The Studies Of Fascin Inducing Epithelial-mesenchymal Transition And Its Signal Pathway In Cholangiocarcinoma

Part1Differential expression of fascin, E-cadherin, and vimentin:proteins associated with survival of cholangiocarcinoma patientsObjective:By measuring the expression level of fascin, E-cadherin, and vimentin in cholangiocarcinoma tissue and benign bile duct tissue, the expression level was compared in these tissues, the relationship between the expression level and the clinicopathological features was analyzed.Methods:This study detected their expressions in142cholangiocarcinoma and20benign bile duct tissue specimens by using immunohistochemistry. Clinicopathological characteristics and survival data were also collected and analyzed for their association with expression of these three proteins.Results:The data showed that both fascin and vimentin proteins were significantly overexpressed in cholangiocarcinoma, whereas E-cadherin expression was reduced in cholangiocarcinoma compared to normal tissues. Fascin protein expression was associated with tumor dedifferentiation, venous invasion, and lymph node or distant metastasis. Similarly, vimentin expression was associated with tumor dedifferentiation and venous invasion as well as hepatitis virus infection. In contrast, loss of E-cadherin expression was associated with tumor dedifferentiation (P<0.05). Since the functions of these three proteins were closely related, the data showed that fascin and E-cadherin protein expression was reversely associated. However, fascin and vimentin protein expression was positively associated in cholangiocarcinoma. Furthermore, overexpression of fascin and vimentin was associated with shorter overall survival of patients with cholangiocarcinoma than without (median survivals of23.0vs.39months and20.0vs.40.0months, respectively). A combination of fascin and vimentin protein expression predicted a very poor median patient survival of only15.0months, which was significantly shorter than45.0months for patients without their expression. The data from the current study demonstrated that overexpression of fascin and vimentin proteins could be further evaluated as biomarkers for the prediction of cholangiocarcinoma patient survival.Conclusions:1. Fascin expression was was significantly associated with tumor dedifferentiation, vascular invasion, and lymph node or distant metastasis; vimentin expression was positive was significantly associated with tumor dedifferentiation, vascular invasion, and hepatitis virus infection; E-cadherin expression was absent in cholangiocarcinoma tissues and was significantly associated with tumor dedifferentiation;2. There was a reverse association between fascin and E-cadherin expression and between E-cadherin and vimentin expression, but there was a positive association between fascin and vimentin expression in cholangiocarcinoma tissues; and expression of fascin and vimentin was associated with poor prognosis of cholangiocarcinoma, and the combination of these two protein expressions predicted very poor patient survival.3. The clinic pathological data indicated that loss of E-cadherin and gain of vimentin may lead to a more aggressive tumor behavior in CC tissues, it may has relation with fascin.Fascin may have relation with EMT in cholangiocarcinoma.Part2Construction and packaging of lentiviral vector of fascin gene and establish with stably transfected cholangiocarcinoma cell lines.Objective:To construct a lentiviral vector of RNA interference (RNAi) of fascin gene in cholangiocarcinoma cell QBC939Methods:To construct an effective fascin specific siRNA lentiviral vector, The target sequence of fascin gene which can be effectively silenced in RNA interfence was confirmed in our previous study. The vector then the results confirmed by RT-PCR and DNA sequencing.293T cells were cotransfectd with lentiviral vector and packing plasmids,to produce lentiviral vector which can transfect other cells.The changes of fascin expression was detected by quantitative Real-time polymerase chain reaction (RT-PCR) and western blot.Results:PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of fascin was constructed successfully. Fascin expression levels of the experimental group were significantly lower than the control group and negative control group.Conclusion:Fascin-shRNA lentivirus vector has been constructed successfully,and it can effectively silence the fascin expression in QBC939cells. Establish with Fascin-shRNA stably lentivirus vector transfected cholangiocarcinoma cell linesPart3Effects of slience the expression fascin by RNA interference in cholangiocarcinoma cellsObjective:To investigate the role of Fascin on human cholangiocarcinoma cells QBC939.Methods:Using MTT test the proliferation of QBC939,and using Transwell test the invasion of QBC939,and using the Scratch experiment test the metastasis of QBC939Results:With MTT,the growth curve of the QBC939was drawn when knockdown of fascin. After knockdown of fascin,the invasion of the QBC939was reduced by Transwell. After knockdown of fascin,the metastasis of the QBC939was reduced by the scratch test.Conclusion:RNAi slience fascin expression redcue the invasion and proliferation on QBC939.Part4The role of fascin to related signal pathway responsible for fascin induced EMT in cholangiocarcinoma cellsObjective:To investigate fascin induced QBC939EMT,and related signal pathway responsible for fascin induced EMTMethods:The expression of the epithelial marker E-cadherin and the mesenchymal marker vimentin were determined by western blotting in experimental group、the control group and negative control group. Immunofluorescence was performed to demonst rate the relocalization of β-catenin,and the expression of MMP7and cyclin D1were determined by western blotting in experimental group、The control group and negative control group.Results:Knockdown of fascin induced E-cadherin upexpression and reduced vimentin expression. Knockdown of fascin reduced MMP7and cyclin D1expression. Both western blot and immunofluoresence results demonstrated that the knockdown of fascin by RNA interference was associated with relocalization of β-catenin from the nucleus to the plasma membrane and decreased p-catenin-mediated transactivation.Conclusion:1.RNA interface slience fascin expression redcued vimentin expression and induced E-cadherin upexpression which show fascin maybe induced EMT in cholangiocarcinoma.2. Wnt/p-catenin signal pathway play a important role to fascin induce EMT in cholangiocarcinoma.Part5The role of fascin to the proliferation of cholangiocarcinoma which were transplanted to athymic miceObjective:To detect the role of fascin on athymic transplanted cholangiocarcinoma.Methods:The model of nude mouse with region injection human cholangiocarcinoma was established. The nude mice were divided into experimental group、 the control group and negative control group.To use Fascin-shRNA lentivirus vector treatment experimental group nude mice through region injection.To investigate fascin, vimentin and E-cadherin expression in three group by western blot.Results:The transplanted cholangiocarcinoma tumor growed more slowly in the experimental group than in the control groups and negative control groups. E-cadherin upexpression and reduced vimentin expression. In vivo tests, Knockdown of fascin induced E-cadherin upexpression and reduced vimentin expression.Conclusion:In vivo, RNA interface slience fascin expression redcued the growth curve of choangiocarcinoma tumor and inducing EMT in cholangiocarcnioma.