Research On The Role Of Mitochondrial Homeostasis Regulators In Radiation Stress

Radiotherapy is one of the commonly used methods to treat tumors.At present,about half of the patients need to receive a comprehensive treatment of radiotherapy combined with other treatments [1].ROS-mediated damage is an important reason for radiotherapy to kill cells.Radiotherapy kills cells are divided into direct action and indirect action.Direct action refers to the killing of cells through DNA damage,and indirect action refers to the production of large amounts of ROS in cells to kill cells.The damage area of the indirect effect is wider and the degree is more serious [2].Mitochondria are the main place where ROS are produced in cells.Radiotherapy and chemotherapy will cause a vicious circle of mitochondrial function and ultimately lead to cell death due to energy metabolism disorders and ROS damage.Mitochondrial homeostasis is the basis for maintaining the normal physiological functions of cells.Abnormal mitochondria will produce excessive ROS,which will cause apoptosis.The dynamic balance of mitochondrial fusion and fission is conducive to clearing mitochondria with abnormal functions and maintaining cell homeostasis.More and more attention has been paid to the molecular mechanism of mitochondrial homeostasis regulation.Enhancing the radiosensitivity of tumor cells through regulation of mitochondrial homeostasis has become a new approach to tumor therapy.Therefore,studying the effect of ionizing radiation on mitochondria and the mitochondrial mechanism of tumor cell resistance to radiotherapy is very important to improve the radiation sensitivity of tumor cells.In the preliminary laboratory experiments,the collected tissue samples of tumor patients were subjected to immunohistochemical staining and analysis.The results showed that the high expression of PB1 in the tissues was positively correlated with radiotherapy resistance,and the laboratory used the si RNA library in the preliminary work to screen.The protein PB1,which is related to the fusion and fission of cell mitochondria and plays a role in cell resistance to radiation stress,was released.Therefore,in order to further study the mechanism of cell tolerance in radiotherapy,we conducted further studies on the PB1 protein.In order to study the effect of PB1 on the radiation sensitivity of cells,flow cytometry was used to detect the ROS and apoptosis of cells with PB1 gene deletion and control cells before and after radiation stimulation.It was found that cells with PB1 gene deletion were exposed to radiation stimulation.The content of ROS in cells was significantly increased,and cell apoptosis was significantly enhanced.Through the detection of GSH/GSSG and MDA levels,the damage to the redox balance in the cells was detected.The results showed that the GSH/GSSG levels in the cells decreased significantly and the MDA levels increased significantly after the cells lacking the PB1 gene were stimulated by radiation.Suffered more severe redox damage.In order to further clarify the regulation mechanism of PB1 on mitochondrial homeostasis and how PB1 affects the molecular mechanism of cellular radiosensitivity by regulating mitochondrial homeostasis,we used PB1 knockout human breast cancer cell lines,human cervical cancer cell lines Hela and PB1 Knockdown Hela cells,under the condition of applying ionizing radiation stimulation,detected the expression levels of the proteins DRP1,MFN1,and FIS1 related to mitochondrial fusion and fission in cells with PB1 gene deletion and control cells.It was found that compared with control cells,In cells with PB1 gene deletion,the level of the protein MFN1 related to mitochondrial fusion decreased under radiation conditions,and the level of the protein DRP1,FIS1,cyto-c related to mitochondrial fission increased.Furthermore,JC-10 staining,transmission electron microscopy,and laser confocal microscopy were used to detect the effect of radiation conditions on mitochondrial morphology after PB1 knockout.It was found that PB1 gene-deficient cells significantly increased mitochondrial fission after being stimulated by radiation and accompanied by mitochondrial cristae.Apparently disappeared.The oxygen consumption of PB1gene-deficient cells and control cells were tested to determine the changes in mitochondrial metabolism.The results showed that the PB1 gene-deficient cells significantly reduced oxygen consumption after being stimulated by ionizing radiation,which proved that the mitochondrial function of the cells was significantly impaired.In order to further study the regulatory mechanism of PB1 protein upstream,we used mass spectrometry to screen out the protein deacetylase Sirt3 that interacts with PB1,and screened out the acetylation site of PB1.The immunoprecipitation experiment further proved that Sirt3 and PB1 There are interactions between proteins.Next,we further verified the regulatory effect of Sirt3 on PB1,using inhibitors and agonists of Sirt3 to detect the expression of PB1 protein in cells,and found that after using Sirt3 inhibitors,the level of PB1 protein in cells increased;After using the Sirt3 agonist,the intracellular PB1 protein level decreased.Further experiments on the radiation sensitivity of cells found that the cells were more tolerant to radiation after the use of Sirt3 inhibitors.Under radiation stimulation conditions,the fission of mitochondria was reduced;after the use of Sirt3 agonists,the cells were more sensitive to radiation,and under radiation stimulation conditions,Mitochondrial fission is obviously aggravated.Prove that Sirt3 can affect mitochondrial homeostasis and the radiation sensitivity of cells through PB1.