Regulation Of Hepatic Sinusoidal Endothelial Cell Adhesion By Cholangiocarcinoma Exosomes Via NF-κ B Signaling Pathway

Background:Cholangiocarcinoma(CCA)is a cancer characterized by cholangiocyte differentiation,which is the most common biliary malignant tumor and can be divided into intrahepatic cholangiocarcinoma,hilar cholangiocarcinoma and distal cholangiocarcinoma,of which hilar cholangiocarcinoma is the most common.CCA is prone to liver,other organ and lymph node metastasis through blood vessels and lymph nodes,and its high fibrinolysis and rich tumor microenvironment around the cancer are helpful for chemoresistance of cholangiocarcinoma.Although the 5-year survival rate of patients after surgery is very low,surgery is still the current treatment of choice for CCA.CCA development is inseparable from changes in oncogenes,signaling pathways and cancer-related cells.Exosomes are small vesicles produced by cells,40-160 nm in diameter,with a bilayer lipid membrane structure.Most cells have now been demonstrated to synthesize and secrete exosomes.Exosomes can carry bioactive substances such as nucleic acids and proteins,therefore,exosomes have become an important way of communication between tumor cells,between tumor cells and other types of cells,and between tumor cells and the tumor microenvironment.Exosomes play a crucial role in the process of tumor growth as well as tumor invasion and metastasis.Hepatic Sinusoidal Endothelial Cell(HSEC)is a highly specific endothelial cell,one side of which is blood cells,the other side is hepatocytes and HSCs.HSECs are both the most permeable endothelial cells in vivo and the cells with the highest endocytic capacity.Under specific conditions,the protective effect of HSEC on the liver will fail and further promote the generation of nutrient vessels in tumor tissue.Objective:1.To investigate the regulation of CCA exosomes on HSEC adhesion function.2.To investigate whether the NF-κB signaling pathway is involved in the above process.Methods:The expression of ICAM-1,VCAM-1 and E-selectin in liver tissue was detected by immunohistochemistry in cholangiocarcinoma specimens from January 2014 to June 2019,and the relationship between these factors and the survival of CCA patients was analyzed.Cholangiocarcinoma QBC939 cell line was cultured,and exosomes were isolated and identified by centrifugation.The shape of the exosomes of QBC939 cells was observed by transmission electron microscopy,and the exosome-specific markers CD9 and TSG101 were analyzed by Western-blot.QBC939 exosomes were labeled using PKH26,and HSEC uptake of exosomes was visualized by confocal microscopy.HSEC culture was used to form monolayer cells added to QBC939 exosomes,and cholangiocarcinoma QBC939 cells were fluorescently labeled and cocultured to analyze the effect of QBC939 exosomes on the ability of HSEC to adhere to QBC939 cells by flow cytometry.QBC939 exosomes were co-cultured with HSEC,and the expression of ICAM-1,VCAM-1,and E-selectin by HSEC was detected by Western-blot and q RT-PCR;the expression of NF-κB subunits p65 and I-κB by HSEC was analyzed by Western blot.Si RNA inhibited NF-κB pathway p65 expression in HSEC,and the expression of ICAM-1,VCAM-1,and E-selectin was detected by Western-blot and q RT-PCR;the ability of HSEC to adhere to QBC939 cells was analyzed by flow cytometry.Results:1.Immunohistochemical detection of ICAM-1,VCAM-1 and E-Selectin expression in cholangiocarcinoma adjacent liver tissue revealed that VCAM-1 and E-Selectin were highly expressed in cholangiocarcinoma adjacent liver tissue with low differentiation and liver metastasis;the mean survival time of cholangiocarcinoma patients with high VCAM-1 and E-Selectin expression in cholangiocarcinoma adjacent liver tissue(6.6 ±2.13 months,6.8 ± 1.73 months)was significantly lower than that of cholangiocarcinoma patients with low VCAM-1 and E-Selectin expression(35.3 ± 6.92 months,39.9 ± 7.67 months).2.HSEC co-cultured with cholangiocarcinoma QBC939 exosomes enhanced QBC939 cell adhesion;meanwhile,HSEC expressed ICAM-1,VCAM-1,and E-selectin with enhanced effect;and the expression of HSECp65 was enhanced.3.After inhibiting NF-κB signaling pathway,the enhancement effect of cholangiocarcinoma QBC939 exosome on HSEC adhesion function was weakened;the enhancement effect of HSEC expression of ICAM-1,VCAM-1,and E-selectin was weakened.Conclusion:1.VCAM-1 and E-Selectin are highly expressed in patients with low differentiation and liver metastasis of cholangiocarcinoma,and high expression of VCAM-1 and E-Selectin is a risk factor for shortened survival.2.cholangiocarcinoma exosomes can enhance the adhesive function of HSECs.3.Cholangiocarcinoma exosomes can enhance ICAM-1,VCAM-1,and E-selectin expression by HSEC.4.cholangiocarcinoma exosomes can regulate HSEC adhesion function through the NF-κB signaling pathway.