UNC5B Mediates G2/M Phase Arrest Of Bladder Cancer Cells By Binding To CDC14A And P53

Introduction:Bladder cancer(BC)is an epithelial tumor that occurs in the bladder mucosa.BC has a high incidence and is the most common urinary tumor in China.BC is not sensitive to traditional radiotherapy and chemotherapy.In view of the diversity of biological phenotypes on BC cells,the therapeutic effect and prognosis of BC with high pathological stage are not ideal.Some recent studies have proved that biotherapy targeting tumor suppressor genes or inhibiting oncogenes and related signaling pathways is promising.UNC5 B is the direct target gene of p53 according to latest studies.UNC5 B can mediate apoptosis and inhibit cell proliferation in the absence of netrin-1 or by its own overexpression.In addition,UNC5 B has the inhibitory effect on the proliferation,migration and invasion of tumor cells,and can enhance the sensitivity of chemotherapeutic drugs to tumors.In this study,lentivirus transfection,mass spectral analysis and co-immunoprecipitation were used to identify UNC5B-binding proteins;bio-informatics was used to analyze the activated molecular pathways;flow cytometry,gene knockout and in-vivo experiments were also conducted to explore the molecular mechanism of UNC5 B regulating cell cycle arrest and inhibiting tumor proliferation on BC cells.Methods:1.Determine the endogenous expression of UNC5 B in BC tissues and cells.Twenty pairs of BC and para-tumor tissues confirmed by pathological report were selected from the pathological specimen database of the Department of Urology of the First Affiliated Hospital of China Medical University.PCR and Western Blot were used to detect UNC5 B in BC specimens.Using the same method,we examined the expression of UNC5 B in 7 BC cells.Suitable cells were selected for further experiment.2.The intracellular domain of UNC5 B was established and transfected into BC cells to verify the anti-tumor effect of UNC5 B.The eukaryotic epitope of UNC5 B intracellular domain and full-length UNC5 B was established and transfected into the selected cells.Fluorescence microscope was used to detect the change of intracellular green fluorescence intensity;PCR was used to detect the m RNA level of UNC5B;Western Blot was used to detect the relative protein level;flow cytometry was applied to detect the fluorescence signal of UNC5 B.The biological effects of UNC5 B were determined by real-time cell assay(RTCA)and flow cytometry.3.Using mass spectrometry and bio-informatics analysis to identify UNC5 Bmediated pathway and related factors.To identify the differentially expressed proteins between groups,BC cells were examined by co-immunoprecipitation(Co-IP),fast silver staining and mass spectrometry.The metabolic pathways related to UNC5 B overexpression were predicted by gene oncology(GO)analysis.Protein-protein-interaction(PPI)network analysis was also applied to evaluate UNC5 B binding proteins.The specific pathways and important target genes activated by UNC5 B were identified by KEGG analysis.4.Using Co-IP,Western Blot,in-vivo experiment and immunohistochemistry to verify the anti-tumor effect of UNC5 B.Using Co-IP,we detected the binding of UNC5 B to CDC14 A and p53.The change of cyclin was examined by Western Blot.The inhibitory effect of full-length UNC5 B and UNC5 B intracellular domain on proliferation was determined by xenograft tumor model in nude mice,and the expression of Ki67 and P53 in implanted tumors was determined by immunohistochemistry(IHC).5.Verify the mechanism of cell cycle arrest mediated by UNC5 B by knockout of CDC14 A.SiRNA and ShRNA against CDC14 A were established to knockout CDC14 A in BC cells.Flow cytometry was applied to detect the change of cell cycle.Western Blot was used to examine the change in related cyclins.Nude mice xenograft models were also established by subcutaneous injection of the sh CDC14 A cells,to identify the effect of CDC14 A on UNC5 B in vivo.IHC was applied to verify the mechanism of cell cycle arrest mediated by UNC5 B and CDC14AResults:1.Both tumor and para-tumor tissues expressed low m RNA levels of UNC5 B.The expression of UNC5 B in para-tumor tissues was slightly higher than that in tumor tissues.The endogenous expression of UNC5 B m RNA on seven BC cell lines were also low expressed,among which 5637 cells expressed the highest m RNA level of UNC5 B,while T24 cells expressed the lowest.PCR,Western Blot,fluorescence microscopy and flow cytometry confirmed the stable transfection of UNC5 B in BC cells.Real time cell analysis(RTCA)verified the inhibitory effect of UNC5 B on proliferation.The cell cycle arrest mediated by UNC5 B was confirmed by flow cytometry.2.Through the analysis of mass spectrometry and bio-informatics,the anti-tumor effect of UNC5 B was found to be closely related to cell cycle arrest,in which CDC14 A and P53 were considered as key factors facilitating G2/M arrest by the interaction with UNC5 B.The binding of UNC5 B with CDC14 A and P53 changed the phosphorylation of P53 at Ser-P53 site,thereby down-regulating the expression of cyclin B1 and inhibiting cell cycle progression.Xenograft tumor model and immunohistochemistry also confirmed the anti-tumor effect of UNC5 B in vivo.3.Knockdown of CDC14 A re-activated the phosphorylation of P53 at Ser-315 site.Cyclin B1 was up-regulated and p-CDK1 was down-regulated in this process,thereby suppressing promoting G1 phase progression and suppressing G2/M phase arrest.The xenograft tumor model indicated that the knockout of CDC14 A increased the volume of the implanted tumor and attenuated the anti-tumor effect of UNC5 B in vivo.Conclusions:1.UNC5 B dephosphorylates P53 at Ser-315 site by combing with CDC14 A and P53 in bladder cancer cells.This dephosphorylation facilitated G2/M phase arrest by reducing the expression of cyclin B1 and increasing the expression of p-CDK1,thus inhibiting tumor proliferation in vitro and in vivo.2.UNC5B-mediated cell cycle arrest may be a potential treatment for bladder cancer.