The Effect Of TLR9/NF-κB Signaling Pathway On The Gastric Cancer Metastasis

Objective In this study the gastric cancer cells MGC803cultured in vitro and micetransplanted with gastric cancer cell line MGC803as the research objects, to observe theexpression of TLR9in gastric cancer cells and in tumor tissues of nude mice transplanted.Atthe same time,TLR9nonspecific inhibitor chloroquine (CQ) was used to intervene TLR9/NF-κB signaling pathway to observe the effect on the proliferation activity and migration ofMGC803,on the influence of IL-6secretion and on the mRNA expression of cytokinesassociated with gastric cancer development and metastasis: matrix metalloproteinases(MMP)and cyclooxygenase-2(COX-2). Discuss the mechanism about the expression of TLR9andTLR9mediated NF-κB signaling pathway in gastric cancer occurrence and development.Providing theoretical basis of new and effective treatment methods for gastric cancer clinicalimmunotherapy.Methods (1) The gastric cancer cells line MGC803were cultured in vitro. The immunofluorescence staining test was used to examine the expression of TLR9in the gastric cancercells MGC803.(2) Detecting tumor cell toxicity after TLR9inhibitors CQ were added to theMGC803cell suspension by CCK assay.(3) The bioactivity of interleukin-6(IL-6) in theMGC803cell suspension treated with different concentration TLR9inhibitors CQ at differenttimes was detected by ELISA.(4) To observe the effect of TLR9inhibitors CQ on themigration ability of the gastric cancer cells MGC803through wound-healing scrape assayafter TLR9inhibitors were applied to the cells.(5) With RT-PCR method to detect expressionof mRNA including COX-2mRNA, MMP-2mRNA, MMP-7mRNA and NF-κB p65mRNAwhen different concentration TLR9inhibitors were used to the cell suspension after24h.(6)Through the establishment of human gastric cancer in animal models to observe TLR9 inhibitor CQ effects on tumor size and tumor metastasis.(7) Dissecting mice spleen and tumortissues of transplanted mice to calculate spleen index and to observe the changes of theimmune system.(8) Immunohistochemical methods were performed to observe the effect ofTLR9inhibitors CQ on tumor tissue structure and the expression of TLR9.Results (1) Gastric cancer cells MGC803express TLR9and there was higher expressionin cytoplasm.(2)50μg·mL(-1),100μg·mL(-1),200μg·mL(-1)CQ could inhibit gastric carcinomaMGC803cells proliferation and had inhibitive effects on gastric cancer cells proliferation in adose and time-dependent manner (p<0.05).(3)200μg·mL(-1)inhibitor CQ decreased thebioactivity of IL-6in24h,48h and72h, but50μg·mL(-1)and100μg·mL(-1)inhibitor CQsuppressed IL-6expression only after72h (p<0.05).(4) TLR9/NF-κB signaling pathwayagonists CpGODN could promote MGC803cell migration (p<0.05),however inhibitor CQcould inhibit MGC803cell migration in a dose and time-dependent manner (p<0.05).(5)TLR9/NF-κB signaling pathway agonists CpGODN could promote gastric cancer cellsMGC803COX-2mRNA,MMP-2mRNA and NF-κB p65mRNA expression, and inhibitorcould inhibit the mRNA expression(p<0.05); for MMP-7mRNA, TLR9agonists and low andmiddle concentration of the inhibitor could promote mRNA expression(p<0.05),but mRNA inthe high concentration inhibition groups were not more statistically significant than thecontrol group.(6) In the early days the TLR9/NF-κB signaling pathway inhibition couldinhibit the growth of the tumor, while the late couldn’t suppress the growth of tumor.Afterdissecting nude mice,cancer metastases did not be seen in the lymph nodes which were closedto the implanted site and in the liver and spleen.(7) SP method tests showed there were morepositive cells which expressed TLR9in the TLR9/NF-κB signaling pathway agonists groupand in the control group than in inhibitor group, and the difference was statistically significant.More obvious focal necrosis in the TLR9agonists group and in the control group could beseen than TLR9inhibitor group.Conclusions (1) MGC803cells express TLR9.(2) TLR9/NF-κB signaling pathway are involved in the proliferation and metastasis of gastric cancer cells.(3) Inhibiting TLR9/NF-B signaling pathway might be new targets for gastric cancer immunotherapy.