To Investigate The Effects And Mechanisms Of NANOG And GSDME On NK Cells Killing Glioma

Background:Natural killer cell(NK)-based immunotherapy provides a new therapeutic strategy for patients with malignant solid tumors,but the efficacy is still not ideal.As a stemness marker,NANOG is especially highly expressed in the general tumor stem cells and also enhanced the stemness of tumor cells.GSDME,as a key molecule of pyroptosis,can promote tumor immunity and inhibit tumor growth by mediating the pyroptosis of tumor cells.However,the effects and mechanisms of NANOG and GSDME in glioma,especially in the killing of glioma by NK cells,is still unclear.Objective:The purpose of this study was to explore the role of NANOG and GSDME in glioma,especially the mechanism of NK cell killing glioma cells,so as to provide experimental basis for enhancing the efficacy of NK-based immunotherapy for glioma.Methods:(1)Firstly,after overexpressing NANOG in glioma cell line GL261,the expression levels of stemness markers were detected by RT-PCR,flow cytometry and western blot and the biological function of NANOG in glioma cells was investigated by sphere-forming experiment,Transwell,Edu and CCK8 proliferation experiment.Then,in order to investigate the effect of NANOG on NK cells killing glioma cells,in vitro,C57bl/6J mice spleen NK cells were extracted by magnetic bead negative screening and MTS assay was used to detect the activity of the glioma cells in control group and overexpressed group after co-culturing with NK cells.In vivo,intracranial in situ model was established by infusion of glioma cells with NANOG overexpressed to detect the influence of NANOG overexpression on the tumorigenicity of glioma cells in the presence or absence of NK cells.(2)As to GSDME,in order to explore the effect of GSDME on NK cells killing glioma cells,after overexpressing or interfering GSDME in glioma cells,Edu、CCK8、flow cytometry apoptosis and cycle were used to detect the effect of GSDME on the proliferation ability of glioma cells.Then,after glioma cells co-cultured with NK92MI cells,the release of lactic dehydrogenase(LDH)was detected in supernatant and the morphological changes of glioma cells were observed by electron microscopy.Lastly,ELISA was used to detect the changes of IL18 and granzyme B,WB was used to detect the changes of Caspase3,GSDME and IL1β.Results:(1)After NANOG overexpression in GL261 cells,the results of RT-PCR and WB showed that the expression of CD 133 and CD44 in overexpression group was higher than that in control group(P<0.01),while Oct4,Sox2 and Nestin showed no significant difference(P>0.05).Flow cytometry showed that CD 133 and CD44 positive cells in the overexpression group were 10%higher than those in the control group.Overexpression of NANOG enhanced cell sphere formation efficiency(P<0.01),but the proliferation and migration ability of the cells did not change significantly.MTS test results showed that compared with the control group,the cell viability of the overexpressed NANOG group was not significantly enhanced.The results of animal experiments showed that the presence of NK cells could significantly inhibit the tumor formation of GL261 glioma cells,but the tumor did not increase significantly with overexpressing NANOG(P>0.05).(2)As to GSDME,the results showed that under normal culture conditions,GSDME expression did not affect the proliferation,apoptosis and cycle of glioma cells.After co-cultured with NK92MI,Caspase3 and GSDME were activated significantly,and granzyme B secretion was increased.Overexpression of GSDME significantly increased the release of lactate dehydrogenase in supernatant(P<0.01),and decreased after interference(P<0.01).The observation of cell morphology showed that pyrosomes and cell membrane pores increased after GSDME overexpression.ELISA results showed that the release of IL18 increased(P<0.001),but decreased after interference(P<0.001).The results of WB showed that the expression of N-GSDME and IL1β was up-regulated after overexpression,but the interference was opposite.These results suggest that GSDME can promote pyroptosis of glioma after co-culture with NK92MI.Conclusion:The overexpression of NANOG enhances the stemness characteristics of glioma cells,but does not affect the proliferation and migration ability of GL261 cells and the killing of NK cells on glioma cells.Whereas GSDME can effectively promote the killing of NK cells on glioma cells by mediating glioma cells pyrophosis,and provide a possible effective target for NK cells to treat glioma.