The Role Of SETD8 In Genome Intergrity Maintenance And Tumoregenesis

Background:Tumor is a disease with multiple genes involved and gradually evolved.It is regulated by both genetic and environmental factors.Genomic instability is the most important and essential feature of tumor cells.Defect congenitally or under the influence of external harmful factors,genomic instability rises,thereby increasing the frequency of acquired mutations.Mutations in many genes are currently known to be closely related to tumors,and these mutations play an important role in the assessment and treatment of cancer.In order to make a deeper and comprehensive understanding of the occurrence of various types of neoplasms,it is crucial to discover unknown tumor-related mutations.Exome suquencing analyzes exomes in the human genome which has been widely used to dig new tumor-related genes.It is applied most frequently in finding rare gene variation and Mendelian inherited diseases.A 10-year-old girl was pathologically diagnosed with pulmonary right bronchial inflammatory fibroblastoma.Retrospective of family history,we found that the patient’s identical twin sister was in good health.Because the identical twins were grown in the same environment and the genetic similarity was highly consistent,a very small number of genetic differences between the sisters may be involved in the occurrence of the disaster.Objective:We aimed to find out a new mutation associated with patient’s tumorigenesis through whole exome sequencing and explore potential mechanisms.Methods:In order to screen potential tumor-associated genes,we took the twins’peripheral blood to extract genomic DNA and sequenced,SNPs(Single nucleotide polymorphism),InDels(Insert and delation),CNVs(Copy number Mutations)and SVs(Structural variation)were analyzed.Compared to gene databases and her healthy twin sister,potential harmful missense mutation sites in the patient were screened out.In order to verify whether the selected mutations were rare tumor-associated gene mutations biologically,we selected 4 genes with a general mutation rate lower than 1/10^5,namely FRG1 SETD8,BEGAIN,and KIR2DL3.Specific siRNA was used to detect the proliferation and apoptosis after gene knockout in U20S(human osteosarcoma)and HBE(human bronchial epithelial)cells.We found that the lack of SETD8 slowed proliferation and increased apoptosis,which is not observed in other genes’ depletion.For further understood the function of SETD8,on one hand we designed two specific setd8 siRNA,transfected U2OS and HBE cells with setd8 siRNA,cell count and colony formation assay were used to detect cell proliferation,cellular senescence was detected by β-Gal staining(cell aging galactosidase staining),cell cycle and apoptosis were detected by flow cytometry,immunofluorescence were used for DNA damage repair markers RPA and gamma-H2AX,DNA replication rate and replication fork were assessed by DNA fiber;On the other hand,we linked setd8 cDNA to eukaryotic vector and overexpressed them in U20S and HBE cells,flow cytometry was used to detect cell cycle,immunofluorescence was used for DNA damage repair markers RPA and y-H2AX.To verify the effect of single nucleotide mutation carried by setd8 of the patient,wildtype and mutant GFP-SETD8 were overexpressed in U20S and HBE cells respectively,GFP-Trap pulled down the GFP-SETD8 fusion protein and interacting proteins,mass spectrometry and western blot were applied to detect H4 and H4K20mel.Results:FRGl,SETD8,BEGAIN and KIR2DL3 were specifically knocked out with siRNA,only the deletion of SETD8 slowed down the cell proliferation and increased apoptosis,indicating that the mutation of setd8 was most likely associated with tumor;Silenced SETD8 by siRNA in cells,cell proliferation was inhibited,senescence and apoptosis were increased,revealing that SETD8 is very important for the maintenance of cell viability;RPA and y-H2AX in cells lacking SETD8 were significantly increased,indicating that the silence of SETD8 induced a spontaneous DNA damage repair response.Also,DNA replication was slowed in the absence of SETD8,indicating that the lack of SETD8 exerted replication pressure,but no increase in the ratio of stalled replication forks was observed.Interfere SETD8-depleted cells with WEE1 inhibitor,cell proliferation was more limited than that of WEE1 inhibitor alone.Combination inhibition both WEE1 and SETD8 also leads to more apoptosis and DNA damage repair response,indicating that SETD8-deficient cells are more sensitive to WEE1 inhibitor.Overexpress exogenous wildtype and mutant SETD8,protein interaction analysis revealed that the mutant SETD8 enhanced histone H4 binding and H4K20mel levels.Conclusion:SETD8 participates in the maintenance of genomic stability by influencing DNA replication and damage repair.Setd8 T904C enhances the binding of H4 to SETD8.Down-regulation of SETD8 synergistically exerts anti-tumor effects with WEEl inhibitor.