Gy 8h after irradiation there The decreased, but still higher (about 5 times); TIGAR gene mRNA expression levels in 4Gy 4h after irradiation compared with the control group increased about 8 times in 8Gy peaked 4h after irradiation, compared with the control group, increased 13 times in 8Gy 8h after irradiation decreased, but still higher (about 7 times). Detected by Western bloting DRAM and TIGAR protein expression level, protein that is found in DRAM clear upward trend,4h after irradiation slightly lower, but still higher than sham control group (compared with sham irradiated group increased by 30%) 32h up to a maximum value (compared with sham irradiated group increased by 100%), DRAM protein 8Gy aging studies have shown that protein expression after irradiation significantly increased DRAM,4h after irradiation, a slight drop, but still higher than sham control group (with fake photos group increased by 50%),32h up to a maximum value (compared with sham irradiated group increased by 150%); TIGAR protein 4Gy aging studies have shown that the protein expression after irradiation TIGAR a clear upward trend,8h up to a maximum value (with increase compared to sham control group 100%), TIGAR protein 8Gy aging studies have shown that protein expression after irradiation TIGAR interval in the 2-32h was time-dependent upward trend,24h up to a maximum value (compared with sham irradiated group increased by 150%),8h somewhat lower, but still higher than sham control group.5 MCF-7 cells in the DRAM siRNA cell model the impact of irradiation on the MAP1LC3BOur exposure by MCF-7 cells and MCF7 cells silent DRAM DRAM compared with LC3 protein known, can be seen from Figure 4.8, DRAM silence model group and sham control group decreased significantly compared to DRAM protein,8Gy irradiation silence after the DRAM DRAM model group and radiation group compared with protein, decreased; DRAM silence model group and sham control group decreased compared with LC3 protein,8Gy irradiation DRAM model group and radiation group silencing protein compared to LC3, decreased; DRAM silence model group and sham irradiated group compared with P53 protein expression increased,8Gy silence after irradiation DRAM model group and radiation group compared with LC3 protein, declined. The results show that in MCF-7 cells, gene knockdown, and when the DRAM to be combined with ionizing radiation means, the two play a synergistic role in inhibiting MCF-7 breast cancer cells autophagy.In this study, MCF7 cells to ionizing radiation can increase the expression of fluorescent proteins of this phenomenon and, based on microarray results, MCF7 cells 4Gy,8Gy irradiation and found that DRAM and TIGAR mRNA expression, which is consistent with the gene chip; the same time we DRAM that ionizing radiation can induce protein expression with TIGAR, consistent with the expression of LC3, indicating that proteins involved in the DRAM and TIGAR MCF7 autophagic process. The ionizing radiation induced by MCF7 human breast cancer DRAM and autophagy-related gene and protein expression studies TIGAR for autophagy induced by ionizing radiation to provide a new theoretical basis.