Co-inhibitory Molecule TIGIT Expression And Regulation In Human Hepatocellular Carcinoma And Its Clinical Significance

Objectives(1)To investigate the expression and clinical significance of TIGIT in tissues and peripheral blood of patients with primary liver cancer;(2)In order to further study the mechanism of regulating the function of TIGIT,the Tigit gene expression plasmid PMIR-TIGIT-WT/mut was constructed,and the TIGIT high expression Jurkat T cell model was established to explore the mechanism and clinical significance of TIGIT related miRNAs in T cells.Methods(1)Immunohistochemical technique(IHC)and real-time fluorescence quantitative PCR(RT-q PCR)were used to detect the expression of Tigit,CD4 and CD155 in 77 cases of primary liver cancer(73 cases of cancer tissue and 47 cases of para cancerous tissue).The correlation between them and clinicopathological characteristics was analyzed by statistical methods.(2)Flow cytometry was used to detect the expression of TIGIT on the surface of peripheral blood mononuclear cells(mainly CD4+T,Treg cells)in 31 patients with HCC before and after operation,and to analyze its clinical significance.(3)Liquid chip technology was used to detect the clinical significance of inflammatory cytokines in patients with primary liver cancer.(4)According to the nucleic acid sequence of TIGIT,miRBase and Targetscan software were used to predict the TIGIT of miRNAs.(5)Q-PCR detected the expression level of TIGIT related miRNAs(miR-206,miR-143,miR-1,miR-203,mir-425 and miR-206)in cancer and adjacent tissues of HCC patients.(6)MiRNAs related to TIGIT expression was screened,and TIGIT3 '-UTR wild type and mutant recombinance were constructed according to the target of its combination,and the targeting effect of related miRNAs and TIGIT was detected by double luciferase reporter system.(7)The acute T cell leukemia cell line(Jurkat T cells)was selected to construct a stable TIGIT overexpressing Jurkat T cell line.(8)The micro RNA-up lentivirus vector was transfected into Jurkat-TIGIT overexpressing cells,and the TIGIT m RNA and protein levels were detected by q PCR and W-B.Results(1)TIGIT protein expression was localized in lymphocytes in the interstitial infiltrate of liver cancer.The positive sites were cytoplasm and cell membrane.The morphological features of these cells are small and round,with small nuclear and large pulps distributed in spots;TIGIT has no or rare expression in adjacent tissues,and infiltrating lymphocytes in cancer tissues and cancer tissues are the main parts of their expression.The positive expression rate was 94%(69/73).TIGIT-positive cells were mostly distributed around the portal area and blood vessels,and were also extensively infiltrated in the hepatic cord space.Through statistical analysis of the average optical density(IOD)and the cumulative positive area value(AREA),with the pathological stage of tumor cells from high to low,TIGIT gradually increased in the liver cancer tissue and participated in the regulation of tumor infiltrating lymphocyte immune function.Mediate the immune escape mechanism of tumor cells;The CD155 protein expression is mainly localized in hepatocellular carcinoma cells(the cancer cells are polygonal,with abundant cytoplasm,large nuclei and obvious nucleoli,and those with better differentiation can see bile granules in the cytoplasm.The cancer cells often line up in nests or cords,and cancer nests.There are abundant sinusoidal cell membranes,sometimes with darker cytoplasm.The positive staining was light yellow to tan,and the nuclei were not stained;the positive expression rate of CD155 was 89%(65/73).The statistical analysis was performed by IOD and AREA.The expression of CD155 was high to low with the pathological stage of tumor cells.The expression level was highest in the differentiated hepatoma cells in high school,but decreased in the poorly differentiated hepatoma cells and increased significantly in the normal hepatocellular carcinoma.CD155 is a ligand of TIGIT,and its expression increases in tumor cells.By binding with TIGIT,it inhibits the activation and proliferation of immune cells and induces apoptosis of immune cells,which mediates immune escape of tumor cells.(2)When the relative expression levels of TIGIT and CD155 m RNA in paracancerous tissue were 1,the relative expression levels of Tigit and CD155 m RNA in hepatocellular carcinoma were 2 and 3,which was significantly higher than that in the paracancerous tissue.The results showed that the change pattern of TIGIT in different pathological stages was from the low differentiation stage to the middle differentiation stage,and gradually decreased from the middle differentiation stage to the high differentiation stage.The expression of CD155 m RNA had no obvious change trend in all pathological stages.(3)Compared with healthy controls,the expression of TIGIT on the surface of CD4+ T lymphocytes in patients with HCC was significantly higher than that in healthy controls(P = 0.001),but it was decreased after surgery,but there was still a significant difference compared with healthy controls(P = 0.004).Tigit expression on the surface of preoperative Treg cells was up-regulated(P = 0.02),postoperative expression decreased,and there was no statistical difference compared with healthy controls.Preoperative TIGIT+CD4+ T cell frequency was positively correlated with AFP(r2=0.327,P=0.0015).Preoperative TIGIT+Treg cell frequency was positively correlated with AFP(r2=0.592,P=0.001).The results suggest that the upregulation of TIGIT expressed on the surface of CD4+ T cells and Treg cells in peripheral blood of HCC patients may be a potential biomarker for early diagnosis and disease assessment of HCC.(4)Liquid-phase chip technology for the detection of plasma inflammatory cytokines in patients with HCC before and after surgery,compared with the secretion of inflammatory cytokines in patients with HCC before surgery,pro-inflammatory cytokines in the plasma of postoperative HCC patients IL-17 a expression was elevated(P=0.047),IL-6 expression was elevated(P=0.043),and anti-inflammatory cytokine IL-10 expression was decreased(P=0.034).The results suggest that the disorder of inflammatory cytokines in plasma participates in the occurrence and development of human primary liver cancer.(5)The potential target relationship between TIGIT3'-UTR region and miR-206,miR-143,miR-203,miR-1,and miR-425 was predicted through a bioinformatics website,and TIGIT-related miRNAs(mir-206)were detected by q-PCR.The expression levels of mir-143,mir-1,mir-203,mir-425,and mir-206 in cancer and adjacent tissues of HCC patients.The results showed that miR-206 and miR-143 were expressed in cancer tissues of HCC patients.The levels were significantly lower than those in adjacent tissues and were negatively correlated with the frequency of TIGIT+ Treg cells.It is suggested that miR-206 and miR-143 can regulate TIGIT expression on Treg cells and mediate tumor immune escape.(6)The TIGIT recombinant plasmid targeting miR-206 was successfully constructed.The results of dual luciferase assay showed that overexpression of miR-206 could inhibit the expression of TIGIT recombinant vector luciferase,and inhibiting the expression of miR-206 could make TIGIT recombinant vector fluorescein Increased enzyme expression.The experimental results confirm the existence of a targeted relationship between miR-206 and TIGIT.(7)TIGIT overexpressing Jurkat T cell stably strain was successfully constructed and infected with micro RNA-206-up lentiviral vector.The TIGIT m RNA and protein levels were detected by q PCR and WB.Compared with the negative control group,the TIGIT m RNA was detected in the micro RNA-206-up lentiviral vector infection group.Protein levels decreased.It is suggested that mir-206 can negatively regulate the expression of TIGIT;Conclusion(1)The expression levels of Tigit and CD155 m RNA and protein in cancer tissue were significantly higher than those in paracancerous tissues.With the pathological stage of tumor cells from high to low,the expression of TIGIT in hepatocellular carcinoma gradually increased;the CD4+ T cells in peripheral blood of patients with HCC,The expression of TIGIT on the surface of Treg cells was up-regulated and was positively correlated with the early diagnosis index of HCC.TIGIT is expected to be a potential biomarker for early diagnosis and disease assessment of HCC;compared with the level of preoperative inflammatory cytokine secretion in HCC patients.The expression of pro-inflammatory cytokine IL-17 a in plasma of patients with HCC after surgery was increased(P=0.047),the expression of IL-6 was increased(P=0.043),and the expression of anti-inflammatory cytokine IL-10 was decreased(P=0.034).).In summary,TIGIT participates in the regulation of tumor infiltrating lymphocyte immune function,mediates the immune escape mechanism of tumor cells,and disturbance of plasma inflammatory cytokines participates in the occurrence and development of human primary liver cancer.(2)TIGIT-associated miRNAs(miR-206,)were significantly lower in HCC patients than in paracancerous tissues and negatively correlated with the frequency of TIGIT+ Treg cells;TIGIT recombinant plasmid targeting miR-206 was successfully constructed.A dual-luciferase reporter system was used to verify the existence of a targeted relationship between miR-206 and TIGIT;a TIGIT-overexpressing stable Jurkat T-cell was successfully constructed,verifying that mir-206 can negatively regulate TIGIT expression.