Expression And Mechanism Of EMR2 In Extrahepatic Cholangiocarcinoma

Objective: Cholangiocarcinoma(CCA)is a highly malignant bile duct tumor.In recent years,its incidence has been increasing and its prognosis is poor.Therefore,this project explores the effect of adhesion G protein-coupled receptor E2(adhesion G protein-coupled receptor E2,ADGRE2),also known as EMR2,on the biological behavior of extrahepatic cholangiocarcinoma and its internal mechanism.Methods:(1)query the expression of EMR2 in TCGA database through TIMER website.(2)40 paraffin samples from patients with extrahepatic cholangiocarcinoma were collected,and immunohistochemical experiment was carried out to verify the difference of EMR2 expression in human tissues.COX analysis and Kaplan-Meier analysis were used to observe the effect of EMR2 on the prognosis of patients with extrahepatic cholangiocarcinoma.(3)the difference of EMR2 expression between human normal bile duct epithelial cells and extrahepatic bile duct cells was verified by Western blot and q RT-PCR experiments using HIBEpic,QBC939,KMBC and SKCHA-1 cells.(4)targeting si RNA was used to knock down the expression of EMR2 in QBC939,and pc DNA3.1 plasmid was used to overexpress EMR2 in KMBC cells.Western blotting and fluorescence quantitative PCR were used to verify the efficiency of knock-down and overexpression.(5)after artificially affecting the expression of EMR2 in QBC939 cells and KMBC cells,Western blot experiment was used to explore the effect of EMR2 on the relative protein expression levels of N-cadherin,Vimentin and E-cadherin in the process of EMT.(6)after transfection of si-EMR2,si-NC,oeEMR2 plasmid and pc DNA3.1 plasmid,the effect of EMR2 on the proliferation of extrahepatic cholangiocarcinoma cells was detected by CCCK-8 reagent.(7)scratch test and Transwell migration test were used to detect the changes of migration ability of EMR2 in QBC939 cells and KMBC cells.(8)Transwell invasion test was used to detect the effect of EMR2 on the invasive ability of extrahepatic bile duct cells.Results:(1)TCGA database showed that EMR2 was highly expressed in cholangiocarcinoma.(P < 0.0001)(2)through the quantitative analysis of paraffin sections,it was found that the expression of EMR2 in human extrahepatic cholangiocarcinoma tissues was higher than that in human normal tissues.EMR2 was highly expressed in 70% of extrahepatic cholangiocarcinoma tissues,while only 32.5%in paracancerous tissues(P < 0.01).COX and Kaplan-Meier analysis showed that the expression of EMR2 was related to the degree of tissue differentiation.We demonstrated the correlation between the increased expression of EMR2 and the poor survival rate of patients with extrahepatic cholangiocarcinoma.The degree of pathological differentiation and the level of EMR2 expression in patients with extrahepatic cholangiocarcinoma can independently affect their survival and prognosis.(3)the expression level of EMR2 in extrahepatic cholangiocarcinoma cells QBC939 and SK-CHA-1 was significantly higher than that in normal bile duct epithelial cells HIBEpic.(4)High expression of EMR2 promotes the migratory ability of extrahepatic cholangiocarcinoma cells in scratch assay and Transwell migration assay.(5)in CCK8 experiment,the proliferation ability of QBC939 cells with low EMR2 expression was decreased,while that of KMBC cells with high EMR2 expression was significantly increased.(6)when EMR2 was knocked down,the relative expression of N-cadherin protein was decreased,and the relative expression level of Vimentin protein was also decreased,but the relative expression of E-cadherin protein was significantly increased.Conclusion: The expression of EMR2 is related to the degree of differentiation and survival prognosis of EHCC,and the expression level of EMR2 and the degree of differentiation are factors that independently affect the survival and prognosis of EHCC.The high expression of EMR2 can promote the invasion and migration of EHCC cells,and its mechanism may be related to the transition of epithelial to mesenchymal.