The Roles And Mechanisms Of BNIP3 In Radiation-induced Autophagy In Breast Cancer Cells

Breast cancer is a common malignant tumor in women,the incidence and mortality increased year by year,as people have the awareness of the radiotherapy technology constantly,and the continuous improvement of radiotherapy technology and the wider range of adaptation to radiotherapy,the radiation therapy has become one of the main treatments for breast cancer.Autophagy is a digestive progress to eliminate damaged proteins or organelles in cells,in recent years the relationship between autophagy and cancer has become a research hotspot,autophagy is a double-edged sword in the process of tumor development,it maybe play a role in protecting cells during hypoxia or starvation,or maybe cause autophagic cell death.Bcl-2 and adenovirus E1B19kDa interacting protein 3?BNIP3?is a member of the Bcl-2 family proteins,it is a gene contains the BH3 domain which could encode the mitochondrial proteins.BNIP3 could activate apoptosis and autophagy,its high expression is closely related to the invasion,metastasis and the prognosis of tumor cells.This study focused on the role of BNIP3 in the radiation-induced autophagy in breast cancer MCF-7 cells.Objective:Human breast cancer MCF-7 cells were used in this study to research the role of BNIP3 in the radiation-induced autophagy and its possible mechanism,providing a new approach for tumor targeting therapy.Methods:?1?Human breast cancer MCF-7 cells were used in this study,employing lentivirus infection fluid to construct stable BNIP3 silencing model cells?shBNIP3?and space carrier cells?shNC?;?2?Irradiation condition:X-RAD 320-iX deep irradiating instrument was used to launch X ray irradiation,voltage 180kV,electric current 20mA,distance between target and X source 70cm,dose rate 1.0 Gy/min,and the dose of irradiation was 8Gy;?3?CCK8 method and colony formation assay were used to evaluate cells viability and radiosensitivity;?4?Western Blot was applied to detect the expression levels of different proteins,co-IP was used to detect the interaction between proteins;?5?Autophagy and ROS expression levels were detectd by the flow cytometry;?6?Western Blot and PCR were applied to detect the silence effect of shBNIP3;?7?SPSS software was used for statistical analysis,GraphPad was used to make statistical figures,and the experimental data were showed by x±s,t-test was applied in the group comparison between two groups,and the difference is statistically significant when P<0.05.Results:1.BNIP3 was participated in the radiation-induced autophagy in MCF-7 cellsThe MCF-7 cells were exposed to 8Gy radiation,CCK8 results showed that cell viability decreased in a dose-dependent manner after 8Gy radiation when compared with 0Gy group.After the cells exposed to 8Gy radiation,the total proteins was extracted respectively at two time points 24h and 48h.The result showed that Beclin-1,MAPLC3 and BNIP3 expression levels increased significantly after exposed to 8Gy radiation and showed a time-dependent manner.ROS could induce autophagy,the flow cytometry result showed that ionizing radiation not only could increase the autophagy in MCF-7 cells,but also could significantly increase the level of ROS.The above results suggested that ionizing radiation could increase the autophagy,and BNIP3 is involved in the radiation-induced autophagy in MCF-7 cells.2.The mechanism of BNIP3 regulates ionizing radiation induced autophagy in MCF-7 cellsConstructed a stable BNIP3 gene silencing model in MCF-7 cells,using Western Blot and PCR to verify the effect of silence,and the results showed that BNIP3silencing model was successfully established,and shBNIP3-2's silence effect was better than shBNIP3-1's silence effect.shBNIP3 were exposed to 8Gy radiation:?1?The CCK8 assay was used to detect the survival of cells after 8Gy radiation on shBNIP3,and the result showed that the cell survival rate of the BNIP3 silencing model group was significantly higher than the cell survival rate of the shNC group after radiation.It means that silencing of BNIP3 could significantly reduce the cell death induced by radiation;?2?The colony formation assay was used to detect the effect of silent BNIP3 on cell radiosensitivity after irradiation,the result showed that silencing of BNIP3 could significantly reduce the radiosensitivity of the MCF-7cells and increase the radiation resistance of the cells;?3?Western Blot was used to detect the effect of silent BNIP3 in the radiation-induced autophagy.The result showed that in shBNIP3 after radiation,the expression level of MAPLC3-II was significantly increased,and the expression level of BNIP3 was also significantly increased.It means that silencing of BNIP3 could significantly increase the radiation-induced autophagy;?4?The flow cytometry result showed that radiation not only could increase the autophagy in the BNIP3 silencing model,but also could increase the level of ROS in the BNIP3 silencing model;?5?Western Blot was used to detect the effect of silent BNIP3 on the radiation-induced iron metabolism after radiation,and the result showed that in the BNIP3 silencing model after radiation,the expression level of Transferrin increased significantly,and the expression level of FTH1 decreased significantly.It means that silencing of BNIP3 could significantly increase the radiation-induced iron metabolism;?6?Western Blot was used to detect the TOM20 expression level,and in the BNIP3 silencing model after radiation,TOM20 increased significantly.It indicates that the mitochondrial autophagy induced by ionizing radiation occurs in the BNIP3 silencing model,and silencing of BNIP3 could increase the radiation-induced mitophagy.3.The relationship between MAPLC3 and BNIP3 mediated mitochondrial autophagyAutophagy flux is a reliable indicator of autophagy activity,its detection method is usually processed by adding a certain concentration of NH₄Cl into the cells to detect the changes in the expression level of MAPLC3-II.The NH₄Cl preprocessing of the concentration of 30mM in the BNIP3 silencing model,and 0Gy or 8Gy doses were performed 1h later,CCK8 method was used to detect the effect of NH₄Cl on the radiation-induced cell death in shBNIP3.The result showed that the survival rate of the cells in the BNIP3 silencing model group treated with NH₄Cl and8Gy radiation was significantly higher than the shNC group treated with NH₄Cl and8Gy radiation.It means that NH₄Cl could inhibit the radiation-induced cell death in the BNIP3 silencing model.The NH₄Cl preprocessing of the concentration of 30mM in the BNIP3 silencing model and 0Gy or 8Gy doses were performed 1h later,and Western Blot was used to detect the BNIP3 and MAPLC3 expression levels.The result showed that the BNIP3 and MAPLC3 expression levels in the BNIP3silencing model group treated with NH₄Cl and 8Gy radiation was significantly higher than the shNC group treated with NH₄Cl and 8Gy radiation.It means that NH₄Cl could increase the BNIP3 and MAPLC3 expression levels,the addition of NH₄Cl amplifies the autophagy signal,and it could increase the radiation-induced autophagy in the BNIP3 silencing model.The total protein in the MCF-7 cells was extracted by 48h after 8Gy irradiation in the BNIP3 silencing model,co-IP was used to detect the interaction between BNIP3 and MAPLC3.The result showed that BNIP3 could interact with autophagy related protein MAPLC3,and radiation could increase the interaction between BNIP3 and MAPLC3.Conclusions:1.BNIP3 could increase ionizing radiation-induced autophagy in MCF-7 cells.2.BNIP3 could increase the level of ionizing radiation-induced ROS.3.BNIP3 could increase the abnormal iron metabolism induced by ionizing radiation.4.BNIP3 could mediate mitochondrial autophagy.5.IR increased mitochondrial autophagy by enhancing the interaction between BNIP3 and MAPLC3.