| Objective:Malignant tumor is a serious threat to human life.Bladder cancer(BCa)is one of the most common malignant tumors of urinary system.BCa typically occurs in the bladder epithelium and is the fifth most common type of cancer in the world.The subtype transition of bladder cancercells from non-muscle invasive to muscle invasive is detrimental to BCa patients.Epithelial-mesenchymal transition(EMT)is reported to possess the most positive correlation with this transition.However,the underlying mechanism is still elusive although EMT-related gene signature has been demonstrated to perform favorably for prognosis prediction and subtype survival analysis.To address this issue,we focused on identifying some deubiquitinases.USP35 acts as a deubiquitinating enzyme that alters the protein stability of a variety of epithelial cell-mesenchymal conversion correlated factors.Therefore,we believe that it plays an important role in the development of bladder cancer.At present,there are no reports on the role of USP35 in bladder cancer,the purpose of this study is to detect the expression of USP35 in bladder cancer,and to study the EMT and potential molecular mechanism of USP35 in bladder cancer cells.Methods:1.Based on bioinformatics analysis,we performed a gene set enrichment analysis(GSEA)to determine the correlation between USP35 and EMT.2.The m RNA expression level of USP35 was detected by real-time quantitative PCR(RT-q PCR)assay.3.The effect of USP35 on Epithelial-mesenchymal transition(EMT)after knockdown was detected by Western blot.4.The expression of USP35 in bladder cancer cells and normal urinary tract cell lines was detected by Western blot.5.Ubi Browser 2.0 analysis predicted the downstream target of USP35.6.Protein interactions between USP35 and MDM2 were analyzed using the Dockeasy molecular docking platform and Py Mol 2.2.0 for protein and protein docking conformation.7.Possible signals involved in USP35 promoting EMT were detected by Western blot.8.mi RNAs that may be involved in USP35 and MDM2 regulation were identified by Target Scan analysis.9.Expression of USP35 in bladder cancer and paracancer tissues based on TCGA database.10.USP35 was analyzed by Western blot to detect the protein expression level of P53 in a dose-dependent manner.11.Cell function tests(CCK8,plate cloning,tumor pellet formation,Transwell chamber and cell scratch assay)were conducted to verify the effects of USP35 knockdown on the biological behaviors of bladder cancer cell proliferation,tumor cell dryness,invasion and migration.Results:1.Through gene collection enrichment analysis(GSEA),USP35 was closely related to EMT process.2.Both sh RNAs significantly decreased the expression of USP35 m RNA.3.After USP35 knockdown,the expression of E-cadherin increased,and the expression of N-cadherin decreased.4.USP35 expression was significantly higher in bladder cancer cell line(T24)than in normal urothelial SV-HUC-1.5.The Ubi Browser 2.0 analysis predicted that the downstream targets of USP35 included P53 and MDM2.6.Molecular docking analysis indicated that USP35 might mediate MDM2/P53 signal transduction through direct interaction with MDM2.7.Western blot analysis confirmed that USP35 triggered EMT by affecting the MDM2 / P53 signaling pathway.8.Nearly half of the mirnas regulating USP35 identified by Target Scan analysis were involved in the expression of MDM2.9.The expression of USP35 was significantly higher in bladder cancer tissues than in paraneoplastic tissues,as verified by the TCGA database.10.Western blot analysis showed that USP35 decreased P53 levels in a dose-dependent manner.11.Knockdown of USP35 promoted the acquisition of bladder cancer stem cells and inhibited the proliferation,invasion and metastasis of bladder cancer.Conclusion:In conclusion,data analysis indicated the role of up-regulation of E-cadherin and P53 and down-regulation of N-cadherin after USP35 knockdown.Simultaneously,we found that almost half of USP35-related mi RNAs are shared with MDM2 by Targetscan analysis and thus USP35 might trigger MDM2 upregulation via mi RNA competition,which in turn leads to P53 attenuation as a well-established negative feedback loop.Furthermore,functional data from both bladder cancer cell colony counting and migration studies confirmed that USP35 promotes EMT induced prolifefration and migration capability of bladder cancer cells,suggesting that the up-regulation of USP35 may serve as a foundational signal for bladder cancer transition to malignant phenotype.Figure [4] Table [36] Reference [82]... |
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