The Function And Mechanism Of CENPU In Regulating The Occurrence Of Breast Cancer

Objective:1.Expression of CENPU in different types of breast cancer tissues and its correlation with prognosis;2.Effects of CENPU on the occurrence,migration,invasion of breast cancer;3.Screening of CENPU targets,establishment of regulatory network;Methods:1.Immunohistochemical staining,Western blotting,PCR were used to detect the expression level of CENP-U in different types of breast cancer.2.Next-generation sequencing was performed.Do differential genes,GO genes and KEGG signaling pathways were enriched according to the NGS.3.CD24-CD44~+ flow cytometry was used to verify the influence of CENPU on the self-renewal ability of BCSC.The Mammosphere formation was used to verify the influence of CENPU on the formation ability of BCSC,Wounding healing and Transwell were used to verify the influence of CENPU on the migration and invasion ability of breast cancer.4.Western blotting and ELISA were used to detect the influence of CENPU on proNGF and NGF expression,and NGF neutralizing antibody was added to detect whether could neutralize CENPU ability to promote self-renewal,migration and invasion of breast cancer.5.Western blotting was used to detect the change of CENPU on the expression of Furin,and the activity of Furin was detected by detecting the fluorescence intensity of the specific substrate.6.Co-IP was conducted to verify whether CENP-U and Furin interact at the protein level,and MG132 and chloroquine were added to explore the pathway through which CENPU inhibited Furin protease degradation.7.The xenograft NSG mouse model was established by the method of multiple dilution into tumor.IHC and Western blotting were used to detect the expression of CENPU,proNGF,NGF and Furin in the experimental group and the control group.8.MDA-MB-231 xenograft was further constructed,and mice were intraperitoneally injected with Furin nhibitor to observe whether it could inhibit CENPU ability to promote breast tumor growth.Results: 1.The expression of CENPU in breast cancer tissues was significantly higher than that in normal breast tissue.The expression level of CENPU was also positively correlated with the degree of invasion of breast cancer.The expression level of CENPU in triple negative breast cancer was significantly higher than that in Luminal breast cancer.High expression of CENPU is corelated with poor survival.2.According to NGS,differential genes were screened out: NGF,PTGS2,NR1D1,IFIT2,CD22 and DDX60.Do enrichment showed a significant correlation between CENPU and breast cancer,and KEGG signaling pathway enrichment showed a significant correlation between CENPU and cancer signaling pathway.3.The CD24-CD44~+ flow cytometry results showed that the proportion of CD44~+ cell population in MDA-MB-231-shCENPU and CAL51-shCENPU was significantly lower than that in the control group.Mammosphere experiment showed CENPU could promote the self-renewal of BCSC.The results of Transwell and wounding healing showed that CENPU could promote migration,invasion of breast cancer.4.CENPU could increase the expression level of NGF and decrease the expression level of proNGF.Adding neutralizing antibodies to NGF could inhibit the ability of CENPU to promote breast cancer migration,invasion and mammosphere formation.5.By detecting the expression and activity of Furin,it was found that CENPU could increase the expression of Furin and increase the affinity of Furin to substrate,thus increasing the activity of enzyme.Furin inhibitor could inhibit CENPU’s ability to promote breast cancer migration,invasion.6.Co-IP verified the interaction between CENPU and Furin at the protein level.CENPU could inhibit the lysosomal degradation of Furin and thus increase the expression of Furin and the activity of the enzyme7.Tumor volume of mice in the CAL51-shCENPU group was significantly smaller,and none of the mice in the CAL51-shCENPU group formed tumors when the tumor cell concentration was 103.8.Tumor tissues showed that the expression of CENPU decreased,the expression of proNGF increased,the expression of NGF decreased,and the expression of Furin decreased in the CAL51-Sh CENPU group.9.Furin inhibitor could inhibit the ability of CENPU to promote breast tumor growth.Conclusion: 1.High expression of CENPU in breast cancer tissues is associated with poor prognosis,and the expression level of CENPU is positively correlated with the degree of invasion.Compared with dysplasia of breast tissues and breast ductal carcinoma,the expression level of CENPU is the highest in invasive ductal carcinoma of breast and the highest in triple negative breast cancer.2.CENPU can promote the occurrence,migration and invasion of breast cancer.3.CENPU interacts with Furin at the protein level to inhibit the lysosomal pathway degradation of Furin,increase the enzyme activity of Furin,and promote the maturation of proNGF to NGF,thus promoting the occurrence,migration and invasion of breast cancer.