Characteristics Study Of Bladder Transitional Cell Carcinoma With E-cadherin And N-cadherin Double-negative Expression

Research background:The bladder cancer is one of the most common human malignant tumors, accounted for 3.2% of all human malignant tumors. The bladder cancer also is one of the most common urinary tumors, and its incidence rate showed an upward trent. According to the American Cancer Society, the bladder cancer ranked fourth in the malignant tumors of American male in 2006, ranked ninth in the malignant tumors of American women. The bladder cancer incidence and fatality rate also are at the top of urinary tumors in China. Bladder cancer including a variety of pathological types, Bladder Urothelial Carcinoma is the most common type. Bladder cancer including normuscle invasive bladder cancer and muscle invasive bladder cancer. The main biological characteristics of bladder cancer are infiltration and metastasis. To study the mechanisms of tumor invasion and metastasis will contribute to learn more about the development of bladder cancer, this will provide more important theoretical basis in the treatment and prognosis of Bladder Urothelial Carcinoma. In recent years, the research of pathomechanism in Bladder Urothelial Carcinoma has made significant progress, at the same time, the study of treatment and prognosis in Bladder Urothelial Carcinoma also have made significant progress. However, invasion and metastasis are still a difficult problem for malignant tumor treatment, it is the major cause of death in cancer patients. The research showed that epithelial mesenchymal transition and the development of bladder cancer have a close relationship. Therefore, make a further study for epithelial mesenchymal transition will help us to know more about the development of bladder cancer, and its helpful to guide treatment and preventive of Bladder Urothelial Carcinoma.epithelial-mesenchymal transition(EMT) that is a complex biological processes which epithelial tumor cells lost polarity and transform into mesenchymal cells under special physiological and pathological condition, the main show that epithelial cells adhesion reduce, loss of epithelial cells polarity, enhance tumor cells ability of invasion and metastasis, at the same time, cells phenotype change and obtain mesenchymal cells phenotype. Epithelial tumor cells obtain ability of invasion and metastasis by EMT that is an important biological processes. E-cadherin and N-cadherin are molecular biomarkers of epithelial cells and mesenchymal cells, respective. They are molecular biomarkers of EMT. In short, the expression of E-cadherin decreased and the expression of N-cadherin increased are the biological features of EMT. The research showed that EMT played a critical role in the development of bladder cancer. The development of EMT is a complex process which involves multiple genes, multi-steps and multi-stages. Large studies indicate that E-cadherin and N-cadherin play important role in EMT, and its relationship with tumors invasion and metastasis. The study to EMT have obtained significant progress, however, characteristics study of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression and its relationship with epithelial-mesenchymal transition are rare to know.The article had showed that E-cadherin negative expression and N-cadherin negative expression could be found in non-muscle invasive bladder cancers by immunohistochemical staining. But they didn’t propose E-cadherin and N-cadherin double-negative expression in bladder cancer cells and study their cells biological characteristics, and didn’t study their relationship with EMT. We suspect wheather E-cadherin and N-cadherin double-negative expression cells can be found in Bladder Urothelial Carcinoma? how are their cells biological characteristics? And what are their relationship with EMT? For this problem, we carried out this research. In this paper, we study the characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression and its relationship with epithelial-mesenchymal transition, learn more about the progress of EMT and the development of bladder cancer. Objective:The purpose of this study was to confirm E-cadherin and N-cadherin double-negative expression cells exist in bladder transitional cell carcinoma, and examine the characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression and investigate its relationship with epithelial-mesenchymal transition. Methods:Immunofluorescence assay was used to detect E-cadherin and N-cadherin expression in infiltrative bladder cancer tissues. Immunofluorescence assay and western blotting were used to detect E-cadherin and N-cadherin expression in bladder cancer cells (5637, UMUC-3, and EJ cells). Cell proliferation assay, migration assay, invasion assay and plate colony formation tests were used to detect the abilities of cells proliferation, migration, invasion and the efficiency of plate colony formation on 5637, UMUC3, and EJ cells, respectively. Tumor xenograft formation assay was used to evaluate tumorigenic abilities of 5637, UMUC-3, and EJ cells in vivo.Result:1. E-cadherin and N-cadherin double-negative expression in bladder urothelial carcinoma tissues.The immunofluorescence analysis of tissue samples showed that E-cadherin and N-cadherin double-negative expression were detected in lower-level, mid-level, and high-level infiltrative bladder urothelial carcinoma. E-cadherin marked of red fluorescence, N-cadherin marked of green fluorescence. Cell nucleus marked of blue fluorescence.2. E-cadherin and N-cadherin expression in bladder cancer cells (5637, UMUC-3, and EJ cells)The result of western blotting and immunofluorescence analysis showed that E-cadherin and N-cadherin double-negative expression were detected in UMUC-3 cells. However, E-cadherin positive and N-cadherin negative expression were identified in 5637 cells, E-cadherin negative and N-cadherin positive expression were identified in EJ cells.3. Functional comparison(1).The cell proliferation assay showed that the ability of cell proliferation had significant difference between the three(5637 cells, UMUC-3 cells and EJ cells)(F=308.583; P<0.01); The effect of different points in time on the ability of cell proliferation in vitro had significant difference(F=1513.910; P<0.01); Interactive effect of different cells groups and different time groups is significant(F=58.889; P<0.01); Except for the first day(F=0.553,; P=0.602) and the second day(F=1.078; P=0.398), the ability of cell proliferation difference between groups had significant difference at other different time. The cell proliferation assay showed that the ability of proliferation in UMUC-3 cells was significantly stronger than in 5637 cells on day 3,4,5,6, and 7 after using CCK8(P<0.05), however, it was significantly weaker than in EJ cells (P<0.05).(2) The plate colony formation assay showed that the ability of cell colony formation had significant difference between the three(5637 cells, UMUC-3 cells and EJ cells)(F=203.304, P<0.01); UMUC-3 cells formed larger and more numerous colonies than 5637 cells(The rate of cell colony formation in UMUC-3 cells is 15.38%, The rate of cell colony formation in 5637 cells is 5%, P<0.05). However, in comparison to EJ cells(The rate of cell colony formation in EJ cells is 24.25%), UMUC-3 cells showed a significant decrease (P<0.05). The results of assay revealed that the proliferative abilities of UMUC-3 cells fell in between EJ cells and 5637 cells.(3) The migration assay showed that migration capability between the three cells(5637 cells, UMUC-3 cells and EJ cells) had significant difference(F=1523.776, P<0.01);With the same number of cells and the same incubation conditions, UMUC-3 cells showed a significant increase in the ability of motility in comparison to 5637 cells(P<0.05). However, in comparison to EJ cells, migration capability of UMUC-3 cells exhibited a significant decrease (P<0.05). Migration assays revealed the transmembrane activity of UMUC-3 cells fell in between EJ cells and 5637 cells.(4) The invasion assay showed that invasion capability between the three cells(5637 cells, UMUC-3 cells and EJ cells) had significant difference(F=2161.376, P<0.01); UMUC-3 cells showed a significant increase in invasion capability in comparison to 5637 cells(P<0.05). However, in comparison to EJ cells, invasion capability of UMUC-3 cells exhibited a significant decrease (P<0.05). Invasion assays revealed the transmembrane activity of UMUC-3 cells fell in between EJ cells and 5637 cells.(5) Nude mice subcutaneously transplanted tumor experiment showed that overall difference of the tumor volume between groups(5637 cells group, UMUC-3 cells group and EJ cells group) had significant difference at seven point in time(F=23.903; P<0.01); The tumor volume gradually increase as time goes on, overall difference of the tumor volume between level of times had significant difference(F=16.295, P<0.01); Interactive effect of different cell groups and times had significant difference(F=2.372; P<0.01); The number of tumor is less and The tumor volume is smaller in 5637cells group than other groups(UMUC-3 cells group and EJ cells group), The tumor volume change of 5637 cells group had not significant difference as time goes on(F=2.292; P=0.138); The tumor volume change of UMUC-3 cells group had significant difference as time goes on(F=12.210; P<0.01); and The tumor volume change of EJ cells group had significant difference as time goes on(F=11.348; P<0.01); Tumor volume from different groups had significant difference after 12 day(P<0.05); The results showed that UMUC-3 cells triggered larger tumor volume than 5637 cells in immune deficient mice(P<0.05). In comparison to EJ cells, the subcutaneous tumor volume of UMUC-3 cells were significantly smaller (P<0.05). The morphology of subcutaneous tumor sections were stained by H&E in a similar fashion as the tumor tissue.Conclusions:1. E-cadherin and N-cadherin double-negative expression cells exist in bladder urothelial carcinoma tissues.2. The biological characteristics of bladder cancer cells with E-cadherin and N-cadherin double-negative expression is significantly stronger than the bladder cancer cells of E-cadherin positive and N-cadherin negative expression, but ignificantly weaker than the bladder cancer cells of E-cadherin negative and N-cadherin positive expression.3. The status of E-cadherin and N-cadherin double-negative expression maybe take part in the process of EMT in the pathogenesis of bladder urothelial carcinoma.Innovations of the research:1. To Point out that E-cadherin and N-cadherin double-negative expression cells exist in bladder urothelial carcinoma tissues.2. To study biological characteristics of bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression.3. To speculate bladder transitional cell carcinoma with E-cadherin and N-cadherin double-negative expression relationship with epithelial-mesenchymal transition.