| Objective: The incidence of bladder cancer ranks first among urological malignancies in China and second only to prostatic carcinoma in western countries.With the study of the mechanism of bladder cancer,the treatment changes rapidly,and the prognostic survival period and survival quality of bladder cancer patients have improved year by year.However,the high recurrence rate and high metastasis rate of high grade muscle-invasive bladder cancer(MIBC)still bring great difficulties to the treatment of bladder cancer.Recently,it has been shown that the epithelial-mesenchymal transition(EMT)and stemness of tumor cells are the important mechanisms for tumor growth,proliferation,invasion and metastasis.Therefore,clarifying the regulatory mechanism of EMT and stemness has great significance for improving the treatment and survival quality,prolonging the survival period of bladder cancer patients.The current study shows that the WNT pathway plays an important role in a variety of tumors,and WNT7 B participates in the development of some tumors.Few mechanisms has been reported for the effect of WNT7 B in bladder cancer cells.In this research,we examined WNT7 B and other regulatory molecules of bladder cancer tissue and bladder urothelial cancer cell lines,clarifying the regulating mechanism of WNT7 B on EMT and stemness characteristics of bladder cancer cells,and providing new therapeutic directions and precise therapeutic targets for MIBC.Methods: 1.The differential pathological grade of WNT7 B expression and the relationship between WNT7 B expression and the survival time in bladder urothelial carcinoma were analyzed in the TCGA database.In the clinical specimens of bladder urothelial carcinoma,22 high grade and 15 low grade pathological tissues were selected,and immunohistochemistry assay was used to determine WNT7 B expression level in clinicopathological specimens.The relationship of WNT7 B expression level and survival time was analyzed.Referring to the CCLE database to analyze expression level of WNT7 B in different bladder urothelial carcinoma cell lines,RT4,J82,and UM-UC-3 were selected for follow-up experiments.2.Stable transfected J82,UM-UC-3 cell lines with WNT7 B overexpression and the RT4 cell line with WNT7 B knockdown were constructed by the lentiviral vector.Stable transfected J82,UM-UC-3 cell lines with WNT7 B overexpressing and WNT7 B knockdown RT4 cell line were examined by Western blot,Real-time PCR,CCK8 and flow cytometry to clarify the effect of WNT7 B on β-catenin pathway,cell growth and apoptosis.3.Investigate the effect of WNT7 B on the EMT and stemness of bladder urothelial carcinoma.The correlation between WNT7 B and EMT related markers in bladder urothelial cancer samples and urothelial cancer cell lines through the TCGA and CCLE database was analyzed.Stable transfected RT4 cell lines with WNT7 B knockdown and J82,UM-UC-3 cell lines with WNT7 B overexpression were used.The transfection efficiency and the effect of WNT7 B expression to the epithelial mesenchymal phenotype and stemness markers were examined by Western blot,immunofluorescence assays,and Real-time PCR.Cell morphological changes were detected by immunofluorescence assays.The effect of WNT7 B in the invasion ability of cells was examined by transwell.The effect of WNT7 B in the migration ability of tumor cells was examined by wound healing assay.The effect of WNT7 B in the stemness was examined by cell spheroidization experiment.The effect of WNT7 B in the drug resistance of tumor cells was examined using the CCK8 kit.4.Clarify the mechanism of WNT7 B in regulating EMT and stemness of bladder urothelial carcinoma.Through the TCGA and CCLE database,correlation of FZD5 with epithelial and mesenchymal markers were analyzed in samples and cell lines of bladder urothelial caricinama.Applied to Co IP experiment,whether the binding relationship between WNT7 B and FZD5 was clarified.The stable transfected RT4 cell line with FZD5 knockdown was constructed using the lentiviral vector.The effect of FZD5 in epithelial mesenchymal phenotypic markers and cell invasion was determined by immunofluorescence assay and transwell assay.The transfection efficiency of FZD5 knockdown and the effect of FZD5 on stemness were tested by Western blot,Real-time PCR,drug resistance assay and cell spheroidization experiment.The stable transfected J82 cell line with FZD5 knockdown and WNT7 B overexpression was constructed using the lentiviral vector.Transfection efficiency was clearly defined by Western blot and Real-time PCR assay.Through immunofluorescence experiment,wound healing experiment,cell spheroidization experiment,and drug resistance test,the effect of WNT7 B in EMT and stemness characteristics of bladder urothelial tumor cell lines with FZD5 knockdown were determined;Through the TCGA and CCLE databases,correlations of ELF3 with WNT7 B,FZD5 and EMT-related markers were analyzed respectively.Using two stable transfected RT4 cell lines with WNT7 B and FZD5 knockdown respectively,the effect of WNT7 B and FZD5 in ELF3 expression in bladder urothelial cancer cells was determined by Western blot and Real-time PCR.Using the stable transfected RT4 cell line with WNT7 B knockdown and the wild-type J82 cell line,after overexpressing ELF3,through Real-time PCR,transwell experiment,cell spheroidization experiment and drug resistance test,the effect of ELF3 in EMT and stemness of the RT4 stable transfer cell lines with WNT7 B knockdown and J82 was determined.Through the CCLE database,the correlations of NOTCH1 with WNT7 B,FZD5,and ELF3 were analyzed.The effect of WNT7 B and FZD5 in NOTCH1 expression of bladder urothelial cancer cells was determined by Western blot and Real-time PCR,using two stable transfected RT4 cell lines with WNT7 B and FZD5 knockdown.The transcriptional regulation of ELF3 on NOTCH1 was analyzed by Ch IP assay.Using the stable transfected RT4 cell line with WNT7 B knockdown and the wild-type J82 cell line,after overexpressing ELF3 and NOTCH1 respectively,the effect of ELF3 overexpression in NOTCH1 expression was determined by Western blot,the effect of NOTCH1 overexpression in EMT was detected by immunofluorescence staining and Real-time PCR.The effect of NOTCH1 overexpression in the stemness of tumor cells was examined by spheroidization experiment and drug resistance test.Results: 1.WNT7 B is highly expressed in low grade bladder urothelial carcinoma,and patients with bladder urothelial carcinoma with high WNT7 B expression can achieve longer survival times.2.WNT7 B is not taken part in the regulation of the classical β-catenin pathway in bladder urothelial cancer cells,and does not affect the proliferation and apoptosis of bladder urothelial cancer cells.3.WNT7 B positively regulates epithelial phenotype-associated factors in different bladder urothelial cancer cell lines.The stable transfected J82 cell line with WNT7 B overexpression showed increased CDH1 and E-cadherin expression,decreased VIM,ZEB1,and Vimentin expression,transformation of cells to epithelial morphology,and decreased migration and invasion.The stable transfected UM-UC-3 cell line with WNT7 B overexpression,showed increased CDH1 expression,decreased VIM and ZEB1 expression,and decreased migration and invasion.The stable transfected RT4 cell lines with WNT7 B knockdown showed decreased expression of CDH1,E-cadherin,increased expression of VIM,ZEB1,and Vimentin,and the transformation of cells to mesenchymal morphology,and enhanced migration and invasion.4.WNT7 B negatively regulates cell stemness-related factors in different bladder urothelial cancer cell lines.The stable transfected J82 and UM-UC-3-cell lines with WNT7 B overexpression decreased CD44 expression,inhibited the IL-6/Stat3 pathway,and diminished cell globulation capacity and drug resistance.The stable transfected RT4 cell line with WNT7 B knockdown,increased CD44 expression,promoted the IL-6 / Stat3 pathway,enhanced cell globulation capacity and drug resistance.5.FZD5 has similar phenotypic characteristics to WMT7 B,and Co IP experiments confirmed that FZD5 binds to WNT7 B in RT4 cells.6.FZD5 positively regulates epithelial phenotype-associated factors in bladder urothelial cancer cells.The stable transfected RT4 cell lines with FZD5 knockdown,reduced E-cadherin expression,increased Vimentin expression,and decreased invasive ability of the cells.7.FZD5 negatively regulates the stemness-related factor of bladder urothelial cancer cells,.The stable transfected RT4 cell line with FZD5 knockdown,had no significant change in WNT7 B expression,but increased CD44 expression,upregulated the IL-6/Stat3 pathway,enhanced cell globulation and drug resistance.8.Overexpression of WNT7 B based on the FZD5 knockdown RT4 cell line showed no significant change in E-cadherin and Vimentin expression of the cells,and no change in cell migration,globulation capacity,and drug resistance.It is suggested that WNT7 B regulates the EMT and stemness characteristics of bladder urothelial cancer cells by binding to FZD5.9.Combined with the TCGA and CCLE databases,ELF3 was positively associated with WNT7 B,FZD5,and negatively associated with ZEB1,ZEB2 in bladder urothelial cancer tissues and cell lines.Knockdown of both WNT7 B and FZD5,respectively,reduced ELF3 expression in RT4 cells.Overexpression of ELF3 in the WNT7 B knockdown RT4 cell lines showed increased VIM and ZEB1 expression,and decreased cell migration,globulation capacity,and drug resistance.The same phenotypic signature was observed in the stable transfected J82 cell lines with ELF3 overexpression,that increased VIM,ZEB1 expression and decreased cell migration,globulation and drug resistance.10.Combined with the CCLE database,NOTCH1 and WNT7 B,FZD5,and ELF3 were positively associated in the bladder urothelial cancer cell lines.Knockdown of both WNT7 B and FZD5,respectively,reduced NOTCH1 expression in RT4 cells.There was theoretical binding sites for ELF3 to the NOTCH1 promoter sequence,and Ch IP experiment confirmed that ELF3 can bind to the NOTCH1 promoter sequence and ELF3 is involved in transcriptional regulation of NOTCH1.Overexpression of ELF3 in RT4 cell lines with WNT7 B knockdown increased NOTCH1 expression.Overexpression of NOTCH1 in RT4 cell lines with WNT7 B knockdown reduced VIM expression,turned to epithelial morphology,and decreased cell globulation and drug resistance.In J82 cells with ELF3 overexpression,the NOTCH1 expression was increased.The stable transfected J82 cells with NOTCH1 overexpression,reduced VIM expression,turned to epithelial morphology,decreased globulation and drug resistance.Conclusions:1.WNT7 B is highly expressed in low grade bladder urothelial cancer tissues,and suggests a good prognosis and longer survival time.2.WNT7 B inhibits EMT and stemness in bladder urothelial cancer cells.3.WNT7B/FZD5 inhibits the EMT and stemness in bladder urothelial cancer cells by regulating the ELF3-NOTCH1 axis,and this pathway can be used as a therapeutic target for bladder urothelial cancer. |
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