Molecular Mechanism Study Of DcR3 In Immune Escape Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the most common tumors in China,accounting for the second leading cause of cancer death.At present,the molecular mechanism of hepatocarcinogenesis and development is still not well understood,especially the molecular mechanism of how to evade the surveillance of the body’s immune system,and how to induce the body’s immune dysfunction and lead to the body’s immune suppression needs to be further studied.DcR3(decoy receptor 3,also known as TNFRSF6B/TR6),is a new tumor necrosis factor receptor found in recent years.The coding gene is located at 20q13.3,and the mRNA length is 1.2kb.DcR3 is composed of271 amino acid residues,and the amino acid sequence lacks the transmembrane domain,which is a secretory cytokine.The results show that DcR3 is highly expressed in most cancer tissues and can induce immune escape in many ways.Therefore,understanding the relationship between liver cancer and immune system,and explaining the mechanism of immune escape of liver cancer are of great significance for the study of the occurrence and development of liver cancer and further anti-tumor immunotherapy.ObjectivesObjective to detect the expression of DcR3 in tumor tissues,normal tissues and T lymphocytes of hepatocellular carcinoma,to find the subtypes of T cells that DcR3 binds to,and to explore the signal pathway that DcR3 regulates T cells.In order to provide new targets and new ideas for immunotherapy of hepatocelluar carcinoma.Methods1.Immunohistochemical staining(IHC)was used to detect the expression of DcR3 in tumor tissues and paracancerous stroma of different hepatocellullar carcinoma patients,and to analyze the difference between cancer tissues and normal tissues,as well as the relationship between the expression level of DcR3 and clinical parameters such as gender,age,tumor size,metastasis and so on.2.Western blotting(WB)was used to detect the expression of DcR3 in hepatoma cell lines and NF-kB signaling pathway.3.Cell counting kit-8(CCK-8)standard curve was drawn;Transwell experiment was used to observe the effect of DcR3 secreted by hepatoma cells on T lymphocyte migration.4.The interaction proteins of DcR3 were analyzed by immunoprecipitation.5.Immunofluorescence(IF)assay was used to detect the location of DcR3 in T lymphocytes.Results1.The expression of DcR3 in liver cancer tissue: immunohistochemical staining showed that the cytoplasm of liver cancer cells was positive,and the expression of DcR3 was statistically different between liver cancer tissue and adjacent normal tissue(P < 0.05).2.WB test was used to detect the expression of DcR3 in T lymphocytes: after stimulation by DcR3 factor,the expression of DcR3 in T lymphocytes was the best at the condition of 0.15ug/ml and 48 h.3.Transwell experiment results: the number of T lymphocyte migration induced by DcR3 factor and hepatoma cells was more than that induced by control group and DcR3 antibody,Statistical analysis showed differences(P < 0.05).4.IP experiment results: DcR3 secreted by hepatoma cells can pull down light protein.5.IF results: DcR3 and light could bind to the surface of T lymphocytes.6.WB assay was used to detect the important proteins in NF-kB signaling pathway.Results: compared with the control group(NC),the expression level of p65 was lower and the expression level of p-p65 was higher in DcR3 stimulation group(P<0.05).Compared with the control group,the expression of p65 and p-p65 in the co-culture group of HepG-2,Huh-7 and T lymphocytes was higher(P65:HepG-2:P<0.05、Huh-7:P<0.01;P-P65:HepG-2:P<0.01、Huh-7:P<0.05).Compared with DcR3 factor stimulation group,the co-culture group of liver cancer cells HepG-2,Huh-7 and T lymphocytes showed higher expression of P65(HepG-2: P<0.05,huh-7:P<0.01),but the expression of P-P65 was not statistically significant(P>0.05).Compared with DcR3 AB group,the expression of p65 and p-p65 in HepG-2,Huh-7 and T lymphocyte co-culture group was higher(P65:HepG-2:P<0.05、Huh-7:P<0.01;P-P65:HepG-2:P<0.01、Huh-7:P<0.01).Conclusions1.DcR3 is highly expressed in hepatocellular carcinoma tissues.DcR3 secreted by hepatocellular carcinoma cells can enhance the migration ability of T lymphocytes and activate NF-kB signaling pathway.2.DcR3 can interact with LIGHT,and both of them can bind to the surface of T lymphocytes.