| Bladder cancer is the most common malignant tumor of urinary tract clinically, tumor resection, postoperative adjuvant chemotherapy were the main treatment strategy of bladder cancer nowadays. However, due to the high metastatic ability, bladder cancer recurrence rate and mortality rate is still high. Therefore, it is necessary to study the transfer mechanism of bladder cancer. Oncogene MTDH was discovered in past recent years, it has been reported that it is highly expressed in malignant tumor. Epithelial mesenchymal Transition(EMT) is that the epithelial cells gained the stroma phenotype, and it is easyly for invasion and metastasis, E-cadherin is one of classic epithelial markers. It has been reported that MTDH can promote EMT in malignant tumor, and EMT is the important reason for bladder cancer invasion and metastasis. At present, no research of MTDH related to EMT has been carried out in bladder cancer. In our Study,the relationship between MTDH and E-cadherin in bladder cancer tissues and cells has been studied,and then the further study of MTDH influence on EMT, and the mechanism of promote bladder cancer cells invasion and metastasis through Specific mechanism were studied by gene silencing and overexpression.PART I The expression and clinical significance of MTDH and E-cadherin in bladder cancer tissues and cellsObjectiveTo test the expression of MTDH, E-cadherin in bladder cancer tissues and cells, Explore the relationship between MTDH,E-cadherin with its clinical pathological features respectively, as well as the correlation of MTDH with E-cadherin.Methods1. To detect MTDH and EMT epithelial markers E-cadherin expression level in bladder cancer tissues by the application of immunohistochemical SP method(immunohistochemistry, IHC). Analyze the relationship between MTDH, E-cadherin and clinical pathologic features respectively,the possible relationship between MTDH and E-cadherin were analyzed by the Spearman’s method.2. The mRNA expression level of MTDH and E-cadherin in bladder cancer cell lines and urinary tract epithelial cell lines were analyzed by the Real-time PCR method.3. The protein expression level of MTDH and E-cadherin in bladder cancer cell lines and urinary tract epithelial cell lines were analyzed by Western blot method.Results1. MTDH protein expression of carcinoma tissue was significantly higher than adjacent tissue, the protein expression level of E-cadherin decreased obviously. There was no significant relationship between MTDH, E-cadherin expression and patient age, gender, tumor size,number, and there was significant relationship between MTDH,E-cadherin expression and tumor distant metastasis, clinical stage,pathological grading. There was a negative correlation between MTDH and E-cadherin protein expression(r = 0.722, p < 0.05).2. The MTDH mRNA expression level in 5637, T24 bladder cancer cell lines were higher than that in the urinary epithelial cell lines SV-HUC-1;E-cadherin m RNA expression level in SV-HUC-1 was more highly than that in 5637, T24 bladder cancer cell lines.3. The MTDH protein expression level in 5637, T24 bladder cancer cell lines were more highly than that in the urinary epithelial cell lines SV-HUC-1; E-cadherin protein expression level in SV-HUC-1 was higher than that in 5637, T24 bladder cancer cell lines.ConclusionMTDH were more highly expressed in bladder cancer tissues and cells,E-cadherin were more highly expressed in adjacent tissue of carcinoma and normal cells. MTDH is closely related to tumor clinical pathological features, and can be used as tumor molecular indicators of clinical pathology. MTDH negatively correlated with E- cadherin.PART II The effect and mechanism of MTDH gene silencing on bladder cancer cellsObjectiveTo study the effect of proliferation, apoptosis, migration, and invasion when knockdown MTDH expression in bladder cancer cells, and study the influence of MTDH silencing on Epithelial mesenchymal transition.Methods1. MTDH mRNA and protein expression after transfection of si RNA-MTDH in 5637 and T24 cells were detected by Real-time PCR and Western blot method, Analyze the inhibition efficiency of si RNA, and determined the most efficient silence si RNA finally.2. The change of T24 and 5637 proliferation capacity after MTDH genesilencing were analyzed by CCK8 method, the impact of 5637 and T24 cell apoptosis were analyzed by flow cytometry method, the influence of T24 and 5637 cell migration ability were analyzed by cell scratch test, the change of T24 and 5637 cell invasion ability were analyzed by transwell test.3. The m RNA expression changes of markers related to epithelial mesenchymal transition were detected by Real-time PCR method, The protein expression changes of markers related to epithelial mesenchymal transition were detected by Western blot, The fluorescence expression changes of epithelial mesenchymal markers were analyzed by cell immunofluorescence technique.Results1. The silencing efficiency of si RNA2-MTDH is more than 70% on the level of gene and protein expression, and then si RNA2-MTDH was determined for follow-up study.2. CCK8 method results show that: compared with control group, the proliferation ability of 5637, T24 cells after MTDH gene silence were significantly decreased, Flow cytometry test results suggest the apoptosis rate of 5637, T24 cells after MTDH gene silence were significantly increased, The migration and invasion ability of 5637 and T24 cells after MTDH gene silence were significantly decreased by cell scratch and transwell analysis.3. The Real-time PCR, Western blot analysis showed that E-cadherin m RNA and protein expression were significantly increased after si RNA2 transfection in 5637 and T24 cells, whereas N-cadherin m RNA and protein expression were significantly decreased; Cell immunofluorescence technique showed that E-cadherin fluorescence and number of positive cells enhanced after si RNA2 transfection in 5637 and T24 cell, whereas N-cadherin, Vimentin fluorescence expression and number of positive cells were decreased.ConclusionMTDH gene silencing inhibit proliferation of bladder cancer cells,promote apoptosis and inhibit bladder cancer cell migration and invasion,promote epithelial marker expression, inhibit expression of mesenchymal markers, inhibit epithelial mesenchymal transition.PART III The effect and mechanism of MTDH high expression on bladder cancer cellsObjectiveTo study the effect of proliferation, apoptosis, migration, and invasion when MTDH over-expression in bladder cancer cells, and the influence of MTDH over-expression on Epithelial-mesenchymal transition.Methods1. MTDH lentivirus expression vector GV341-MTDH and GV341-NC were purchased, the lentivirus concentrate of LV-MTDH and LV-NC were prepared, and then infected in T24 and 5637 cells, LV-MTDH monoclonal cell lines and the negative control group LV-NC cell lines were built.MTDH m RNA and protein expression were detected by Real-time PCR and Western blot methods.2. The changes of T24 and 5637 proliferation capacity after infected with LV-MTDH were analyzed by CCK8 method, the impact of 5637 and T24 cell apoptosis were analyzed by flow cytometry method, the influence of T24 and 5637 cell migration ability were analyzed by cell scratch test,the change of T24 and 5637 cell invasion ability were analyzed by transwell test.3. After infected with LV-MTDH in T24 and 5637 cells, The m RNA expression changes of markers related to epithelial mesenchymal transition were detected by Real-time PCR method, The protein expression changes of markers related to epithelial mesenchymal transition were detected by Western blot, The fluorescence expression changes of epithelial mesenchymal markers were analyzed by cell immunofluorescence technique.Results1. The lentivirus of MTDH vector has been built successfully,established an overexpressed LV-MTDH monoclonal cell lines and the negative control group LV-NC cell lines based on the level of gene and protein expression.2. CCK8 method results show that: compared with control group, the proliferation ability of 5637, T24 cells after infected with LV-MTDH were significantly increased, Flow cytometry test results suggest the apoptosis rate of 5637, T24 cells after over-expression of MTDH were significantly decreased, The migration and invasion ability of 5637 and T24 cells after infected with LV-MTDH were significantly increased by cell scratch and transwell analysis.3. The Real-time PCR, Western blot analysis showed that E-cadherin m RNA and protein expression were significantly decreased after infected with LV-MTDH in 5637 and T24 cells, whereas N-cadherin m RNA and protein expression were significantly increased; Cell immunofluorescence technique showed that E-cadherin fluorescence and number of positive cells weakened when over-expression of MTDH in 5637 and T24 cells,whereas N-cadherin, Vimentin fluorescence expression and number of positive cells were increased.ConclusionOver-expression of MTDH gene accelerate proliferation of bladder cancer cells, inhibit apoptosis and promote bladder cancer cell migration and invasion, suppress epithelial marker expression, Promote expression of mesenchymal markers, Promote epithelial mesenchymal transition.PART IV MTDH over-expression regulating the function of T24, 5637 cells via Wnt/β-catenin signaling pathway and its downstream target genesObjectiveExplore the effect of MTDH over-expression on Wnt/β-catenin signaling pathway and its downstream target genes in T24 and 5637 cellsMethods1. Wnt/β-catenin signaling pathway, downstream target genes and MMP9 protein expression after infected with LV-MTDH in 5637 and T24 cells were analyzed by Western blot analysis.2 The β-catenin fluorescence expression after infected with LV-MTDH in 5637 and T24 cells were analyzed by immunofluorescence test.Results1. Western blot experiments show that Wnt/β-catenin signaling pathway were activated after infected with LV-MTDH in 5637 and T24 cells, Totalβ-catenin protein expression increased, Nuclear β-catenin protein expression increased dramatically, Whereas cytoplasmic β-catenin protein expression decreased, Meanwhile, c-Myc and MMP9 protein expression increased significantly.2. Immunofluorescence test show that β-catenin protein expression enhanced dramatically after infected with LV-MTDH in 5637 and T24 cells, number of positive cells also increased compared with LV-NC group,and prompting a significant increase in the translocation of cytoplasmicβ-catenin to the nucleus.ConclusionWnt/β-catenin signaling pathway was activated by MTDH gene over-expression, promoting the epithelial mesenchymal translation, and enhanced the capacity invasion of bladder cancer cells. |
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