Study On The Mechanism Of TROP2 Regulating The Proliferation,invasion And Metastasis Of Gallbladder Cancer Cells

Human trophoblast cell-surface antigen 2(TROP2)overexpressed in many epithelial malignancies and involved in malignant tumor progression.But the mechanism of proliferation,invasion and metastasis in gallbladder cancer cells is unknown.In this study,we investigated the role of TROP2 in regulating the proliferation,invasion and metastasis of gallbladder cancer cells in three aspects: clinical specimens,in vitro and in vivo experiments.In our study,we found that TROP2 was highly expressed while in paracarcinoma tissues TROP2 expression was low,or undetectable in chronic cholecystitis tissues in gallbladder cancer(GBC)tissues.And the expression of TROP2 was closely related to the degree of tumor differentiation,tumor invasion,lymph node metastasis and TNM staging(P<0.05).On another hand,this was not associated with age,sex,and tumor size(3cm)(P>0.05).Survival analysis showed that patients with high expression of TROP2 had a very poor survival(P<0.05).The expression of TROP2 is an independent predictor of prognosis in patients with gallbladder cancer(PGBC).The proliferation,invasion and metastasis of gallbladder cancer cells were significantly decreased after silence TROP2 expression in GBC cells.The further mechanism research found that silence TROP2,the expression of key protein vimentin in epithelial mesenchymal transition would increase;the expression of epithelial marker protein E-cadherin would decrease.On another hand the PI3K/AKT signal pathway key general PTEN protein,PTEN and PDK-1 expression would increased,and p-AKT expression would decreased.To rise the expression of TROP2 would find the opposite phenomenon.Finally,in the animal transplanted tumor model experiments,we found same result as the experiment in vitro,silence TROP2 gallbladder cell lines of transplanted tumors had lighter weight and overexpression of TROP2 gallbladder cell lines of transplanted tumors had more weight.TROP2 expression was same as the expression of vimentin?p-AKT,and E-cadherin,total PTEN expression in the opposite trend.Therefore,TROP2 could be a cancer gene,By regulating the transformation of epithelial mesenchymal(EMT)and PI3K/AKT signaling pathway mediated gallbladder cell proliferation,invasion and metastasis,lead to the poor prognosis of PGBC,this provides a theoretical basis for targeted intervention therapy on the clinical.Part ? The expression of TROP2 and clinical significance in the gallbladdercancerObjective: Investigate the expression of TROP2 in gallbladder cancer and analyze its relationship with clinicopathological parameters and its prognostic value.Method: Take 4 cases of fresh gallbladder carcinoma,3 cases of paracancerous tissues,and chronic cholecystitis tissues in 3 cases.Than use Western blot to detect the expression of TROP2 protein.Take 28 cases of Fresh gallbladder carcinoma and adjacent tissues,than use Real-time PCR method to detect the expression of TROP2 mRNA.Collected a total of 105 patients with gallbladder carcinoma who were diagnosed and received pathological specimens from the Changzheng Hospital of Second Military Medical University from 2005 to 2010.Immunohistochemical method was used to detect the expression of TROP2 protein in 105 cases of gallbladder carcinoma and the adjacent tissues of 15 cases,combined with clinical data and follow-up data,and Chi square(?2)test was used to analyze the relationship between the expression of TROP2 protein and clinicopathological parameters;To produce survival curves of patients with gallbladder carcinoma by Kaplan-Meier;Cox multivariate proportional regression model To evaluate the prognostic significance of each index.Result: Trop2 protein had a high expression in fresh gallbladder carcinomak Low expression or no expression in adjacent tissues.No expression was found in 3 cases of chronic gallbladder carcinoma.In 28 cases of fresh gallbladder carcinoma,Trop2 mRNA was significantly higher than that in adjacent tissues(P<0.05).Immunohistochemical results showed that the positive expression rate of ROP2 protein in gallbladder carcinoma was 61.90%.It was significantly higher than that in adjacent tissues(6.67%).The difference was statistically significant(P<0.05).The expression of TROP2 protein was closely correlated with the degree of tumor differentiation,tumor invasion,lymph node metastasis and TNM stage(P<0.05).It was not associated with age,sex,and tumor size(3 cm)(P>0.05).It had a Single factor cue for the tumor size(3 cm),tumor differentiation,lymph node metastasis,TNM stage and TROP2 expression were closely related to prognosis(P>0.05).Cox multivariate survival analysis showed that TNM staging(HR:0.352,95%CI:0.183-0.678,P=0.002)and TROP2 expression(HR:0463.95% CI: 0.274-0.782 P=0.004)were independent prognostic factors in patients with gallbladder cancer.Conclusion: It was confirmed that TROP2 was highly expressed in gallbladder carcinoma tissues,and was poorly expressed or not expressed in adjacent tissues.The expression of TROP2 was closely correlated with the degree of tumor differentiation,tumor invasion,lymph node metastasis and TNM staging.High expression of TROP2 in pa-tients with gallbladder cancer has a very poor survival.The expression of TROP2 protein is an independent predictor of prognosis in patients with gallbladder cancer.Part ? TROP2 affect gallbladder cell proliferation,invasion and metastasisObjective: To investigate the effect of TROP2 on the proliferation,invasion and metastasis of gallbladder cancer cell line,and to observe the effect of TROP2 on tumor weight.Method: Gather Common gallbladder cancer cell lines: NOZ,GBC-SD,OCUG-1,SGC-996,and EH-GB-1.Western blot and Real-time PCR were used to detect the expression of TROP2 protein and mRNA.Build gallbladder cancer ShRNA-TROP2 cell lines and over expressing TROP2 cell lines: GBC-SD ? SGC-996.MTT was used to detect the proliferation activity of cells in 1,2,3,4,5,6,7.Cell viability was observed under inverted microscope.The ability of cell invasion and metastasis was detected by Transwell plate.Build gallbladder cancer ShRNA-TROP2 cell lines and over expressing TROP2 cell lines: GBC-SD ? SGC-996.In nude mice model,the growth of tumor was observed and the weight of tumor was calculated.Result: The protein level and mRNA level were confirmed: The expression of TROP2 was higher in Gallbladder carcinoma cell line GBC-SD and SGC-996 than that in EH-GB-1,OCUG-1,lowest expression in NOZ.Silencing and over expressing cell lines of gallbladder cancer cell lines GBC-SD?SGC-996 were successfully established.Detection of TROP2 expression in GBC-SD-Scramble/GBC-SD-shTROP2 ?SGC-996-Scramble/SGC-996-shTROP2 ? GBC-SD-Vector/GBC-SD-TROP2 ?SGC-996-Vector /SGC-996-TROP2 by RT-PCR.The cell model was successfully constructed from the mRNA level.After silencing TROP2 expression in gallbladder cancer cell lines GBC-SD?SGC-996,compared with the control group Scramble.The ability of cell proliferation decreased significantly.Especially in the first 5,6,7 days,the difference was statistically significant(P<0.05).There was a similar phenomenon in cell proliferation,cloning,migration and invasion(P<0.05).In the gallbladder cancer cell line GBC-SD?SGC-996 and the expression of tumor in nude mice,The tumor weight of over expression group was higher than that of the normal control group,the tumor weight of the control group was higher than that of the control group,the difference was statistically significant(P<0.05).Conclusion: The overexpression of TROP2 is associated with tumor cell proliferation,invasion,and metastasis in epithelial tumors.In vivo xenograft model also confirmed that the expression of TROP2 in nude mice was more severe.This may be an important cause of malignant and malignant progression of gallbladder carcinomaPart ? The mechanism of TROP2 regulate proliferation,invasion and metastasis in gallbladder cellsObjective: The mechanism of TROP2 regulating the proliferation,invasion and metastasis of gallbladder cancer cells was investigated in vitro and in vivo.Method: Build silencing and over expressing cell lines for gallbladder cancer cell line GBC-SD,SGC-996.Western blot was used to detect the expression of key proteins vimentin and E-cadherin in epithelial mesenchymal transition.To detect the total expression of PTEN,p-PTEN,PDK-1,akt,p-akt in the key proteins of PI3K/AKT signaling pathway.Result: After silencing the expression of TROP2 in gallbladder carcinoma cell line GBC-SD,SGC-996 in vitro.EMT vimentin had low expression;E-cadherin had high expression.After silence the expression of TROP2,in pI3 K signaling pathway,high expression of key molecule PTEN;high expression of PDK-1;low expression of P-AKT.On another hand,increase the expression of TROP2,in pI3 K signaling pathway,low expression of key molecule PTEN;low expression of PDK-1;high expression of P-AKT.In the transplanted tumor,Immunohistochemical results showed After silencing the expression of TROP2 EMT vimentin had low expression;E-cadherin had high expression;in pI3 K signaling pathway,high expression of key molecule PTEN.On another hand,increase the expression of TROP2,vimentin had high expression;E-cadherin had low expression;in p I3 K signaling pathway,low expression of key molecule PTEN;high expression of P-AKT.Conclusion: In vivo experiments confirmed that TROP2 mediated the proliferation,invasion and metastasis of gallbladder cancer cells through EMT pathway and PTEN/AKT signaling pathway,leading to malignant progression of gallbladder carcinoma.This provides a theoretical basis for clinical targeted intervention therapy.