Construction Of Gene Signature For Predicting Bladder Cancer Progression And Prognosis As Wellas The Effect Of CTHRC1 In Bladder Cancer Progression

PART1 CONSTRUCTION OF A 13-MRNA SIGNATURE TO PREDICT BLADDER CANCER DISEASE PROGRESSION AND PROGNOSISObjective: There is currently no reliable standard for evaluation of the risk of progression of non-muscle invasive bladder cancer.The purpose of this study was to identify potential biomarkers based on gene expression profiles to better predict disease progression and prognosis in patients with bladder cancer.Methods: Transcriptome expression profile from GEO array was used to identify differential genes between primary non-muscle invasive bladder cancer and progressive bladder cancer,and a predictive model based on mRNA was constructed by univariate COX regression analysis and LASSO regression analysis.ROC curve was used to evaluate the diagnostic efficacy of the model.Kaplan-Meier curve,univariate and multivariate COXregression were used to analyze the correlation between gene models and prognosis of bladder cancer.Gene model combined with other clinicopathological parameters was applied to construct a nomogram.GSEA was used to analyze gene model-related biological functions and signal pathways.Construct a protein-protein interaction network to find Hub genes in the model.Results: We screened 73 differentially expressed genes by analyzing bladder cancer samples with or without progression.47 prognosis? related genes were screened through differential gene analysis and univariate analysis,and 13 of which were identified to build a progression-associated gene signature using the LASSO regression method.Based on this13? mRNA signature,patients were divided into high-and low-risk groups,with different prognostic outcomes.After validated in an independent GEO cohort and TCGA cohort,the 13-mRNA scoring model has good diagnostic and prognostic value.It was also an independent prognostic factor for overall survival.Receiver operating characteristic analysis suggested that its predictive power outperformed other several published signatures.The nomogram based on the 13 gene model can well predict the progress of NMIBC.GSEA analysis found that “CHEMOKINES SIGNALING PATHWAY”,“FOXM1 PATHWAY”,“EPITHELIAL MESENCHYMAL TRANSITION”,and “INFLAMMATORY RESPONSE” were enriched inhigh-risk groups.CTHRC1,MMP11,AEBP1,SNCAIP,COL1A1 and S100A8 were identified as central genes in the protein interaction network.Conclusion: The 13-mRNA-based prediction model contributes to better predict the progress and prognosis of bladder cancer,and may become a potential target for therapy of bladder cancer or an important biological marker for early screening of bladder cancer.PART2 CTHRC1 PROMOTES INVASION AND METASTASIS OF BLADDER CANCER AND ITS MOLECULAR MECHANISMObjective: To investigate the expression level of CTHRC1 in different types of bladder cancer,and to study the biological function and related signal pathways of CTHRC1 in bladder cancer cells through in vitro and in vivo functional experiments.Methods: Open public database analysis combined immunohistochemistry,RT-q PCR and Western blot to analyze the expression of CTHRC1 in human bladder cancer and adjacent tissues,as well as human bladder cancer cell lines and normal urothelial cells.The correlation between CTHRC1 and clinicopathological characteristics was analyzed by Chi-square test.The Kaplan-Meier curve and univariate and multivariate COX regression were used to analyze the effect of CTHRC1 on overall survival.RNAi and lentiviral vectors were used to interfere with or stably overexpress CTHRC1 levels in bladder cancer cells.The effects of CTHRC1 on migration and invasion of bladder cancer cells were detected by scratch and Transwell assays.A tail vein metastasis model was constructed to evaluate the effect of CTHRC1 on lung metastasis of bladder cancer cells in vivo.CTHRC1-related potential molecular mechanisms were analyzed using GSEA.Western blot was used to detect changes in related signaling pathways and their corresponding functions.Results: Analysis of TCGA sequencing data and Array Express transcriptome data analysis,immunohistochemistry,RT-q PCR and Western blot results showed that compared with normal urothelial and non-muscle invasive bladder cancer tissues and cells,CTHRC1 expression was significantly up-regulated in muscle invasive bladder cancer tissues and cells.High expression of CTHRC1 was closely related to TNM stage(P<0.001),p T stage(P = 0.010)and p N stage(P = 0.002).The overall survival of patients with CTHRC1 high expression was significantly shorter than that with CTHRC1-low(HR = 3.23,95% CI: 1.82 to 5.73,P<0.001).Multivariate COX regression analysis found that CTHRC1 was an independent prognostic factor for predicting OS(HR = 2.91,95% CI: 1.54 to 5.50,P = 0.001)in patients with bladder cancer.Subsequent functional experiments showed that migration and invasion of bladder cancer cells were reduced after CTHRC1 knockdown,while overexpression of CTHRC1 enhanced the malignant biological behaviors of bladder cancer cells in vitro and promote lung metastasis in vivo.GSEA analysis showed that cell focal adhesion kinase and FAK pathway were enriched to the CTHRC1 high expression group.Western blot confirmed that recombinant CTHRC1 can promote the phosphorylation of FAK and ERK1/2 pathways in bladder cancer cells.After blocking FAK and ERK1/2 by specific inhibitors,CTHRC1-induced cell migration and invasion were attenuated,with decreased MMP9 m RNA expression.Conclusion: Compared with normal urothelial and low-invasive bladder cancer,CTHRC1 expression is up-regulated in invasive bladder cancer.CTHRC1 expression is closely related to the prognosis of bladder cancer and can be used as an independent prognostic factor for bladder cancer.Regulation of CTHRC1 can affect the invasion and metastasis of bladder cancer cells.On mechanism,CTHRC1 can activate phospho-FAK,a key protein of focal adhesion kinase,and its downstream phospho-ERK1/2.The invasion and metastasis abilities of bladder cancer cells induced by CTHRC1 are mediated by FAK and ERK1/2 signaling pathways.PART3 UPSTREAM REGULATORY MECHANISM OF CTHRC1 OVEREXPRESSIONObjective: To further investigate the upstream molecular mechanism that regulates the transcriptional activity of CTHRC1,and elucidate the reason for the up-regulation of CTHRC1 expression in bladder cancer.Methods: The correlation between CTHRC1 and TGFB1 was analyzed using the open database.RT-q PCR and Western blot were used to detect the effect of TGF-?1 on CTHRC1 expression.JASPAR and PROMO were performed to predict transcription factors that bind to the CTHRC1 promoter region.The expression of CTHRC1 was detected after transfection of pc DNA3.1-Sp1 plasmid into bladder cancer UMUC-3 cells.The double luciferase reporter assay,Ch IP,RT-q PCR and Western blot experiments were performed to explore the binding of Sp1 to the CTHRC1 promoter and the effect of TGF-?1 on the binding of the two.Results: Correlation analysis of TCGA-derived sequencing data revealed that CTHRC1 was positively correlated with TGFB1 expression,with a Spearman correlation coefficient of 0.41.Expression of CTHRC1 m RNA and protein in UMUC-3 cells was increased after treatment with different concentrations of TGF-?1.JASPAR and PROMO predicted the presence of Sp1-binding elements in the CTHRC1 promoter region.Expression of CTHRC1 m RNA and protein in UMUC-3 cells after Sp1 transfection was higher than that in Vector control group.Ch IP assay confirmed the binding of Sp1 to the CTHRC1 promoter region.The results of double luciferase reporter gene experiment indicated that transfection with Sp1 increased luciferase activity,and the deletion of Sp1 induced luciferase activity was reduced after the mutation was deleted.TGF-?1-induced CTRHC1 expression was reduced after Sp1 expression was reduced with specific inhibitors.Compared with the control group,TGF-?1 stimulation enhanced luciferase activity and the ability of Sp1 to bind to DNA.Conclusion: TGF-?1 induces CTHRC1 expression in bladder cancer cells.Sp1 can bind to the CTHRC1 promoter region,resulting in transcriptional activation.TGF-?1 enhances CTHRC1 transcriptional activity through a Sp1-dependent pathway.