Study On Telomere Damage Induced By Ionizing Radiation And The Role Of Telomere Replication Related Protein STN1 In DNA Damage Repair

Objective:Telomeres are fragile sites located on chromosomes,which can protect chromosomes.Epidemiological studies have shown that exposure to adverse environment leads to telomere dysfunction and related diseases.Ionizing radiation causes DNA damage,and telomere length changes when it is exposed to ionizing radiation.In our study,the peripheral blood of workers engaged in radiation-related industries in Tianjin were collected for fluorescence in situ hybridization(FISH)to detect telomere terminal signals,which evaluates the effect of radiation on telomere function in order to clarify the relationship between occupational exposure and telomere damage.Methods:Collect 5ml(heparin anticoagulant)from peripheral blood of low dose occupational exposed people and normal people in Tianjin;culture peripheral blood with blood cell culture medium,then make metaphase chromosome,which has been used for follow-up experiment;use FISH technique to detect telomere terminal signal.The telomere terminal signal was recorded by fluorescence microscope,and the telomere terminal signal was counted by TELO-TEF software.The telomere damage in the control group and the experimental group was compared by linear regression and analysis of variance to clarify the effect of occupational radiation exposure on telomere.Results:According to the statistics of the telomere signal detected by immunofluorescence microscope and TELO-TEF software,it was found that the telomere signal free ends and multiple telomere signals in the experimental group(n=258)were higher than those in the control group(n=300).Further analysis of the results showed that the telomere signal free ends was related to length of workage and type of work,while multiple telomere signals was correlated with length of workage and type of work,but there was no statistical significance.Conclusion:Occupational radiation exposure leads to genomic instability,telomere dysfunction,which may induce related diseases.Objective:The damage response of cells to ionizing radiation,especially the repair of DNA damage,directly determines the fate of cells,which also affects the effect of radiotherapy for cancer.It has been reported that a variety of intracellular proteins are involved in DNA damage response and repair induced by ionizing radiation.Telomere replication-associated protein CST complex(CTC1-STN1-TEN1)not only participates in telomere replication at the end of chromosomes,but also plays an important role in the stagnation and restart of genomic DNA replication forks.However,the role of telomere replication-related proteins such as CST in cell response to ionizing radiation damage and repair remains to be studied.In this study,we analyzed the relationship between STN1 protein in CST complex and carcinogenesis,studied the effect of STN1 on cell ionizing radiation sensitivity,determined its role in cell ionizing radiation injury and response,and discussed its mechanism,in order to provide theoretical basis for cancer radiotherapy.Methods:In this project,bioinformatics method was used to analyze the expression of STN1 protein in tumor cells and its effect on the prognosis of tumor patients;western blot method was used to detect the expression of STN1 protein in different tumor cells induced by ionizing radiation to observe the effect of ionizing radiation on the expression of STN1.Cervical cancer cell hela,cultured in vitro was used to observe cell survival and proliferation by colony formation experiment.The effects of ionizing radiation on cell cycle and ROS production of STN1 under different expression conditions were observed by flow cytometry,and the damage and repair of DNA were observed by comet assay and immunofluorescence assay(γ-H2AX,RAD51).Western blotting was used to detect the expression of DNA damage repair related proteins,and RNA-Seq sequencing was used to analyze the signal pathway of STN1 involved in cell response to ionizing radiation,in order to explore the possible mechanism of STN1 in DNA damage induced by ionizing radiation.Results:Through bioinformatics analysis,we found that the expression of STN1 is abnormal in many kinds of tumor cells,and the change of its expression level is closely related to the prognosis of tumor.In the cell experiment,we found that ionizing radiation could affect the expression of STN1 protein in many kinds of tumor cells;after knocking down the STN1 protein specifically,we found that the cell clone formation decreased after ionizing radiation,and the cell survival rate decreased significantly compared with the control group after the increase of radiation dose;when the irradiation dose was 4Gy,the cell cycle of STN1 knockout group had obvious G2/M phase arrest.After knockout of STN1 protein,DNA damage increased,y-H2AX foci increased and RAD51 foci decreased after irradiation.Compared with the normal group,after knocking down STN1,the expression of DNA damage-related protein changed after irradiation.In addition,overexpression of STN1 could effectively reverse DNA damage induced by ionizing radiation,such as comet tail distance decreased,cycle arrest slowed down,γ-H2AX foci formation decreased,and the level of intracellular reactive oxygen species decreased significantly after overexpression of STN1.The analysis of sequencing results showed that the expression of STN1 could affect the damage repair response of cells after ionizing radiation,in which the change of CtIP protein in the homologous recombination(HR)signal pathway was more significant.Through the HR report detection system,we found that STN1 may be involved in the HR signal pathway.Conclusion:STN1 protein is expressed in a variety of tumor cells,affecting the prognosis of tumors,and its expression is affected by ionizing radiation.The radiation resistance of STN1 protein was further verified by comet assay.According to the phenotypic experiment,different groups of cells treated with ionizing radiation for 12 hours were selected for transcriptome analysis.It was found that STN1 protein participated in DNA damage repair response induced by ionizing radiation,and its deletion would reduce the expression of CtIP protein.Because CtIP protein was involved in HR signal pathway,we verified whether STN1 was involved in HR signal pathway.The results showed that STN1 was involved in HR signal pathway.STN 1 participates in HR signal pathway,so we speculate that STN 1 protein may affect the expression of DNA damage repair signal pathway protein and promote the expression of related genes,so as to reduce radiation-induced DNA damage and enhance the radiation resistance of tumor cells.