| Part 1 Correlation between thymosinβ4 and epithelial-mesenchymal transition in human bladder transitional cell carcinomaObjective:To investigate the relationship between human thymosinβ4 (T(34) and epithelial-mesenchymal transition (EMT) in bladder transitional cell carcinoma (BTCC), explore the association with clinicopathologic features. Methods:60 BTCC specimens from surgically resected were selected. For each specimen, the cancerous tissue,peri-cancer tissue and its remote normal mucosa were analyzed and compared. The expression of Tβ4,ILK,E-cadherin andβ-catenin proteins in resected cancer tissues were detected by using Real-time RT-PCR,Western blot and immunohistochemistry, and compared with clinicopathologic data. Results:(1) The expression rate of Tβ4 was significantly higher in BTCC tissues than in normal tissues (91.7% vs 15.0%, P＜0.05), and the expression rate of Tβ4 in deeply infiltrating group was significantly higher than that in superficially infiltrating group(97.4% vs 81%, P＜0.05); the level of ILK was significantly higher in BTCC than in normal tissues(80.0% vs 18.3%, P＜0.05); the expression rate ofβ-catenin was higher in BTCC tissues than in normal tissues(76.7% vs 26.7%, P＜0.05); the expression of E-cadherin in BTCC tissues were significantly lowered in comparison with those in normal bladder epithelium(30.0% vs 90.0%, P＜0.05); (2) Tβ4 expression was inversely correlated to E-cadherin, but positively to ILK andβ-catenin. Conclusion: Thymosinβ4 is related to the differentiation and metastasis of bladder transitional cell carcinoma, and may induce EMT to promote BTCC metastasis. Part 2 The reverse effect of thymosinβ4 gene silencing on EMT in human bladder transitional cell carcinomaObjective:To investigate the reverse effect of epithelial-mesenchymal transition (EMT) by silencing human gene thymosinβ4(Tβ4), and further reseach for its role on invasion and metastasis of cancer. Methods:Lentiviral shRNA vector encoding Tβ4 was transfected into T24 cell line. The expression of Tβ4,ILK and epithelial markers E-cadherin,β-catenin were detected by Real-time RT-PCR and Western blot assay; the expression of ILK,E-cadherin andβ-catenin in T24 cells were detected by immunofluorescence after transfection via lentiviral vector; the metastatic potential and apoptosis were examined by in vitro cell wound model,Boyden chamber invasion assay and acridine orange-ethidium bromide fluorescent staining. Results:The expression of Tβ4,ILK andβ-catenin were decreased in the T24 cells after transfected with Tβ4 shRNA, and the expression of E-cadherin was significantly up-regulated (p<0.05); the level of ILK in cytoplasm and level ofβ-catenin in nuclear were obviously decreased by immunofluorescence analysis, and the higher level of E-cadherin was observed, the morphology of T24 Cells was transformed into a normal epithelial phenotype; the motility,invation of T24 cells were inhibited and apoptosis was enhanced. Conclusion:The silencing of Tβ4 may reverse fibroblastoid morphology into a normal epithelial phenotype, and suppress tumor metastatic potential.Part 3 The study on reversion of epithelial-mesenchymal transition (EMT) in bladder transitional cell carcinoma (BTCC) by using lentivirus-mediated Tp4-siRNA in vivoObjective:To establish xenografted tumor and lung metastasis BALB/C mouse models of bladder transitional cell carcinoma (BTCC) expressing green fluorescent protein, and investigate effect of Tumor Growth, Metastasis and Apoptosis in vivo by inhibiting Tβ4 expression. so as to lay a foundation for future study. Methods:(1)EJ cell line was used in the present study. The cells were proliferated and the cell suspension was subcutaneously injected into the BALB/C mice. Histologic and immunohistochemical analysis were performed for Tβ4, ILK,β-catenin and E-cadherin. The formed tumor cells apoptosis was detected by TUNEL in the BALB/c nude mice and images were taken using light microscopy. Dark-brown apoptotic bodies were located in nucleus. (2)The in situ tumor information, lymphatic and pulmonary metastasis were all recorded in detail. EJ cells transfected with lenti-GFP (Cont) or lenti-Tβ4 shRNA (lenti-Tβ4) were injected into the tail veins of mice (20 mice per group), and the mice were killed 20 days after injection. The number of metastatic lung nodules was determined by direct counting of the nodules on the lung. Dark arrows indicate the nodules on the lung. Quantification of antiangiogenic activity was calculated by measuring the length of tube walls formed between discrete endothelial cells in each well. Results:(1)Note the E-cadherin stain in the adjacent epidermis of lenti-Tβ4 group xenografts. Note the cytoplasmic staining of the Tβ4 patterns, while ILK showed a corresponding pattern. This staining pattern stands in sharp contrast to the expression profile of E-cadherin, which was absent in the majority of cases in lenti-Tβ4 group, while positivity in staining was observed in control group. (2)The lung metastasis was observed in BALB/C mice, two weeks after mice were injected intravenously with lentivirus-infected cells, the mean number of metastatic lung nodules was 2 for mice injected with EJ cells infected with Tβ4 shRNA and 6 for mice injected with EJ cells infected with control lentivirus (P<0.05). Extensive tube formation of endothelial cells was observed in non-transfection and lenti-GFP (Cont) groups. However, when the endothelial cells were treated by the medium preconditioned with Tβ4 shRNA-transfected EJ cells, the tube formation was markedly suppressed. Conclusion:These data suggested that overexpression of Tβ4 was associated with increases in primary tumor growth and in the number of lung metastases, and transfection of Tβ4 shRNA remarkably inhibited cancer cell malign differentiation in vivo and decreased the angiogenesis of urothelial carcinoma cells in vitro. |
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