| Colorectal cancer(CRC)is a common malignant cancer in the digestive tract and a disease caused by many factors.Aspirin(ASA)is a classic antipyretic analgesic and anti-inflammatory drug.Cardiovascular disease prevention test data showed that routine use of aspirin can reduce the number of colorectal cancer by about 40%,but the mechanism of preventing colon cancer is still unclear.T cell immunoglobulin and IT AM domain(TIGIT)is synergistic co-suppressor molecules discovered in recent years,which play a role in inducing T cell immune tolerance and tumor immune escape.Importantly,CD 155 acts as a receptor for TIGIT,and TIGIT-CD155 signaling pathway mediates immunoregulatory regulation.This study investigated the mechanism of action of aspirin to prevent colorectal cancer by regulating the biological characteristics of tumor cells and immune cells through the TIGIT-CD155 signaling pathwayPrat One:Aspirin Regulates the Biological Function of Colorectal Cancer Cells through TIGIT-CD155 Signaling PathwayObjective To investigate the expression of TIGIT and CD155 in colorectal cancer tissues and colorectal cancer cells,and to study the effect of aspirin on the biological function of colorectal cancer cells by regulating TIGIT-CD155 signaling pathwayMethods Paraffin-embedded tumor tissues and paracancerous tissues from CRC patients from the Department of Pathology,Ningxia Medical University General Hospital was collected and made into paraffin sections.The expression of TIGIT,CD226 and CD155 was detected by immunohistochemical staining(IHC)The expression of TIGIT and CD155 in different colorectal cancer HT-29,SW480,SW620 and HCT116 cell lines was detected by Western Blot(WB).CCK8 assay,scratch test,Trans well migration assay and flow cytometry were used to detect different concentrations of ASA(2 mM,4 mM,8 mM)in colorectal cancer cell line HT-29 for 0 h,24 h,48 h and 72 h,observed the effects of ASA on colon cancer cell activity,migration ability and apoptosis.WB was used to detect different concentrations of ASA in HT-29 cells for 48 h,and to observe the expression of TIGIT,CD155 proteins,in migration-associated proteins MMP9 and FAK,apoptosis-related proteins BAX and Bc12,TIGIT-CD155 signaling pathway,and ASA concentration of 8 mMol/L(IC50)observed the expression of TIGIT and CD 155 proteins in the migration-related proteins MMP9 and FAK,apoptosis-related proteins BAX and Bcl2,and TIGIT-CD155 signaling pathways after 24 h,48 h and 72 h,respectively.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of TGF-?1 and IL-10 in the supernatant of colon cancer cells HT-29 after different concentrations of ASAResults IHC results showed that TIGIT and CD226 were significantly higher on CRC tumor tissue infiltrating lymphocytes than in invasive adjacent lymphocytes,and CD155 was not detected on colon cancer tissues and adjacent tissues infiltrating lymphocytes.The results of CCK8 showed that the cell viability of Jurkat cells decreased with the increase of ASA concentration at the same time.At the same concentration,ASA could significantly inhibit the cell viability of Jurkat cells with the prolongation of action time.The results of flow cytometry showed that the apoptosis rate of Jurkat cells increased significantly with the increase of ASA concentration and time.The results of WB showed that the expression of proapoptotic protein BAX was up-regulated and the expression of Bcl2 was down-regulated after 48 hours of ASA intervention.The expression of TIGIT was down-regulated in TIGIT-CD155 pathway.When ASA concentration was 4mMol/L,The expression of pro-apoptotic protein BAX was up-regulated and the expression of Bcl2 protein was down-regulated in TGFIT-CD155 pathway.The ELISA results showed that at the same time,the secretion levels of cytokines TGF-?1 and IL-10 cytokines decreased as the concentration of ASA increasedConclusion The expression of TIGIT and CD155 is related to the biological function of the tumor.Aspirin inhibits the proliferation,apoptosis and migration of tumor cells by inhibiting TIGIT-CD155 signaling pathway,thereby achieving the purpose of preventing tumorsPart Two:Aspirin Regulates the Biological Function of Immune Cells through TIGIT-CD155 Signaling PathwayObjective To investigate the expression of TIGIT and CD 155 on immune cells,and to study the effect of aspirin on the biological function of T lymphocyte leukemia cell line(Jurkat cell)regulated by TIGIT-CD 155 signaling pathwayMethods Paraffin-embedded tumor tissues and paracancerous tissues from CRC patients from the Department of Pathology,Ningxia Medical University General Hospital was collected and made into paraffin sections.IHC was used to detect the expression of TIGIT,CD226 and CD 155 on tumor infiltrating lymphocytes.CCK8 assay and flow cytometry were used to detect the effects of different concentrations of ASA(1 mM,2 mM,4 mM)on cell viability and apoptosis in Jurkat T cells.WB was used to detect the expression of apoptosis-related proteins BAX and Bcl2 under different concentrations of ASA,and the expression of TIGIT and CD155 in TIGIT-CD155 pathway.The concentration of ASA was 4 mM(IC50)for 24 h,48 h and 72 h respectively,observed the expression of apoptosis-related proteins BAX and Bcl2,expression of TIGIT and CD155 in the TIGIT-CD155 pathway.ELISA was used to detect the expression of TGF-?1 and IL-10 in the supernatant of Jurkat cells after different concentrations of ASAResults IHC results showed that TIGIT and CD226 were significantly higher on CRC tumor tissue infiltrating lymphocytes than in invasive adjacent lymphocytes,and CD155 was not detected on colon cancer tissues and adjacent tissues infiltrating lymphocytes.The results of CCK8 showed that the cell viability of Jurkat cells decreased with the increase of ASA concentration at the same time.At the same concentration,ASA could significantly inhibit the cell viability of Jurkat cells with the prolongation of action time.The results of flow cytometry showed that the apoptosis rate of Jurkat cells increased significantly with the increase of ASA concentration and time.The results of WB showed that the expression of proapoptotic protein BAX was up-regulated and the expression of Bcl2 was down-regulated after 48 hours of ASA intervention.The expression of TIGIT was down-regulated in TIGIT-CD155 pathway.When ASA concentration was 4mMol/L,The expression of pro-apoptotic protein BAX was up-regulated and the expression of Bcl2 protein was down-regulated in TGFIT-CD155 pathway.The ELISA results showed that at the same time,the secretion levels of cytokines TGF-?1 and IL-10 cytokines decreased as the concentration of ASA increasedConclusion Aspirin inhibits the proliferation and apoptosis of Jurkat cells by down-regulating the expression of TGF-?1 and IL-10,and inhibiting the expression of TIGIT in the TIGIT-CD 155 pathway. |
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