The Mechanism Of CSMD2 Promoting The Proliferation And Progression Of Bladder Cancer By Regulating PI3K/AKT Signal Pathway

Part I Study on the influence of CSMD2 expression in bladder cancer tissue on the prognosis of bladder cancer patientsObjective: There is a lack of reliable molecular markers to evaluate the prognosis of bladder cancer.The purpose of this study was to explore whether CSMD2 could become a molecular marker of bladder cancer to predict the prognosis of bladder cancer patients after surgery.Methods: The transcriptome expression profile in TCGA and clinical data were used to analyze the expression of CSMD2 m RNA in bladder cancer,and then the expression of CSMD2 protein in bladder cancer was verified by IHC.Kaplan Meier curve was used to analyze the relationship between CSMD2 and the prognosis of bladder cancer,and single factor and multivariate COX regression analysis were used to predict the independent factors of the prognosis of bladder cancer.Using GSEA gene set enrichment analysis,it was found that the signal pathway may be related to CSMD2.Results: In the TCGA group,the high CSMD2 m RNA group was associated with OS events,DSS events,and PFI events;Among the patients with bladder cancer whose pathological stage was II-IV,T stage T2-T4,N stage N1-N3,and histology was high grade,the OS and PFI of the high CSMD2 m RNA group were worse than those of the low expression group(P<0.05);The DSS in the high expression CSMD2 group was significantly lower than that in the low expression group in different subgroups,such as pathological stage II-IV,T stage T2-T4,N stage N0,N1-N3,and histological grade high(P<0.05).In univariate COX regression analysis,it was found that CSMD2 expression(P=0.039),T stage(P<0.001),N stage(P<0.001),M stage(P=0.002),and pathological stage(P<0.001)were associated with overall survival.In the multivariate Cox model,the expression of CSMD2 m RNA in the group with high expression(P=0.033)and N stage(P=0.009)were independent prognostic factors.In the IHC group,it was verified that the survival prognosis of the group with high CSMD2 protein expression was poor.In univariate COX regression analysis,it was found that CSMD2 protein expression(P=0.039),age(P=0.033),N stage(P<0.001),pathological stage(P<0.001),and histological grade(P<0.001)were correlated with the overall survival prognosis.In the multivariate Cox model,CSMD2 protein expression(P=0.033),age(P=0.024),N-stage(P=0.013),pathological stage(P<0.001),and histological grade(P=0.002)were independent prognostic factors.In TCGA data,using GSEA gene set enrichment analysis,it was found that "PI3K/AKT pathway" and "PI3K/AKT/m TOR pathway" were enriched in high-risk groups.Conclusion: The expression of CSMD2 is helpful to predict the postoperative prognosis of bladder cancer,and can become an important molecular marker to predict the prognosis of bladder cancer,and may become a potential target for targeted treatment of bladder cancer.Part II The mechanism of CSMD2 promoting the proliferation,migration and invasion of bladder cancerObjective: To explore the expression of CSMD2 in human bladder cancer and adjacent tissues and in different types of human bladder cancer cells.Methods: TCGA database,RT q PCR and IHC methods were used to analyze the expression level of CSMD2 in human paracancerous and bladder cancer tissues,normal urothelial cells and human bladder cancer cell lines.The expression of CSMD2 in bladder cancer cells was interfered by si RNA and lentivirus vector,the effect of CCK-8 and monoclonal formation on the proliferation of bladder cancer cells,the effect of flow cytometry on cell cycle and apoptosis,and the effect of CSMD2 on migration and invasion of bladder cancer was detected by cell scratch and Transwell test.To construct subcutaneous tumorigenesis in nude mice and evaluate the effect of CSMD2 on the proliferation and tumorigenesis of bladder cancer cells in vivo.GSEA enrichment analysis predicts the molecular mechanisms associated with CSMD2.Western blot was used to detect changes in relevant signal pathways.Results: The analysis of TCGA sequencing data,RT q PCR and IHC data showed that the expression of CSMD2 was significantly up-regulated in bladder cancer tissues and cells compared with adjacent tissues.The cell function experiment showed that the ability of proliferation,migration and invasion of bladder cancer cells was weakened,cell cycle was mainly blocked in S phase,cell apoptosis was significantly increased,and the ability of subcutaneous tumorigenesis in nude mice was reduced after interfering with the expression of CSMD2.GSEA enrichment analysis showed that the PI3K/AKT pathway was enriched in the highly expressed CSMD2 group.Western blot confirmed that CSMD2 knockdown can inhibit the phosphorylation of PI3 K and AKT in bladder cancer cells,and the use of specific AKT agonists can partially reverse the reduction of cell proliferation,migration and invasion induced by CSMD2 knockdown.Conclusion: The expression of CSMD2 in bladder cancer is higher than that in normal urothelium.Regulation of CSMD2 can affect the proliferation,migration and invasion of bladder cancer cells.In terms of mechanism,CSMD2 can activate PI3 K and AKT phosphorylation of PI3K/AKT pathway.The proliferation,migration and invasion of bladder cancer cells induced by CSMD2 are mediated by PI3K/AKT signaling pathway.Part III The mechanism of CSMD2 inhibiting the proliferation,migration and invasion of bladder cancer by regulating PROM2/AKTObjective: To explore the downstream molecules regulated by CSMD2,and to further study how CSMD2 regulates AKT in bladder cancer.Methods: The total RNA extracted from UMUC-3 cells in knockdown CSMD2 group and negative control group was sent out for transcriptome sequencing analysis.The genes significantly down regulated by knockdown CSMD2 were intersected with the genes up-regulated in TCGA bladder cancer tissue.RT-q PCR Western blot detection was used to verify the amount of relevant gene table,and the expression of relevant genes in bladder cancer was verified by transfection of overexpression plasmid.CCK-8 Monoclonal formation was used to detect the effect of PROM2 on the proliferation of bladder cancer cells,Transwell test was used to detect the effect of PROM2 on migration and invasion of bladder cancer,and Western blot was used to detect the changes of related signal pathways.Results: In bladder cancer cells,PROM2 decreased by knocking down CSMD2,and in TCGA data,PROM2 was highly expressed in bladder cancer;The AKT phosphorylation level of bladder cancer cells overexpressing PROM2 was significantly increased,and the ability of proliferation,cloning,migration and invasion of bladder cancer cells was significantly enhanced;Overexpression of PROM2 can block the decrease in AKT phosphorylation induced by CSMD2 knockdown,as well as the increase in BAX and the decrease in BCL-2 induced by CSMD2 knockdown.In addition,overexpression of PROM2 can block the proliferation,clonal formation,migration and invasion of bladder cancer caused by CSMD2 knockdown.Conclusion: PROM2 is highly expressed in bladder cancer,and CSMD2 can regulate the expression of PROM2.CSMD2 regulates AKT signaling pathway by regulating PROM2 expression.