The Role And Mechanism Of X-linked Inhibitor Of Apoptosis Protein In Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

Background and ObjectiveEsophageal cancer is one of the malignant tumors ranking the eighth in morbidity and sixth in mortality in the world.There are two common pathological types of esophageal cancer,esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).China has the highest incidence of esophageal cancer in the world,and the main pathological type is ESCC.Since esophageal cancer is asymptomatic in early stage,most of patients have lymph node infiltration and metastasis and lost the opportunity of surgery before diagnosis.Although the postoperative survival rate of esophageal cancer patients has been improved in the past decades,the overall survival rate of esophageal cancer is still not ideal due to the high incidence of invasion and metastasis.Tumor metastasis is an important biological characteristic of malignant tumors,which is considered as the biggest obstacle to the progress of tumor treatment.Studies have shown that the 5-year survival rate of patients with localized esophageal cancer is 40%,but the 5-year survival rate of patients with metastatic esophageal cancer is only 4%.Therefore,it is the main direction of current research to explore the mechanism of the invasion and metastasis of esophageal cancer,and the relationship between them and prognosis.To provide new therapeutic targets for the treatment of esophageal cancer is very important.X-linked inhibitor of apoptosis protein(XIAP)has been shown to be involved in cell death,cell cycle,cell migration,immunity,inflammation and other cell biological process.XIAP gene is found to locate in the Xq25 region of the X chromosome,which translated into a protein composed of 497 amino acids with three BIR domains,one UBA domain and one RING domain.XIAP is highly expressed in various types of tumors and is associated with chemotherapy tolerance,disease progression,and poor prognosis.Recently,XIAP has been revealed to play an important role in tumor invasion and metastasis.Some researchers found that XIAP can promote tumor invasion and metastasis in bladder cancer by its downstream effector RhoGDI.On the other hand,XIAP was also found to be able to prevent tumor invasion and metastasis in cervical cancer by inhibiting C-RAF /MAPK signaling pathway.These studies suggest that XIAP may function differently in tumor invasion and metastasis based on the tumor cell lines.However,such diverse mechanism regarding to XIAP biology in tumor invasion and metastasis,particularly in esophageal cancer,remains still unclear.In order to study the mechanism of invasion and metastasis in ESCC,Firstly,we employed immunohistochemical method to detect the expression of XIAP in the tissue samples of ESCC to delineate the relationship between XIAP and the clinicopathology as well as the prognosis of ESCC patients.Subsequently,in vitro experiments,the effect and mechanism of XIAP on cell migration and Epithelial-Mesenchymal Transition(EMT)was detected by interfering with the expression of XIAP in ESCC cell lines.In vivo experiments,we investigated the relationship between XIAP and lung metastasis of ESCC in nude mice.Then the specific mechanism of XIAP on the invasion and metastasis of ESCC was determined in vitro.Finally,the correlation between XIAP and downstream signaling pathway protein was verified in the patient tissue samples.This study is mainly divided into the following three parts:Part ?: Correlation between XIAP expression and clinical characteristic and prognosis in ESCCObjective: To study the correlation between the expression of XIAP and its clinical characteristic and prognosis in ESCC.Methods: The expression of XIAP in 185 cases of ESCC was detected by immunohistochemical staining.The correlation between the intensity of XIAP expression in ESCC patients and clinical characteristic was analyzed,the correlation between XIAP and overall survival rate was performed by Kaplan-Meier analysis,and the correlation between XIAP and prognosis was analyzed by Cox regression analysis.Results: High expression of XIAP in ESCC patients was significantly correlated with lymph node metastasis(P=0.018).There was no significant correlation between the expression of XIAP and patients' age,gender,tumor differentiation,tumor size and TNM stage(P > 0.05).Following up 185 patients,we found that the overall survival rate of patients with high XIAP expression was significantly lower than that of low XIAP patients(P=0.004).Cox regression analysis showed that the high expression of XIAP was an independent risk factor for the prognosis of ESCC patients(P=0.028).Conclusion: High expression of XIAP in ESCC patients was associated with lymph node metastasis and,thus,high level of XIAP was an independent risk factor that predicated the poor prognosis of ESCC patients.Part ?: Effects of XIAP on EMT,invasion and metastasis in ESCC.Objective: To investigate the effects of XIAP on EMT,invasion and metastasis of ESCC.Methods: 1.ESCC cell lines EC9706 and TE13 were transfected with sh-XIAP mediated by lentivirus to establish the down-regulation group(sh-XIAP),while EC9706 and TE13 cell lines were transfected with sh-Ctrl as the control group(sh-Ctrl).Real-time PCR and Western-blot were used to detect the expression levels of XIAP mRNA and protein in the interference group and the control group to confirm successful transfection.2.In vitro experiments,scratch test and Transwell test were performed on EC9706 and TE13 cells in sh-XIAP and sh-Ctrl groups to detect the changes in cell invasion and migration ability of the two groups.Then we used Real-time PCR to detect the mRNA expression of EMT-related proteins E-cadherin,N-cadherin and Vimentin in sh-XIAP and sh-Ctrl groups.Meanwhile,protein expression level and immunofluorescence intensity of EMT-related proteins in sh-XIAP and sh-Ctrl groups were detected by Western-blot and immunofluorescence staining.3.In vivo experiment,lung metastasis model of ESCC in nude mice was established by tail vein injection.The experimental group was injected with EC9706 cells transfected with sh-XIAP,while the control group was injected with sh-Ctrl.The metastatic events of ESCC in the lung was examined in two months.Results: 1.TE13 and EC9706 of ESCC cell lines interfered by XIAP were successfully established.The results showed that the viral transfection efficiency of both the XIAP interference group and the control group were over 90%,and the mRNA and protein levels of XIAP in interference group were significantly lower than those of the control group.2.The in-vitro experiments showed that the invasion and migration ability of ESCC cell lines significantly decreased when XIAP was at lower level,and EMT-related markers were also significantly altered.The results showed that the epithelial markers E-cadherin significantly increased,while the mesenchymal markers N-cadherin and Vimentin significantly decreased.These results suggested that XIAP can induce EMT in ESCC cells.3.Through the establishment of the lung metastasis model of ESCC in nude mice,sh-XIAP injection caused almost no lung metastasis,while the lung metastasis was obvious in the control group.XIAP can significantly promote the lung metastasis after intravenous injection of ESCC.Conclusion: After down-regulation of XIAP in ESCC cell lines,epithelial markers E-cadherin significantly increased,while mesenchymal markers N-cadherin and Vimentin significantly decreased.XIAP can promote lung metastasis of ESCC.These results suggested that XIAP can induce EMT in ESCC cells and promote tumor invasion and metastasis.Part ?: The mechanism of XIAP on EMT,invasion and metastasis in ESCC.Objective: To explore the mechanism of XIAP promoting EMT and invasion and metastasis in ESCC.Methods: 1.The expression of TGF-? in sh-XIAP and sh-Ctrl group was detected by Real-time PCR and Western-blot.Subsequently,TGF-? inhibitor was added into the ESCC cells as the experimental group,while the control group was untreated.Real-time PCR and Western-blot were used to detect the mRNA and protein expression levels of XIAP in the two groups respectively.2.The experimental group of sh-XIAP +TGF-? was established.Transwell assay was used to detect the invasion and migration ability of cells in the groups of sh-XIAP,sh-Ctrl and sh-XIAP +TGF-?.We used Real-time PCR and Western-blot to detect the mRNA and protein expression levels of EMT-related proteins(E-cadherin,N-cadherin and Vimentin)in the three groups respectively.3.The expression of XIAP and TGF-? in clinical tissue samples of ESCC was detected by immunohistochemistry,and the correlation between XIAP and TGF-? was analyzed.Results: 1.We found that the protein expression of TGF-? was significantly reduced after XIAP down-regulation.When TGF-? expression was inhibited,there was no significant change in the mRNA and protein levels of XIAP.2.Transwell assay results indicated that the number of transmembrane cells in the sh-XIAP +TGF-? group was significantly higher than that in the sh-XIAP group.Real-time PCR and Western-blot results indicated that compared with the sh-XIAP group,the expression of epithelial marker E-cadherin decreased while the expression of mesenchymal markers N-cadherin and Vimentin increased in sh-XIAP +TGF-? group.The difference between the two groups was statistically significant.3.There was a positive correlation between XIAP and TGF-?expression in ESCC(r=0.3241,P<0.001).Conclusion: XIAP can induce EMT and promote tumor metastasis through TGF-?signal transduction pathway in ESCC.SummaryThe high expression of XIAP in ESCC tissues was found to be associated with lymphatic metastasis,and the expression level of XIAP represented an independent riskfactor of ESCC patients.In addition,the experiments results further demonstrated that XIAP can significantly promote lung metastasis of ESCC,which may be caused by the induction of EMT through TGF-? signal transduction pathway.Finally,we found a positive correlation between XIAP and TGF-? expression in ESCC tissues.It was further verified that XIAP can promote the invasion and metastasis by regulating the expression of TGF-?in ESCC.