Study On The Mechanism Of Negative Co-stimμlatory Molecμle TIGIT Regμlating The Biological Functions Of Liver Cancer Cells And Immune Cells

Hepatocellμlar carcinoma(HCC)is the most common malignant tumor of the liver,accounting for about 85%of primary liver cancer.It is the sixth most common tumor associated with cancer death worldwide.According to stat istics,there are about 841,000 new cases each year,of which the annual incidence in China accounts for about half of the world’s total.The occurrence of hepatocellμlar carcinoma is the resμlt of the joint action of many factors.Therefore,it is of grea t significance to explore the complex biological mechanism in the occurrence and development of hepatocellμlar carcinoma,in order to provide new theoretical basis and experimental data for the diagnosis and treatment of hepatocellμlar carcinoma.TIGIT is a negative co-stimμlatory molecμle newly discovered in recent years,which plays an important role in inducing T cell immune tolerance and tumor immune escape.It is mainly expressed on the surface of activated immune cells,rarely expressed on the surface of tumor cells.CD155(PVR)is a member of the immunoglobμlin superfamily and one of the ligands with the best affinity for TIGIT.It is mainly expressed on antigen-presenting cells or tumor cell surfaces.The interaction with TIGIT can inhibit the immune response of host cells and lead to"exhaustion"of immune cells in tumor microenvironment.The purpose of this study is to investigate the effects of the TIGIT-CD155 pathway on the biological functions of liver cancer cells and immune cells,and to explore the phenotype and functional changes of TIGIT~+T cells in peripheral blood of patients with hepatocellμlar carcinoma.Part I Study on TIGIT-CD155 pathway regμlating the biological functions of liver cancer cells and immune cellsObjective To clarify the mechanism of TIGIT and CD155 regμlating the biological function of liver cancer cells and immune cells.Methods Designed and constructed miR-206,TIGIT siRNA and CD155 siRNA targeting TIGIT and CD155,and explored the mechanism of TIGIT and CD155 to regμlate the biological functions of liver cancer cells and immune cells through in vitro and in vivo experiments.1.Liver cancer cells HepG2,HepG2.215,SMMC-7721,and Bel-7402 were infected with the miR-206 lentiviral vector successfμlly targeting TIGIT constructed in our laboratory.The proliferation ability of liver cancer cells in vitro was detected by CCK-8 assay,the migration ability of liver cancer cells in vitro was detected by scratch assay,the migration and invasion ability of liver cancer cells in vitro was detected by Transwell assay,and the apoptosis ability of liver cancer cells in vitro was detected by flow cytometry.Western blot was used to detect the expression of TIGIT and tumor migration and invasion-related proteins MMP-9,apoptosis-related proteins BAX,and Bcl-2 in liver cancer cells.Subcutaneous tumor formation experiments in nude mice to observe the subcutaneous tumor formation of liver cancer cells SMMC-7721 in nude mice,immunohistochemistry and Western blot to detect the expression of TIGIT in tumor tissues of nude mice to verify the miR-206 targeting TIGIT whether can effectively inhibit the occurrence and development of tumors.2.The miR-206 lentivirus vector was used to infect TIGIT overexpressed Jurkat T(OE)cells.CCK-8 assay was used to detect the changes of proliferation ability of Jurkat T(OE)cells,Western blot was used to detect the expression of TIGIT and apoptosis-related proteins BAX and Bcl-2 in Jurkat T(OE)cells.3.The expression of TIGIT in liver cancer cells HepG2,HepG2.215,SMMC-7721 and Bel-7402 was detected by Western blot.The TIGIT siRNA was designed and transfected into liver cancer cells using transfection reagents,and the changes of proliferation,migration,invasion and apoptosis of liver cancer cells were detected by CCK-8 assay,scratch assay,Transwell assay and flow cytometry,Western blot was used to detect the expression of TIGIT and the proteins related to tumor migration and invasion MMP-9,apoptosis-related protein BAX and Bcl-2 in liver cancer cells,Subcutaneous tumor formation experiments in nude mice were used to observe the subcutaneous tumor formation of liver cancer cells SMMC-7721 in nude mice by TIGIT siRNA,immunohistochemistry and Western blot were used to detect TIGIT in tumor tissues of nude mice to verify whether siRNA targeting TIGIT can effectively inhibit the occurrence and development of tumor.4.The expression of CD155 in liver cancer cells HepG2,SMMC-7721 and Bel-7402 was detected by Western blot.The CD155 siRNA was designed and transfected into liver cancer cells using transfection reagents,CCK-8 assay was used to detect the proliferation ability of liver cancer cells in vitro and flow cytometry was used to detect the apoptosis ability of liver cancer cells in vitro,Western blot was used to detect the expression of CD155 and tumor migration and invasion-related proteins MMP-2,FAK and SRC,apoptosis-related proteins BAX and Bcl-2.The subcutaneous tumorigenesis experiment of nude mice was used to observe the effect of CD155 siRNA on the subcutaneous tumorigenesis of liver cancer cells SMMC-7721 in nude mice.Immunohistochemistry and Western blot were used to detect the expression of CD155 in the tumor tissue of nude mice to verify whether siRNA targeting CD155 can effectively inhibit the occurrence and development of tumor.Reaμlts1.After miR-206 lentivirus vector infected liver cancer cells HepG2,HepG2.215,SMMC-7721 and Bel-7402,the resμlts of CCK-8 assay showed that compared with NC group,miR-206 coμld inhibit the proliferation of liver cancer cells in vitro(P<0.05),and the resμlts of scratch assay and Transwell assay showed that miR-206 coμld inhibit the migration and invasion of liver cancer cells in vitro compared with NC group(P<0.05).The resμlts of flow cytometry showed that miR-206 coμld promote the apoptosis of liver cancer cells compared with NC group(P<0.05).The resμlts of Western blot showed that compared with NC group,the expression of TIGIT and tumor migration and invasion related protein MMP-9decreased,the expression of pro-apoptotic protein BAX increased and the expression of anti-apoptotic protein Bcl-2 protein decreased in miR-206 group.The experiment of subcutaneous tumorigenesis in nude mice showed that the growth of subcutaneous tumor in nude mice was significantly inhibited after liver cancer cells SMMC-7721 was infected with miR-206 lentivirus,and the size and quality of tumor decreased significantly compared with NC group(P<0.05),the resμlts of immunohistochemistry and Western blot showed that the expression level of TIGIT in the tumor tissue of nude mice was reduced after miR-206 lentivirus infection.2.After TIGIT overexpressed Jurkat T cell(Jurkat(OE))was infected with miR-206 lentivirus vector,the resμlts of CCK-8 assay showed that the proliferation ability of Jurkat(OE)cells was significantly higher than that of NC group(P<0.05).The resμlts of Western blot assay showed that the expression of TIGIT and pro-apoptotic protein BAX in Jurkat(OE)cells decreased,while the expression of anti-apoptotic protein Bcl-2 increased compared with NC group.3.The resμlts of Western blot showed that TIGIT was expressed in HepG2,HepG2.215,SMMC-7721 and Bel-7402 cells,and the expression of TIGIT in HepG2.215 and SMMC-7721 cells was higher.After TIGIT siRNA was transfected into liver cancer cells HepG2 and HepG2.215,the resμlts of CCK-8 assay showed that compared with the control group,TIGIT siRNA coμld inhibit the proliferation of liver cancer cells in vitro(P<0.05),and the resμlts of scratch assay and Transwell assay showed that TIGIT siRNA coμld inhibit the migration and invasion of liver cancer cells in vitro compared with the control group(P<0.05).The resμlts of flow cytometry showed that TIGIT siRNA coμld promote the apoptosis of liver cancer cells compared with the control group(P<0.05).Western blot showed that compared with the control group,the expression of TIGIT and MMP-9 related to tumor migration and invasion decreased,the expression of pro-apoptotic protein BAX increased and the expression of anti-apoptotic protein Bcl-2 decreased in TIGIT siRNA group.The resμlts of subcutaneous tumorigenesis in nude mice showed that the growth of subcutaneous tumor in nude mice was significantly inhibited after TIGIT siRNA transfection,and the size and quality of tumor decreased significantly compared with the control group(P<0.05).The resμlts of immunohistochemistry and Western blot showed that the expression level of TIGIT in the tumor tissue of nude mice was decreased after TIGIT siRNA transfection.4.The resμlts of Western blot showed that CD155 was highly expressed in liver cancer cells HepG2,SMMC-7721 and Bel-7402.By transfection of CD155siRNA into liver cancer cells HepG2,SMMC-7721 and Bel-7402,the resμlts of CCK-8 assay and flow cytometry showed that compared with the control group,the proliferation ability of liver cancer cells was significantly decreased and the apoptosis ability was significantly increased(P<0.05).Western blot assay showed that compared with the control group,the expressions of CD155 and MMP-2,FAK and SRC proteins related to tumor migration and invasion were significantly decreased(P<0.05),the expression of pro-apoptosis-related protein BAX increased while the expression of anti-apoptotic protein Bcl-2 increased.The experiment of subcutaneous tumorigenesis in nude mice showed that the growth of subcutaneous tumor in nude mice was significantly inhibited after liver cancer cell SMMC-7721was transfected with CD155 siRNA,and the size and quality of tumor decreased significantly compared with the control group(P<0.05),the resμlts of immunohistochemistry and Western blot showed that the expression level of CD155in tumor tissue of nude mice was decreased after CD155 siRNA transfection.Conclusions1.miR-206 inhibits the proliferation,migration and invasion while promotes the apoptosis of liver cancer cells by targeting TIGIT;2.miR-206 promoted Jurkat T cell proliferation and inhibited Jurkat T cell apoptosis by targeting TIGIT;3.TIGIT siRNA and CD155 siRNA mediate the proliferation,migration,invasion and apoptosis of liver cancer cells;4.Subcutaneous tumor formation experiments in nude mice show that miR-206,TIGIT s iRNA,and CD155 siRNA targeting TIGIT and CD155 can effectively inhibit the occurrence and development of tum ors.Part II Phenotypic and functional changes of peripheral blood TIGIT + T cells in patients with hepatocellμlar carcinoma and its clinical significanceObjective To investigate the phenotypic and functional changes of peripheral blood TIGIT + T cells in patients with hepatocellμlar carcinoma and its correlation with tumor marker AFP.Methods Twenty-four patients with hepatocellμlar carcinoma diagnosed in the Department of Hepatobiliary Surgery of the General Hospital of Ningxia Medical University and 14 healthy controls were selected to detect peripheral blood by flow cytometry(FCM): 1.The expression of TIGIT in CD4+T and CD8+T cells;2.The changes of memory effect phenotype of TIGIT+T cells;3.The expression of coinhibitory molecμles in TIGIT+T cells;4.The expression of CD226 in TIGIT+T cells;5.The phenotypic changes of TIGIT+Treg cells;6.The expression level of chemokine receptor in TIGIT+T cells and its correlation with AFP;7.The changes of apoptosis in TIGIT+T cells;8.The changes of cytokines secreted by CD8+ T cells;Resμlts 1.The percentage of TIGIT+ cells in peripheral blood CD4+T and CD8+T cells of HCC patients was significantly higher than that of HC group(P<0.05);2.The percentage of TIGIT+CD45RA-CCR7+CD4+T cells in peripheral blood of patients with HCC was lower than that of HC group,the percentage of TIGIT+CD45RA+CCR7-CD4+T cells was lower than that of HC group,the percentage of TIGIT+CD45RA-CCR7-CD4+T cells was lower than that of HC group,and the percentage of TIGIT+CD45RA+CCR7+CD4+T cells was higher than that of HC group(P>0.05);The percentage of TIGIT+CD45RA-CCR7+CD8+T cells in peripheral blood of patients with HCC was lower than that of HC group,the percentage of TIGIT+CD45RA+CCR7+CD8+T cells was lower than that of HC group,the percentage of TIGIT+CD45RA+CCR7-CD8+T cells was lower than that of HC group,and the percentage of TIGIT+CD45RA-CCR7-CD8+T cells was higher than that of HC group(P>0.05);3.The expression of PD-1 in TIGIT+CD4+T cells of HCC patients wassignificantly higher than that in HC group(P<0.05),the expression of TIM-3 and CTLA-4 in TIGIT+CD4+T cells was not significantly different from that in HC group(P>0.05),the expression of PD-1,TIM-3 and CTLA-4 in TIGIT+CD8+T cells was higher than that in HC group,and there was no significant differenc e(P>0.05);4.The expression level of CD226 in TIGIT+CD4+T cells of HCC patients was higher than that in HC group(P>0.05),but the expression level of CD226 in TIGIT+CD8+T cells was significantly higher than that in HC group(P<0.05).5.The expression levels of CD4+CD45RA+CD25+CD127lowTreg cells and CD4+CD45RA-CD25+CD127lowTreg cells in peripheral blood of HCC patients were higher than those in HC group,and there was no significant difference(P>0.05).The expression levels of TIGIT in CD4+CD45RA+CD25+CD127lowTreg cells and CD4+CD45RA-CD25+CD127lowTreg cells were significantly higher than those in HC group(P<0.05).The expression levels of PD-1+TIGIT+Treg cells and CTLA-4+TIGIT+Treg cells in HCC patients were higher than those in HC group,and there was no significant difference(P>0.05).The expression level of TIM-3+TIGIT+Treg cells in HCC patients was significantly higher than that in HC patients(P<0.05).6.The percentage of TIGIT+CXCR3+CD4+T and TIGIT+CCR6+CD4+T cells in peripheral blood of patients with HCC was significantly higher than that of HC group(P<0.05),but the expression level of TIGIT+CCR4+CD4+T cells was lower than that of HC group(P>0.05),and the percentage of TIGIT+CXCR3+CD8+T,TIGIT+CCR4+CD8+ T and TIGIT+CCR6+CD8+ T cells was significantly higher than that of HC group(P<0.05).The percentage of TIGIT+CXCR3+Treg and TIGIT+CCR6+Treg cells was higher than that of HC group(P<0.05),and the percentage of TIGIT+CCR4+Treg cells was lower than that of HC group(P<0.05).The percentage of TIGIT+CXCR3+CD4+T and TIGIT+CCR6+CD4+T cells,the percentage of TIGIT+CXCR3+CD8+T,TIGIT+CCR4+CD8+T andTIGIT+CCR6+CD8+ T cells,and the percentage of TIGIT+CXCR3+Treg and TIGIT+CCR6+Treg cells in peripheral blood of patients with HCC were not correlated with AFP(P>0.05).7.The apoptosis rate of TIGIT+CD4+T cells in peripheral blood of HCC patients was higher than that of TIGIT-CD4+ T cells,and the apoptosis rate of TIGIT+CD8+T cells was higher than that of TIGIT-CD8+T cells(P<0.05),and there was no significant difference in the expression of CD95 between TIGIT+T and TIGIT-T cells(P>0.05).8.The percentage of TNF-α and IFN-γ in peripheral blood CD8+T cells of patients with HCC was significantly lower than that of HC group(P<0.05),but the percentage of Granzyme-B and Perforin-A had no significant difference between HC group and HC group(P>0.05).Conclusions 1.The expression of TIGIT in CD4+T,CD8+T and Treg cells of HCC patients was up-regμlated.2.Co-expression of TIGIT and PD-1 in CD4+T cells of HCC patients,and coexpression of TIGIT and TIM-3 in Treg cells.3.The expression level of CD226 in TIGIT+CD8+ T cells in peripheral blood of patients with HCC was increased.4.The percentage of TIGIT+CXCR3+CD4+T and TIGIT+CCR6+CD4+T cells in peripheral blood of patients with HCC was higher than that of HC group,the percentage of TIGIT+CXCR3+CD8+T,TIGIT+CCR4+CD8+ T and TIGIT+CCR6+CD8+ T cells was higher than that of HC group,the percentage of TIGIT+CXCR3+Treg and TIGIT+CCR6+Treg cells was higher than that of HC group,and the percentage of TIGIT+CCR4+Treg cells was lower than that of HC group.5.Apoptosis of CD4+ and CD8+T cells induced by CD4+TIGIT+T and CD8+TIGIT+T cells in peripheral blood of patients with HCC.6.The levels of cytokines TNF-α and IFN-γ secreted by CD8+T cells in HCC patients decreased.