The Role And Mechanism Of HBx Induced PD-L1 Expression In Hepatocellular Carcinoma

Background Primary hepatocellular carcinoma is one of the most common malignant tumors in the worldwide,and ranks second for the tumor related mortality in our country.Surgical resection,liver transplantation,radiotherapy,chemotherapy and palliative care may make patients recover,but their effect is limited.In recent years,the rapid development of immunotherapy has seen it become one of the most promising methods of tumor therapy after surgery,radiotherapy and chemotherapy.Therefore,it is of great significance to carry out further research on immunotherapy of liver cancer.Programmed death ligand 1(PD-L1),as an immune detection point,was up-regulated in a great many tumor cells.So PD-L1 can inhibit the immune activity of T cells and induce immune escape.The expression of PD-L1 is regulated by a variety of mechanisms,some of which have been found to be associated with viral infection.But the specific mechanism is still unclear.Hepatitis B virus(HBV)infection is one of the most common causes,and HBx that is X gene expression product of HBV plays a key role in the occurrence and development of liver cancer.Objects This study aimed to elucidate the role of HBx and PD-L1 in hepatocellular carcinoma(HCC),to explore the effect of HBx on PD-L1 expression in HCC,to study the potential mechanism,and to investigate the clinical significance of HBx protein in HCC patients.Methods Immunofluorescence staining assay was used to detect the HBx and PD-L1 expression levels and localization in SMMC7721(HBV-)and PLC/PRF/5(HBV+)cells.Expression of HBx and PD-L1 m RNA revealed by real-time PCR in SMMC7721 and PLC/PRF/5 cell lines.Expression of HBx and PD-L1 protein in SMMC7721 and PLC/PRF/5 cells,demonstrated by western blot.The expression of HBx and PD-L1 gene by correlation curves at the m RNA and protein level.HBx gene and empty plasmid were transfected into SMMC7721 cells,and the changes of HBx and PD-L1 in m RNA and protein level were detected by real-time PCR and western blot.si RNA silencing technique was applied to PLC/PRF/5 cells and the expression of HBx and PD-L1 in m RNA and protein levels were detected by real-time PCR and western blot.Expression of NF-κB,p-NF-κB,JAK2,p-JAK2,STAT3 and p-STAT3 in various cells was shown by western blotting.The ability of cell migration and invasion was confirmed by Transwell.PD-L1 expression was tested by immunohistochemistry in HCC samples.Results HBx up-regulates the expression of PD-L1 in SMMC7721 cell line,and both are positively correlated.Inhibition of HBx,the expression of PD-L1 is decreased in PLC/PRF/5 cell line.The expression of p-JAK2,p-STAT3 and p-NF-κB in the pathways are varied with the change in HBx,but JAK2,STAT3 and NF-κB are unchanged when HBx was transfected or silenced.Cell migration and invasion is significantly enhanced in SMMC7721 cells after transfection with HBx,but the ability is decreased after silence with HBx in PLC/PRF/5 cells.Expression of PD-L1 in HBV-related HCC cases is higher than that in cases without HBV infection.Conclusion HBx could induce the expression of PD-L1 in HCC cells,while silencing the gene could decrease the expression of PD-L1.HBx may mediate the expression of PD-L1 in HCC cells by activating some key factors in NF-κB and JAK/STAT signaling pathways.The migration and invasion of HCC cells is enhanced after HBx transfection,suggesting that the prevention of HBV infection may reduce the migration and invasion of HCC cells.The prevention of HBV infection as well as blocking the PD-1/PD-L1 pathway are of great value for the clinical immunotherapy of HCC.