| Cholangiocarcinoma(CCA)is one of the most lethal gastrointestinal malignancies originating from bile duct epithelial cells.Due to the hidden incidence of cholangiocarcinoma,the difficulty of early diagnosis,the high invasiveness,and strong metastatic potential of cholangiocarcinoma,most patients are already in the middle and late stages of the disease when they are definitively diagnosed,resulting in low surgical resection rate,high postoperative recurrence rate,and extremely poor long-term prognosis.Therefore,it is very important to further study the molecular changes and related mechanisms of cholangiocarcinoma invasion and metastasis and explore new strategies for the treatment of cholangiocarcinoma to improve the prognosis of patients.High concentrations of bile acids(BAs)are a special microenvironment of the biliary system.Changes in the composition and concentration of bile acids and activation of related receptors can affect cell proliferation,apoptosis,inflammatory responses,and the secretion of pro-fibrotic factors in vivo.It plays a crucial role in the occurrence and development of biliary diseases.In recent years,studies have reported that bile acids play an important role in the invasion and metastasis of cholangiocarcinoma,but the molecular mechanism remains unclear.The previous research results of our team showed that bile acids can promote the expression of transforming growth factor-β(TGFβ)in bile duct epithelial cells,and play an important role in the occurrence and development of peribiliary fibrosis and ischemic cholangiopathies.Moreover,previous studies have confirmed that TGFβ plays an initiating role in the process of epithelial-mesenchymal transition(EMT)of tumor cells.However,EMT is an important mechanism of tumor invasion and metastasis,and the high invasion and metastasis characteristics of cholangiocarcinoma are closely related to the occurrence of EMT.TGR5 is a bile acid-activated transmembrane G protein-coupled receptor(GPCR),which is widely distributed in multiple organs throughout the body and play an important role in regulating bile acid metabolism,glucose and lipid metabolism,and inflammation in the body.Recent research reports showed that TGR5 may play an important role in the occurrence and development of tumors,but its molecular changes and related mechanisms have not been elucidated,and related research is lacking.The previous research results of our group showed that the expression of TGR5 in cholangiocarcinoma tissue was significantly increased,and its expression was significantly correlated with the clinicopathological grade of cholangiocarcinoma and the prognosis of patients,which suggested that the expression and activation of TGR5 may be involved in the invasion and metastasis of cholangiocarcinoma.Based on the above viewpoints,this topic proposes that bile acids may promote the EMT of cholangiocarcinoma cells through its membrane receptor TGR5,thereby promoting the invasion and migration of cholangiocarcinoma cells.In order to confirm this hypothesis,this project will first observe the effect of TGR5 expression and activation on the invasion and migration ability of cholangiocarcinoma cells,and then analyze the relationship between TGR5 expression and activation and the expression of EMT-related markers in cholangiocarcinoma cells.The role and mechanism of “αvβ6-TGFβ” axis in TGR5 promoting EMT in cholangiocarcinoma cells.This subject provides an important experimental basis for the preliminary elucidation of the mechanism of TGR5 promoting the invasion and metastasis of cholangiocarcinoma cells,and provides new ideas for exploring clinical treatment strategies for cholangiocarcinoma.Part 1: Experimental study on the effect of TGR5 expression on the invasion and migration ability of cholangiocarcinoma cellsObjective: To investigate the effect of TGR5 expression on the invasion and migration ability of cholangiocarcinoma cells.Methods: TGR5 interference lentiviral vector and overexpression plasmid were constructed and transfected into RBE cells.The experimental group was divided into 5 groups,namely blank group(Mock),overexpression control group(Control A),overexpression group(GPBAR1),and silence control group(Control B)and silence group(sh RNA),each group was incubated with 100μm TCA.The changes of cell motility in each group at 24 h and 48 h were evaluated by wound healing experiments.The invasion and migration ability of cells in each group was evaluated by Transwell assay.Results: There was no significant difference in the motility and invasion and metastasis ability of cholangiocarcinoma cells between the blank group,the silent control group and the overexpression control group,and there was no statistical significance(P > 0.05).Compared with the control group,the motility and invasion and migration abilities of the cells in the silence group were decreased(P < 0.05);the motility and invasion and migration abilities of the cells in the overexpression group were enhanced and significantly stronger than those in the silence group(P < 0.05).Conclusion: Changes in the expression level of TGR5 affect the invasive and metastatic ability of cholangiocarcinoma cells.With the increase of the expression level of TGR5 in cholangiocarcinoma cells,the ability of cell invasion and metastasis was gradually enhanced,which suggested that bile acids may promote the invasion and metastasis of cholangiocarcinoma cells through its membrane receptor TGR5.Part 2: Experimental study on the effect of TGR5 expression on EMT markers in cholangiocarcinoma cellsObjective: To clarify the changes in the expression of EMT-related markers in cholangiocarcinoma cells with different TGR5 expression levels.Methods: Groups as above.After each group was incubated with 100μm TCA for 24 h,total cell protein was extracted.The expression levels of EMT-related markers(E-cadherin,CK-19,N-cadherin and Vimentin)in cholangiocarcinoma in each group were detected by western blotting and cellular immunofluorescence.Results: There was no significant difference in the expression levels of EMT-related markers in cholangiocarcinoma cells of blank group,silence control group and overexpression control group(P > 0.05).Compared with the control group,the expression levels of epithelial cell markers(E-cadherin,CK-19)in the silence group were increased,and the expression levels of mesenchymal cell markers(N-cadherin,Vimentin)were decreased(P < 0.05).However,in the overexpression group,the expression levels of epithelial cell markers(Ecadherin,CK-19)were decreased,and the expression levels of mesenchymal cell markers(Ncadherin,vimentin)were increased,and the difference was statistically significant(P < 0.05).Conclusion: Changes in the expression level of TGR5 affected the expression of EMTrelated markers in cholangiocarcinoma cells.The higher the expression level of TGR5,the lower the expression level of epithelial cell markers(E-cadherin,CK-19),and the higher the expression level of mesenchymal cell markers(N-cadherin,Vimentin).It indicated that the change of TGR5 expression level may affect the process of EMT,thereby promoting the invasion and metastasis of cholangiocarcinoma cells.Part 3: Experimental study on the effect of TGR5 expression on the expression of "integrin αvβ6-TGFβ" axisObjective: To preliminarily explore the molecular mechanism of the changes in the expression level of TGR5 promoting EMT in cholangiocarcinoma cells.Methods: Groups as above.After each group was incubated with 100μm TCA for 24 hours,total cell protein and total RNA were extracted,and the expression of αvβ6 and TGFβ1m RNA in each group was detected by RT-qPCR.The expressions of αvβ6 and TGFβ1 in each group were detected by Western blot,and cellular immunofluorescence.Results: There was no significant difference in the expression levels of αvβ6 and TGFβ1in cholangiocarcinoma cells of blank group,silence control group and overexpression control group(P > 0.05).Compared with the control group,the expressions of αvβ6 and TGFβ1 in the silence group decreased(P < 0.05);the expressions of αvβ6 and TGFβ1 in the overexpression group increased(P < 0.05);and the expressions of αvβ6 and TGFβ1 in the silence group and the overexpression group were significantly decreased(P < 0.05).Conclusion: The increase in the expression level of TGR5 promoted the expression and activity of αvβ6 and TGFβ1 in cholangiocarcinoma cells,suggesting that TGR5 may affect the process of cholangiocarcinoma invasion and metastasis by affecting the expression of "integrin αvβ6-TGFβ" axis. |
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