Cytotoxic Necrotizing Factor 1 Promotes Bladder Cancer Angiogenesis Through Activating RhoC

Uropathogenic Escherichia coli(UPEC)is the main pathogenic factor of urinary tract infections(Urinary tract infections,UTIs).UTIs caused by UPEC can cause cystitis,prostatitis and pyelonephritis.Studies have shown that UTIs are closely related to the occurrence and development of bladder cancer(BC).However,the molecular mechanism of UTIs caused by UPEC to participate in the occurrence and development of BC is currently unclear.Cytotoxic necrotizing factor 1(CNF1)is a key toxin protein produced by UPEC and plays an important role in UTIs caused by UPEC.Studies have shown that the cnf1 gene can be detected in UPEC isolated from bladder tissue samples of patients with cystitis;the presence of Escherichia coli expressing the cnf1 gene in biopsy was higher in colorectal cancer patients than in diverticulosis patients.Our previous research found that CNF1 can promote the development of prostate cancer.However,the mechanism of CNF1 in BC progression has not been reported yet.This paper mainly studies the role of CNF1 produced by UPEC in the development of BC and expounds the molecular mechanism of CNF1 involved in the development of BC.Method:1.Transwell and gelatin zymography experiments were used to detect the effect of CNF1 on the motility of bladder cancer and vascular endothelial cells(HUVEC).2.The influence of CNF1 on the ability of HUVEC to form tubular structures was detected by ELISA and in vitro angiogenesis.3.Using q PCR and western blotting experiments to detect whether CNF1 regulates the expression of VEGF in bladder cancer cells and the role of HIF1α in the regulation of VEGF expression by CNF14.SiRNA technology was used to detect the role of Rho C(Ras homolog gene family member C)in CNF1’s effect on bladder cancer cell expression of HIF1α and secretion of VEGF,and the activation of RhoC by CNF1 was verified by western blotting and Pull-down.Activate Rho C(Q63E)was Overexpressed or RhoC was knocked down in bladder cancer cells,and then the effects of Q63 E and RhoC on the expression of HIF1α and VEGF were examined.5.Using transcriptome sequencing(RNA seq)technology to analyze the changes in the expression profile of HIF1α degradation-related genes in bladder cancer cells overexpressing Q63 E under hypoxic conditions,and the expression of HSP90α was verified by qPCR and western blotting.6.Western blotting was used to detect the HSP90α expression in bladder cancer cells overexpressing Q63 E or treatmented with CNF1.Co-Immunoprecipitation(Co-IP)technology was used to detect whether Q63 E overexpression affect the interaction between HSP90α and HIF1?.7.Western blotting and luciferase reporter gene experiment were used to detect whether HSF1 and NF-κB are involved in regulating the expression of HSP90α in bladder cancer cells and the effects of Q63 E and CNF1 on the expression level and phosphorylation level of HSF1.By using inhibitors or si RNA to inhibit HSF1 or NF-κB signaling pathways respectively,the effects of CNF1 or Q63 E on HSP90?,HIF1α and VEGF expression were detected.8.The effect of Q63 E on the development of bladder cancer was detected by subcutaneously xenotransplantation of human bladder cancer cells in nude mice.Furthermore,the effects of Q63 E on the expression levels of VEGF,HSP90α,HIF1α and angiogenesis in tumor tissue were detected by immunohistochemistry(IHC)and immunofluorescence experiments(IF).9.Through analysis of clinical bladder tissue samples,the correlation between the expression levels of VEGF,HSP90α and HIF1α and the development of bladder cancer was demonstrated,and the correlation between the m RNA levels of HSP90α and HIF1α as well as the expression of HSP90α and HIF1α in human bladder cancer tissues and the survival rate of bladder cancer patients were analyzed through a database.Result:1.CNF1 promotes the migration and invasion of bladder cancer cells and vascular endothelial cells.2.CNF1 induces bladder cancer cells to secrete VEGF to promote HUVEC angiogenesis.3.HIF1α participates in CNF1 induced VEGF secretion by bladder cancer cells.4.CNF1 regulates the expression of HIF1α and the secretion of VEGF by activating RhoC of bladder cancer cells.5.The activation of RhoC induced by CNF1 regulates the stability of HIF1α in bladder cancer cells by up-regulating HSF1-HSP90?.6.Activated RhoC promotes angiogenesis associated with bladder cancer tumors in xenograft mouse model in vivo.7.HSP90α,HIF1α and VEGF are associated with the progression of human bladder cancer.Conclusion:We found that CNF1 produced by UPEC promotes the motility of bladder cancer cells and vascular endothelial cells in vitro experiments,and clarified that CNF1 increased the phosphorylation level of HSF1 by activating RhoC of bladder cancer cells,induces increased expression of HSP90α,and thus regulates the stability of HIF1α under hypoxic conditions,thereby promoting VEGF expression and angiogenesis.We have shown in vivo experiments that activated RhoC can promote bladder cancer development and tumor tissue angiogenesis.Further,through the analysis of clinical bladder tissue samples and databases,it was confirmed that HSP90α,HIF1α and VEGF are related to the malignant development of bladder cancer.