Nucleolar Protein FBL Promotes Tumorigenesis Of Colorectal Cancer Through Regulating Oxidative Damage Response

Background: Colorectal cancer ranks the third malignant tumors in the world.In-deepth understanding of its pathogenesis is paramount for the early diagnosis and treatment of colorectal cancer.Several studies have shown that oxidative stress leads to DNA damage,mutation and chromosomal instability,which play an important role in the occurrence and development of colorectal cancer.Objective: Abnormal DNA damage repair is one of the vital factors in tumorigenesis.In this study,by analyzing the proteomics of the PARylation substrates under oxidative damage,we found that the nucleolar protein FBL as its substrate was highly expressed in colorectal cancer.We further explored the mechanism of FBL involved in tumorigenesis of colorectal cancer through regulating oxidative damage response.Methods: In this study,the database was used to find out the potential PARylation substrates under oxidative damage.The expression of FBL in colorectal cancer was analyzed by bioinformatics methods.The expression of FBL in clinical samples of colorectal cancer were detected by western blotting and immunohistochemistry.The effect of FBL expression in cell growth and proliferation were analyzed by CCK8 and clony formation assay.Cell-derived xenograft in nude mice was used to analyze the effect of FBL on the tumorigenic.AOM/DSS induced colitisassociated cancer mouse model was employed to analyze the expression of FBL,and the correlation with the oxidative damage marker 8OHd G in the process of tumorigenesis.In addition,the effect of FBL on DNA damage repair was investigated by CCK8,western blotting,immunofluorescence and alkaline comet assay under treatment of oxidative damage drugs.Using 405 nm laser to detect whether FBL was recruited to DNA damage sites.The interaction between FBL and PARP1 was detected by bimolecular fluorescence complementation,co-immunoprecipitation,in vitro binding assay and in vitro PARylation assay.The BER efficiency in the FBL knockdown cells was verified by BER assay.The downstream proteins of FBL were examined by using mass spectrometry and validated by co-immunoprecipitation and 405 nm laser experiments.HR and NHEJ assays were used to verify the repair efficiency of FBL overexpressing.Finally,the chromosome aberration assay was used to analyze whether FBL affected the genome instability.The correlation between the expression of FBL and CIN,MSI subtypes were analyzed by database.Results: Our study found that FBL as the PARylation substrate was significantly up-regulated in colorectal cancer,and the low expression of FBL inhibited the cells proliferation and growth in vivo and in vitro.In the colitis-associated cancer mouse model,the expression of FBL was high in tumor and was positively correlated with 8OHd G.Under the treatment of oxidative damage drugs,the FBL affected the sensitivity of cells to the drugs and the efficiency of DNA damage repair.Further,we found that FBL was recruited to DNA damage sites through the GAR domain,and PARP1 regulates the DNA repair function of FBL through PARylation.Importantly,the low expression of FBL enhanced the DNA damage response of OGG1 and reduced the efficiency of BER.The downstream of FBL in DNA damage was mainly through regulating Ku70/80 and then affected the NHEJ pathway.The overexpression of FBL could increase chromosome aberration that led to genome instability.The database showed that the expression of FBL was correlated with CIN.Conclusion: This study found that FBL promotes tumorigenesis of colorectal cancer through regulating oxidative damage response.FBL was regulated by PARP1 under oxidative damage,and promotes BER by facilitating the dissociation of OGG1 from DNA.When the SSB is turned into DSB,FBL affects the NHEJ repair pathway through Ku70 and Ku80,and cause genome instability,which promotes the tumorigenesis of colorectal cancer.