Downregulation Of 67kDa Laminin Receptor On MICA/B Induced Bile Duct Cancer Cells Escaping From NK Cells

Aim : To investigate the expression of MICA/B down regulated by 67 LR in cholangiocarcinoma,which leads to cholangiocarcinoma cells escaping from the surveillance of NK cells.Method: IHC staining of 67 LR,MICA and MICB was performed on paraffin embedded specimens from 101 patients with bile duct carcinoma,detecting the expression of 67 LR and MICA/B in cholangiocarcinoma.Sh RNA and overexpressed lentiviral vector of MICA/B was transfected into cells in bile duct cancer cells to interfere with the expression of MICA/B,detecting the killing rate of NK cells against cholangiocarcinoma cells.Sh RNA and overexpressed lentiviral vector of 67 LR was transfected into cells in bile duct cancer cells,the expression of MICA/B and the killing rate of NK cells against cholangiocarcinoma cells were detected.The expression of ADAM9 was detected by interfering with the expression of 67 LR in cholangiocarcinoma cells,and the content of sMICA/B in the culture supernatant was determined.Main results:1.IHC and correlation analysis of clinical features of cholangiocarcinoma1.1 Immunohistochemical results showed that 67 LR was high expression in 27.7%(28/101)bile duct cancer.The high expression of 67 LR was closely related to the histological grade(P=0.010),the T classification(P=0.017),the lymph node metastasis(P=0.000),as well as TNM staging(P=0.042),suggesting that the high expression of 67 LR was significantly correlated with tumor invasion and metastasis.1.2 Kaplan-Meier analysis showed that the expression level of 67 LR was significantly correlated with overall survival time(P=0.0001)and metastasis-free survival time(P=0.0002).1.3 Immunohistochemical results showed that MICA was low expression in 39.6%(40/101)bile duct cancer,while MICB was low expression in 54.5%(55/101)bile duct cancer.The low expression of MICA and MICB was closely related to the histological grade(P=0.020,P=0.020),the T classification(P=0.000,P=0.042),the lymph node metastasis(P=0.001,P=0.002),as well as TNM staging(P=0.000,P=0.001).1.4 Kaplan-Meier analysis showed that the expression level of MICA/B was significantly correlated with overall survival time(P=0.0001,P=0.0001)and metastasis-free survival time(P=0.0001,P=0.0002)1.5.Immunohistochemical results showed that the expression of 67 LR was negatively correlated with the expression of MICA/B(r=-0.419,P=0.000;r=-0.443,P=0.000).2.Through RT-PCR,WB,FCM,ELASIA,NK cytotoxicity test to explore the mechanism of bile duct cancer cells escaping NK cells2.1Bile duct cancer cells co-culture with NK cells in vitro,over-expression of MICA/B can enhance the killing effect of NK cells on cholangiocarcinoma and interfer ring the expression of MICA/B can weaken the killing effect of NK cells in cholangiocarcinoma.2.2 Bile duct cancer cells co-culture with NK cells in vitro,over-expression of 67 LR can weaken the killing effect of NK cells on cholangiocarcinoma and interfer ring the expression of 67 LR can enhance the killing effect of NK cells in cholangiocarcinoma.2.3 In vitro,we found that 67 LR can down regulate the expression of MICA/B in mRNA level and protein level,so as to achieve escaping from NK cells.In the model of orthotopic liver transplantation in mice,it was found that the diameter of ex-67 LR group(1.36 + 0.23cm)was significantly higher than that of ex-CONTROL group(0.5 + 0.08cm),and liver metastasis.2.4 The expression of ADAM9 was up-regulated by 67 LR in cholangiocarcinoma cells.67 LR can down regulate the expression of MICA/B by ADAM9 shearing membrane molecule MICA/B.Conclusion: caused by 67 LR cholangiocarcinoma can escape the monitoring of NK cells by down regulating the expression of MICA/B,thereby promoting the survival and early distant metastasis of cancer cells.