The Function And Mechanism Of Primary Cilia In Regulating Radiation Responses Of Tumor Cells

In clinical radiotherapy,radiation tolerance of tumor cells often occurs,and the mechanism of this phenomenon is related to the biological effects of radiation on tumor cells.Ionizing radiation causes DNA damage and cells initiate a radiation stress response.Current studies have found that the biological effects of radiation in cells are not only reflected in the damage to the genome,but also play an important role in organelles.As an important organelle that senses extracellular signals,primary cilia have been shown to play important regulatory roles in DNA damage repair,cell cycle arrest,autophagy and senescence,as well as activation and transduction of various signaling pathways.Some studies have reported that ionizing radiation promotes the formation of primary cilia in some normal cells,but whether ionizing radiation affects the formation of primary cilia in tumor cells has not been reported,and whether primary cilia can affect the response process of tumor cells to radiation stress is unclear.Therefore,in this study,two tumor cells,human non-small cell lung cancer A549and primary glioblastoma GS1910,were used as the main research objects.X-ray（X-ray）and carbon ion beam（Carbon ion）irradiation were used.The morphology and incidence of primary cilia were detected by immunofluorescence,and the length of cilia was measured by Image J software.Western blotting and real-time quantitative PCR were used to detect the expression changes of key proteins in ciliogenesis and related signaling pathways.Flow cytometry was used to detect cell cycle and cell apoptosis,Ed U incorporation,cell counting,CCK8 and other methods to detect cell proliferation and growth ability,SA-β-gal staining to detect cell senescence,clone survival assay to detect si RNA and kinase inhibitors interfere with cilia and related pathways after the radiation resistance of tumor cells sexual influence.The main results are as follows:（1）Primary ciliary structures were detected in A549 cells,GS1910 cells and various other tumor cells,and the incidence of cilia was different among cell lines.The incidence of cilia in A549 and GS1910 was 20.65±1.71%and 8.12±3.21%,respectively.（2）Ionizing radiation can promote the formation and lengthening of primary cilia in tumor cells in a dose and time-dependent manner.On the third day after 10 Gy X-ray irradiation,the incidence of cilia in A549 and GS1910 increased to 77.30±3.46%and 70.79±1.50%,respectively.The average length of cilia also increased by more than 2μm.（3）The process of ionizing radiation promoting primary ciliogenesis in A549 and GS1910 cells is related to the inhibition of YAP/AURKA pathway.（4）While ionizing radiation promotes the formation of primary cilia in A549 and GS1910 cells,the cells appear G₂/M phase arrest,stop proliferation,increase SA-β-gal activity,and have persistentγH2AX foci,indicating that cells have entered senescence.The use of si IFT88 to effectively inhibit the occurrence of cilia will affect the response process of cells to radiation stress,promote senescent cells to activate the apoptosis pathway,interfere with the normal DNA damage repair process,and reduce the survival rate of cell clones.（5）Ionizing radiation activated the primary cilia-related Hedgehog signaling pathway of A549 cells,and inhibiting the activation of Hedgehog pathway could increase the lethal effect of ionizing radiation on A549 cells.In conclusion,this study found that ionizing radiation can promote the formation of primary cilia in tumor cells,and proved that the primary cilia have the function of regulating the response of tumor cells to radiation stress.In addition,the mechanism of this phenomenon was preliminarily explored.The experimental results provide new ideas for the subsequent research on the regulation mechanism of tumor cells in response to radiation stress,and provide a new theoretical basis for solving the problem of radiotherapy tolerance.