The Role Of NRBP1 In The Tumorigenesis And Progression Of Bladder Cancer

Objective:To investigate the expression of Nuclear Receptor Binding Protein 1(NRBP1)in bladder cancer and analyze its correlation with clinicopathological data and survival of the patients.In addition,knockdown NRBP1 expression in bladder cancer cell lines in vitro and in vivo to investigate its role in tumorigenesis and progression of bladder cancer.Methods:1.First,we constructed a bladder cancer tissue microarray(including 56 bladder cancer tissues and 10 paracancerous tissues),and then used immunohistochemistry to detect the expression of NRBP1 protein in tissue microarray.Finally,the correlation between NRBP1 expression and clinical pathological data and survival was analyzed.2.Real-time quantitative PCR was used to analyze the expression of NRBP1 mRNA in 20 patients with bladder cancer,and the NRBP1 transcriptional difference between cancer and paracancerous tissues in bladder cancer patients was analyzed.3.The NRBP1 down-regulated and over-expressed bladder cancer cell models were constructed firstly.Then,western blot and q PCR were used to verify the NRBP1 expression in the cell models.Finally,the impact of NRBP1 knockdown on cell proliferation,migration,invasion and apoptosis were detected in vitro and the underlying mechanisms were also studied.4.NRBP1 silenced EGFP-Luciferase double-labeled bladder cancer cell line was constructed,and then the effect of NRBP1 on proliferation and apoptosis of bladder cancer cells was verified by subcutaneous tumor formation assay.The xenograft proliferation and apoptosis markers were then detected by immunohistochemistry.5.Immunoprecipitation-mass spectrometry and transcriptome sequencing were used to predict possible mechanisms in NRBP1 overexpressing bladder cancer 5637 cells.In addition,the possible mechanism of NRBP1 in the development of bladder cancer was analyzed by bioinformatics methods.Results:1.(1)NRBP1 protein and mRNA are relatively high expression in bladder cancer tissues,but low expression in normal bladder epithelial tissues,and the expression of NRBP1 is mainly localized in the cytoplasm and is extremely low in the nucleus.(2)High NRBP1 expression is associated with clinical stage of tumors and lymph node metastasis.The results of univariate Cox proportional regression analysis indicated that high expression of NRBP1,tumor pathological grade,clinical stage and lymph node metastasis were unfavorable factors affecting the overall survival of patients.Multivariate Cox proportional regression analysis showed that high NRBP1 expression,age,tumor pathological grade and lymph node metastasis were predictors of poor prognosis in patients with bladder cancer.(3)Survival analysis indicated that high expression of NRBP1 was associated with poor prognosis in patients with bladder cancer.2.(1)Down-regulation of NRBP1 inhibited proliferation,migration and invasion of human bladder cancer cell lines(T24 and 5637),and induced intrinsic pathway apoptosis;(2)Silence of NRBP1 inhibited the activation of NF-?B signaling pathway,in which the expression of IKK?,IKK?,p-I?B?,p-p65,and MMP-9 was down-regulated;(3)The results of mass spectrometry indicated that NRBP1 may interact with some cytoskeletal proteins and cell migration and invasion-related proteins.Bioinformatics analysis of transcriptome sequencing also suggested that the differentially expressed genes after overexpression of NRBP1 are enriched in biological processes such as cytoskeleton formation,signal transduction,transport,and transcriptional regulation.3.The results of subcutaneous tumor formation in nude mice showed that compared with the control group,tumor growth was significantly slowed down in the NRBP1down-regulated group,and the xenograft weight in the NRBP1 down-regulated group was also significantly reduced.The In Vivo Imaging System(IVIS)showed that the Luciferase signal was significantly stronger in the control group than in the NRBP1 downregulated group.Immunohistochemical staining of xenograft tissue in NRBP1down-regulated group showed lower expression of Ki-67 and higher expression of Cleaved-caspase3.Conclusions:1.Overexpression of NRBP1 is closely related to the progression of bladder cancer,suggesting that NRBP1 is a potential marker for bladder cancer diagnosis and prognosis;2.Silencing of NRBP1 could decrease the proliferation,migration and invasion of bladder cancer cells and induce apoptosis in the intrinsic pathway,suggesting that NRBP1 plays an important role in the development of bladder cancer;3.Down-regulation of NRBP1 inhibits the migration and invasion of bladder cancer cells,and its mechanism may be related to the inhibition of NF-?B signaling pathway;4.NRBP1 may affect the development of bladder cancer by participating in biological processes such as cytoskeleton formation,signal transduction,and transcriptional regulation.