| BackgroundAs a multipotent cytokine,IL-15 not only regulates innate immunity,but also regulates adaptive immune responses and stimulates the activation and proliferation of memory CD8+T cells,NK cells and NKT cells.It controls the homeostasis and growth of different non-immune cells and tissues.IL-15 receptor（IL-15R）consists of three subunits,namely,the specific high affinity receptor IL-15Rα,and the intermediate affinity receptors IL-2Rβandγshared with IL-2.IL-15Rαis widely expressed in a variety of immune and non-immune cells and tissues,but IL-2Rβand IL-2Rγare mainly expressed on the surface of T and NK cells.Therefore,IL-15 receptors are mostly distributed on the surface of the cell membrane as IL-15Rαmonomer and IL-2Rβ/γdimer,altogether they function as a trimer.Gastric cancer is a malignant tumor with high incidence in China,especially in Gansu province.According to the2017 Global Burden of Disease survey report,the standardized death rate of gastric cancer in China was 25.16 per 100,000 persons,far higher than the world standard,ranking second in the world.Our previous study found that EBV positive gastric cancer showed significantly increased proliferation and prognosis than EBV negative gastric cancer due to lower expression of IL-15Rα,suggesting that IL-15Rαplays an important role in gastric cancer progression.In this study,two other subunits of IL-15R were found also expressed on the surface of gastric cancer cells,and they could inhibit IL-2Rβ/γon the surface of NK and T cells to bind to IL-15/IL-15Rαcomplex.These results suggest that IL-15 super agonist developed based on IL-15/IL-15Rαmay not be effective in treating cancers which expresses IL-2Rβ/γ.Therefore,it is important to study the expression patterns of IL-15R on gastric cancer cells,and its effect on activation of gastric cancer cells and infiltrating immune cells in the tumor microenvironment.ObjectiveIn this study,we first explored the effect of IL-15Rαon the phenotype of gastric cancer cells by knockdown of IL-15Rαgene,then we observed the effect of IL-15/IL-15Rαsignal axis on the proliferation of gastric cancer cells by the addition of exogenous recombinant IL-15.On the basis of IL-15Rαknockdown,we continued to knockdown IL-2Rβ/γgenes to investigate whether the growth of cancer cells and activation of infiltrating immune cells is regulated by IL-15R expression on tumor cells.Furthermore,we transplanted gastric cancer cells to nude mice to observe how the IL-15R expression influence the proliferation of gastric cancer cells in vivo.Finally,we tried to explore the mechanisms by which IL-15R expressed on the surface of gastric cancer cells compete ligand and interfere with the activation of immune cells by various signaling pathways.Methods1.The expression levels of IL-15Rα,IL-2Rβandγon gastric cancer cell lines and clinical gastric cancer samples were detected by WB/Immunohistochemistry.2.CRISPR-Cas9 system was used to knockdown IL-15Rα,IL-2Rβandγof AGS and MKN-45 gastric cancer cells.The efficiency of knockdown was verified by gene sequencing and indirect immunofluorescence staining,then monoclonal cell was screened（limited dilution method）to get the strain with best knockdown efficiency.3.CCK8 proliferation assay,migration assay and colony formation assay were used to determine the effects of IL-15Rαon the proliferation and migration of gastric cancer cells.4.CRISPR-Cas9 gene knockdown technology was further used for gene knockdown of IL-2Rβandγreceptors in AGS gastric cancer cells,and the knockdown efficiency was verified by WB/indirect immunofluorescence staining.5.Exogenous IL-15 was added to the culture system and CCK8 assay was used to detect the proliferation of gastric cancer cells with different IL-15R expression.6.Gastric cancer cells with different IL-15R expression were co-cultured with PBMCs with/without the addition of exogenous IL-15,then flow cytometry analysis was conducted to detect the activation of PBMCs and apoptosis of gastric cancer cells.7.MKN-45 gastric cancer cells（control group,empty carrier group,IL-15Rαknockdown group）were transplanted to nude mice by subcutaneous injection at fat mat to establish the xenograft tumor model.The size of the transplanted tumors were measured to evaluate the proliferation of gastric cancer cells in vivo.Results1.Gastric cancer cells/tissues express IL-15Rα,IL-2Rβ,IL-2Rγat different levels with positive correlation（P<0.05）.2.After IL-15Rαgene knockdown on the gastric cancer cells AGS and MKN-45,the cells showed significantly decreased proliferation and migration（P<0.01）.3.After Il-15Rα,IL-2Rβand IL-2Rγgenes knockdown,the proliferation ability of gastric cancer cells was significantly decreased compared with that of IL-15Rαgene knockdown alone or blank control cells（P<0.01）.4.When AGS cells were co-cultured with PBMCs,the exogenous IL-15 was able to stimulate the proliferation of gastric cancer cells but unable to activate PBMCs.However,when IL-15 receptors were knockdown the exogenous IL-15 could activate PBMCs efficiently（P<0.05）.5.The growth trend of subcutaneously transplanted tumor cells in nude mice was consistent with that observed in vitro（P<0.05）.Conclusion1.The expression levels of IL-15Rα,IL-2Rβand IL-2Rγin gastric cancer cell lines and cancer tissues were correlated with each other.2.Gastric cancer cell-derived IL-15R can promote the proliferation of tumor cells by IL-15/IL-15R axis,but inhibit the activation of co-cultured immune cells;These effects were reversed when IL-15R was struck low.3.Gastric cancer cells with high IL-15Rαexpression were more likely to form tumors in nude mice than those with low IL-15Rαexpression,and the tumor growth rate was faster,and they were not sensitive to local injection of IL-15 into the lesions. |
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