| Part1Correlation between transcription factor FOXQ1and epithelial-mesenchymal transition in human bladder transitional cell carcinomaObjective:To investigate the relationship between transcription factor FOXQ1and epithelial-mesenchymal transition(EMT)in bladder transitional cell carcinoma(BTCC), explore the association with clinicopathologic features.Methods:65BTCC 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 FOXQ1, TGF-β1, E-cadherin and Vimentin genes in resected cancer tissues were detected by using reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry, and compared with clinicopathologic data.Results:(1)The expression rate of FOXQ1was significantly higher in BTCC tissues than in normal tissues(89.2%vs13.8%, P<0.05), and the expression rate of FOXQ1in deeply infiltrating group was significantly higher than that in superficially infiltrating group(97.3%vs78.6%, P<0.05); the level of TGF-β1was significantly higher in BTCC than in normal tissues(76.9%比26.2%, P<0.05); the expression of E-cadherin in BTCC tissues were significantly lowered in comparison with those in normal bladder epithelium (27.7%vs89.2%, P<0.05); the expression rate of Vimentin was higher in BTCC tissues than in normal tissues(83.1%vs20.0%, P<0.05);(2) The expression of FOXQ1which as one new positive control factor of EMT was inversely correlated to E-cadherin, but positively to TGF-β1and Vimentin. Conclusion:FOXQ1is related to the differentiation and metastasis of bladder transitional cell carcinoma, and may induce EMT to promote BTCC metastasis. Part2The reverse effect of shRNA targeting silence FOXQ1gene on epithelial-mesenchymal transition in bladder cancerObjective:To investigate the reverse effect of epithelial-mesenchymal transition(EMT) by silencing transcription factor FOXQ1in human bladder cancer cell line T24, and further reseach for its role on invasion and metastasis of cancer.Methods:shRNA eukaryotic expression vector (FOXQ1-shRNA) targeting human FOXQ1gene, was transfected into T24cells with high metastatic potential by lipofectamine2000. The expression of FOXQ1and epithelial mesenchymal markers E-cadherin, N-cadherin, vimentin were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot after transfection48h. The migration and metastatic potential of T24cells were examined by cell wound model and transwell chamber assay in vitro respectively, the proliferation of T24cells were determined by methyl thiazol tetrazolium (MTT) assay. AnnexinV-FITC/PI test on flow cytometry was performed to detect apoptosis.Results:In the FOXQ1-shRNA transfection group, the expression of FOXQ1, N-cadherin, vimentin were decreased in the T24cells remarkably, and the expression of E-cadherin was significantly up-regulated(P<0.05); the morphology of T24cells was transformed into a normal epithelial phenotype; the motility, invasion of T24cells[(15.4±1.4)%vs (76.5±1.1)%, P<0.05] were inhibited, and the abilities of proliferation were inhibited significantly(57.8%vs82.7%, P<0.05)in vitro, and apoptosis rate(37.6%vs14.2%, P<0.05) was increased notablely as compared with the negative control group(NC-shRNA). Conclusion:The silencing of FOXQ1may reverse mesenchymal morphology into a normal epithelial phenotype, inhibit cancer cell migration and invasion, and suppress tumor metastatic potential. Part3Role of FOXQ1in Transforming Growth Factor betal-induced Epithelial Mesenchymal Transition in BIU-87CellsObjective:This study was aimed to investigate the effect of FOXQ1gene silencing on TGF-β1induced epithelial mesenchymal transition, analysis role of TGF-β1and FOXQl in the signal transduction pathways for epithelial mesechymal transition in bladder cancer and further clarify the molecular mechanism of tumor invasion and metastasis.Methods:shRNA eukaryotic expression vector (FOXQ1-shRNA and nc-shRNA) targeting human FOXQ1gene, was transfected into BIU-87cells by lipofectamine2000. BIU-87cells were treated with5ng/ml TGF-β1after transfection8h to set up four groups of working cell line as follows:(nc-shRNA);(nc-shRNA+5ng/ml TGF-β1);(FOXQ1-shRNA);(FOXQ1-shRNA+5ng/ml TGF-β1). After40h changes in cell morphology were observed with microscope. Then the expression of FOXQ1and epithelial mesenchymal markers E-cadherin, vimentin were detected by reverse transcription-polymerase chain reaction(RT-PCR) and western blot in four groups of working cells. We surveyed the regulation of TGF-β1on FOXQ1expression and BIU-87cell morphological phenotypes and the effect of shRNA interfere on TGF-β1induced EMT markers.Results:Compared with the negative control group(NC-shRNA), the morphology of BIU-87cells was transformed into a mesenchymal phenotype, the mRNA and protein expression of FOXQ1was upregulated under the effect of exogenous TGF-β1in the (nc-shRNA+5ng/ml TGF-β1)group. But compared with the group (nc-shRNA+5ng/ml TGF-β1), the mRNA and protein of FOXQ1was downregulated(P<0.05), accompanying with E-cadherin upregulated and vimentin downregulated, BIU-87attained a normal epithelial phenotype in the group(FOXQ1-shRNA+5ng/ml TGF-β1).Conclusion:TGF-β1induces epithelial mesenchymal transition in human bladder cancer cells and regulates the mRNA and protein expression of FOXQ1, FOXQ1is an essential downstream transcription factor of TGF-β1-induced EMT. ShRNA interfere could repress TGF-β1-induced expression of FOXQ1from the level of gene and protein, and prevent epithelial mesenchymal transition process in BIU-87cells. |
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