| Objective:To investigate the association between the expression of bile acid membrane receptor TGR5 in extrahepatic cholangiocarcinoma and the prognosis of extrahepatic cholangiocarcinoma and epithelial-tomesenchymal transition(EMT).And we analyze the relationship between bile acid membrane receptor TGR5 which is under the action of bile acid and the morphological changes of cholangiocarcinoma cells and the EMT markers.Methods:We retrospectively analyzed the data of 88 patients with extrahepatic cholangiocarcinoma who admitted to the General Surgery Center of the Western Theater General Hospital from January 2015 to January 2020.We collected the pathological tissues of cholangiocarcinoma,analyzed the difference of TGR5 expression between extrahepatic cholangiocarcinoma and paracancerous tissues,as well as the relationship with clinicopathological features and clinical prognosis.Secondly,lentivirus RNA interference was used to reduce the expression level of TGR5 in human cholangiocarcinoma cell QBC-939.In addition,we used TGR5 agonist deoxycholic acid(DCA)to stimulate cells and observed the morphological changes.And we used the Western blotting(WB)to detect the expression level of EMT-related marker.Results:1)Comparison of relative expression level of TGR5 between extrahepatic cholangiocarcinoma and paracancerous tissues:Compared with the paracancerous tissues,the relative gray value of TGR5 in extrahepatic cholangiocarcinoma tissues decreased significantly,and the relative expression level of TGR5 in extrahepatic cholangiocarcinoma tissues was higher than that in paracancerous tissues(P<0.001).2)Clinical and pathological characteristics of patients with extrahepatic cholangiocarcinoma:Gender:55 males 55(62.5%)and 33 females(37.5%);Age:the median age was 65 years old,with 38 cases(43.2%)above 65 years old and 50 cases(56.8%)below 65 years old;Pathological differentiation:22 cases(25%)were poorly differentiated,53 cases(60.2%)were moderately differentiated,and 13 cases(14.8%)were highly differentiated.Nerve involvement:62 cases(70.5%)had nerve involvement and 26 cases(29.5%)had no nerve involvement.Vascular involvement:there were 58 cases(65.9%)with vascular involvement and 30 cases(34.1%)without vascular involvement.Lymph node metastasis:there were 24 cases(27.3%)with lymph node metastasis,46 cases(52.3%)without lymph node metastasis,and 18 cases(20.5%)could not be evaluated for lymph node metastasis.TNM staging:8 cases(11.4%)in stage I,36 cases(51.4%)in stage Ⅱ,25 cases(35.7%)in stage Ⅲ and 1 case(1.4%)in stage Ⅳ.3)Correlation analysis between the relative expression level of TGR5 and clinicopathological features of extrahepatic cholangiocarcinoma:The relative expression level of TGR5 was negatively correlated with the degree of pathological differentiation(r=-0.304,P<0.01).However,there was no significant correlation with gender,age,nerve involvement,vascular involvement,lymph node metastasis and TNM stage(P>0.05).4)Correlation analysis between relative expression level of TGR5 of extrahepatic cholangiocarcinoma and prognosis:The tumor-free survival time(20.45±12.75)and total survival time(23.32±12.48)of patients with high expression level of TGR5 group were significantly lower than those with very low expression level of TGR5 group(tumor-free survival time:29.00±11.65,total survival time:32.95±12.35)and low expression level of TGR5 group(tumor-free survival time:30.87±8.50,total survival time:34.61±9.41)(P<0.05).Furthermore,the tumor-free survival time(24.48±10.34)and total survival time(27.62±1.04)of patients with medium expression level of TGR5 group were also significantly lower than those with low expression level of TGR5 group(tumor-free survival time:30.87±8.50,total survival time:34.61±9.41)(P<0.05).In addition,the relative expression level of TGR5 was negatively correlated with the prognosis of patients(tumor-free survival time b=-0.230,P<0.01;total survival time b=-0.244,P<0.01).5)Correlation analysis between TGR5 relative expression level of extrahepatic cholangiocarcinoma and EMT marker proteins expression level:The expression level of TGR5 in extrahepatic cholangiocarcinoma was negatively correlated with E-cadherin and CK19 expression level(E-cadherin rs=-0.780,P<0.001;CK19 rs=-0.795,P<0.001).In addition,the expression level of TGR5 in extrahepatic cholangiocarcinoma was positively correlated with the expression level of vimentin and N-cadherin(vimentin rs=0.816,P<0.001;N-cadherin rs=0.820,P<0.001).6)Compared with the control group,the morphological changes of cholangiocarcinoma cell QBC-939 in deoxycholic acid-induced group are mainly characterized by cell dispersion and more fibroblast morphology.In addition,after inhibiting the expression of TGR5 in the interference group,the above changes were not significant.The expression level of E-cadherin in deoxycholic acid induced group was significantly lower than that in control group and TGR5 interference group(P<0.05),but there was no significant difference between control group and TGR5 interference group(P>0.05).In addition,the expression of N-cadherin in deoxycholic acid induced group was significantly higher than that in control group and TGR5 interference group(P<0.05),but there was no significant difference between control group and TGR5 interference group(P>0.05).Conclusion:1)The expression level of TGR5 in extrahepatic cholangiocarcinoma tissues is higher than that in the corresponding paracancerous tissues,which suggested that the high expression level of TGR5 may be associated with the increased risk of extrahepatic cholangiocarcinoma.2)In extrahepatic cholangiocarcinoma,the higher the expression level of TGR5,the lower the degree of tumor differentiation.In addition,the higher the expression level of TGR5,the worse the clinical prognosis of patients with extrahepatic cholangiocarcinoma.Therefore,the relative expression level of TGR5 has the potential to warn the malignant degree of extrahepatic cholangiocarcinoma.3)The expression level of TGR5 in extrahepatic cholangiocarcinoma is related to the expression of EMT specific proteins,which indicates that TGR5 may be involved in the EMT process of cholangiocarcinoma.4)High expression of TGR5 induced by deoxycholic acid can promote the expression of EMT specific proteins in cholangiocarcinoma cells.In addition,the high expression of TGR5 induced by deoxycholic acid can promote the morphological changes of cholangiocarcinoma cells.It is suggested that bile acid may promote EMT process of cholangiocarcinoma cells by mediating TGR5,and then may promote invasion and migration of cholangiocarcinoma cells.To sum up,the expression level of TGR5 is related to the prognosis of cholangiocarcinoma,and it can be used as a potential biomarker to evaluate and predict extrahepatic cholangiocarcinoma.Bile acid may promote EMT in cholangiocarcinoma cells and may promote tumor invasion and metastasis by mediating TGR5. |
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