The Impact Of SPOP Serine 119 Mutation On Genomic Stability And Radiosensitivity In Prostate Cancer

Background:Prostate cancer is a common tumor in the urinary system of men and the second most common cancer in men worldwide.In previous reports,the incidence of prostate cancer was higher among men in Europe and the United States,but in recent years,the incidence of prostate cancer among men in China has also shown an upward trend.Prostate cancer is associated with many genetic changes,the most common somatic mutation is SPOP.Cancer is often accompanied by genetic mutations that cause genome instability.Therefore,it is important to investigate how SPOP mutations affect the stability of prostate cancer genomes.Objectives:1.To investigate the effect of SPOP S119 mutation on the stability of prostate cancer genome.2.To investigate the effect of SPOP S119 site mutation on radiosensitivity of prostate cancer.3.To investigate the effect of SPOP S119 site mutation on tumor formation and the sensitivity of radiotherapy in mice.MethodsIn vitro binding experiments and in vitro kinase experiments proved that the SPOP S119 site is an ATM phosphorylation site;co-immunoprecipitation experiments demonstrated that ATM phosphorylates the SPOP S119 site under the induction of ionizing radiation;and constructed PC3 Crisper S119 N and S119 A point mutation cell lines;Western Bloting to detect the formation of mutant and wild-type cell lines γH2Ax after ionizing radiation,and compare the ability of different groups of cells to repair DNA damage;flow cytometry analysis of mitotic index and nuclear micronucleation formation of mutant and wild-type cell lines To compare the cell cycle checkpoints in different groups for abnormalities;MTT and clone formation experiments to detect radiotherapy sensitivity in different groups;Protein spectrum detection SPOP mutations after the change of protein interaction;use the Cre-loxp system to construct conditional SPOP-(flox-S119A)mutant mice;MEF experiments to obtain hybrid mouse cells and immortalize;The genomic stability of MEF cells was detected by immunofluorescence experiments.Results:1.SPOP interacts with ATM under the induction of ionizing radiation,and ATM phosphorylates the SPOP site at S119.2.DNA damage repair was delayed after SPOP S119 mutation.3.SPOP S119 mutation,resulting in abnormal G2/M phase of cell cycle examination.4.SPOP S119 mutation caused chromosome aberration and increased nuclear micronucleation.5.SPOP S119 site mutation,resulting in increased cell sensitivity to radiotherapy.6.SPOP S119 mutation,delayed DNA damage repair in MEF cells.Conclusion:SPOP S119 mutations cause genomic instability and increased sensitivity to ionizing radiation.