Inhibitory Effect And Mechanism Of UNC5B-ICD/SIRT2/P53 Signaling Axis On Bladder Cancer Cell Proliferation

Objective: Bladder cancer is one of the common urinary tumors,which is a serious threat to human health.Its prognosis is poor,and it has a serious risk of recurrence and metastasis.Early stage bladder cancer can be treated with surgery,with good results,but for patients with middle and advanced bladder cancer,the surgical results are not good.Currently,chemoradiotherapy and immunotherapy are mainly relied on.We found that biologic therapies targeting the activation of tumor suppressor genes or inhibiting oncogenes and their related signaling pathways have good effects on tumor control.Among them,determining how to regulate cell proliferation signaling pathway has become a hot spot in the molecular therapy of bladder tumor.Among candidate genes for bladder tumors,UNC5 H gene,Coordinated-5Homologue family,has attracted the attention of researchers.A large number of previous studies showed that the expression of UNC5 B was significantly reduced in kidney,bladder and prostate cancer,and it was found that the expression of UNC5 B affected the prognosis of bladder cancer.Therefore,UNC5 B may be a potential anticancer target for urinary tumors.In view of the effect of tumor suppressor genes of UNC5 B,we further explored the effect of tumor suppressor active fragment of UNC5 B and intracellular cleavage domain of UNC5B(UNC5B-ICD)on the proliferation of bladder cancer cells.Methods:1.To construct a lentivirus containing the intracellular domain of UNC5B(UNC5B-ICD)using a eukaryotic expression vector,transfect it into T24 and UMUC3 bladder cancer cells,and then culture the bladder cancer cells containing the UNC5B-ICD plasmid in complete medium with puromycin.Following successful screening,further identification will be performed.To verify the effectiveness of culturing tumor cells containing UNC5B-ICD,when the tumor cells are growing normally in the puromycin-containing medium,extract total RNA and proteins,and use PCR and western blot techniques to confirm the successful establishment of the UNC5B-ICD transfected tumor cell line.2.Function of UNC5B-ICD protein in bladder tumor cell lines.The cell model of UNC5B-ICD protein overexpression was confirmed,and the inhibition of UNC5B-ICD on the proliferation of bladder cancer cells was confirmed by CCK8,Real Time Cellular Analysis,Ed U and other methods.3.Co-immunoprecipitation and western-blot were used to prove the functional combination of UNC5B-ICD and SIRT2.It was suggested that UNC5B-ICD can increase the acetylation of P53 and inhibit the proliferation of bladder cancer cells through functional binding with SIRT2.4.Animal models were used to elucidate the molecular biological mechanism of bladder cancer cell proliferation mediated by UNC5B-ICD/SIRT2 and its diagnostic and therapeutic significance: The nude mouse implantation model was comprehensively used to investigate whether UNC5B-ICD/SIRT2-mediated cell biological effect could indeed inhibit the proliferation of bladder cancer cells in vivo,and on this basis,the activation synergistic effect of UNC5B-ICD/SIRT2 signaling pathway and the changes of related proliferation pathways in vivo were analyzed.5.The response experiment further verified the synergistic effect of UNC5B-ICD/SIRT2 signaling pathway activation and the mechanism of cell cycle arrest caused by changes in bladder cancer cells and related proliferation pathways in vivo.SIRT2 was up-regulated in the original UNC5B-ICD cell line,and the acetylation level of P53 was detected and its effect on the proliferation and invasion of bladder cancer cells was detected.Through upregulation of SIRT2 in stable UNC5B-ICD overexpressing bladder cancer cells,the effect of upregulation of SIRT2 in original UNC5B-ICD cell lines on tumor was verified by tumor formation experiments in nude mice,so as to clarify the mechanism of UNC5B-ICD/SIRT2 functional combination regulating P53 acetylation and thus affecting bladder cancer proliferation.Results:1.Confirm the inhibitory effect of UNC5B-ICD on the proliferation of bladder cancer cells through the cell model of UNC5 B intracellular cleavage domain(UNC5B-ICD)overexpression,cell cycle analysis,proliferation activity analysis,western-blot and other methods were used to confirm the inhibitory effect of UNC5B-ICD on the proliferation of bladder cancer cells.Protein spectrum detection showed that UNC5B-ICD combined with SIRT2,and further in vitro and in vivo experiments were used to verify that knockdown of SIRT2 inhibited the proliferation of bladder tumors.2.In the original UNC5B-ICD cell line,SIRT2 was up-regulated,P53 acetylation was inhibited,and the proliferation of bladder cancer cells was promoted.By upregulation of SIRT2 in stable UNC5B-ICD overexpressed bladder cancer cells,and tumor formation experiments in nude mice verified that upregulation of SIRT2 in original UNC5B-ICD overexpressed cell lines reduced the tumor inhibitory effect of UNC5B-ICD,further clarifying the mechanism of UNC5B-ICD/SIRT2 regulating P53 acetylation and thus affecting bladder cancer proliferation.3.It was suggested that UNC5B-ICD can increase the acetylation of P53 and inhibit the proliferation of bladder cancer cells through functional binding with SIRT2.Conclusions:Overexpression of the intracellular segment of UNC5 B has an important correlation with the progression of bladder tumors and poor prognosis.Overexpression of the intracellular segment of UNC5 B inhibits tumor proliferation,and UNC5B-ICD promotes the acetylation of P53 through functional binding with SIRT2,thus inhibiting the proliferation of bladder cancer cells.