| Background and Objective:Currently,lung cancer is one of the most common tumors and the leading cause of cancer-related deaths worldwide,most of which are non-small cell lung cancer(NSCLC).Recent studies have revealed that tumor-associated macrophages(TAM)are an important component of the tumor-associated microenvironment and play an important role in tumor development and invasion.TAM are highly plastic and can polarize into M1/M2 subpopulations with different phenotypes and functions or even diametrically opposed under the action of different environmental factors.Among them,M1-type macrophages can promote inflammation through Th1 cell response and mainly exert anti-tumor effects;M2-type macrophages can promote tumor growth and metastasis by secreting various inflammatory cytokines,stromal degradation enzymes and vascular endothelial growth factor.Currently,the targeting of tumor-associated macrophages has become a new direction in the immunotherapy of non-small cell lung cancer,but its mechanism is still unclear.The literature shows that macrophage polarization is more closely related to P53;ribosomal protein L11(RPL11),represented by ribosomal protein(RP),can regulate cell proliferation through the MDM2-P53 signaling pathway under nucleolar stress conditions.Therefore,we suggest that macrophage polarization may affect the development of non-small cell lung cancer through the RPL11-MDM2-p53 signaling pathway.In this experiment,we will observe the effects of M1/M2 type macrophages on the cell cycle,proliferation,migration and invasion of non-small cell lung cancer cells by constructing M1/M2 type macrophage model,further explore the role of nucleolar stress RPL11-MDM2-p53 signaling pathway in it,and elucidate the molecular mechanism of the role of macrophage polarization on non-small cell lung cancer.Methods:In this project,we firstly induced macrophages with LPS+IFN-γ and IL-4,respectively,and detected polarization-related indexes by Western blot and ELISA to establish a model of M1/M2 macrophage polarization.And based on this,M1/M2 macrophages were co-cultured with non-small cell lung cancer cells.Subsequently,cell scratch assay,cell invasion assay by Transwell,CCK-8 and flow cytometry were used to detect the role of polarized macrophages in non-small cell lung cancer cells in terms of migration,invasion situation,proliferation and cycle,etc.,Western blot was used to detect p53,p21 Western blot was used to detect changes in p53 and p21;realtime fluorescence PCR was used to determine changes in pre-r RNA and immunofluorescence was used to determine changes in the location of RPL11 protein in the nucleus;Western blot was used to detect changes in RPL11 and MDM2expression;immunoprecipitation was used to determine the effect of RPL11 and MDM2;Western blot was used to detect the effect of cytokines IL-12 and IL-10 on RPL11 protein,IL-10 on RPL11 protein;the expression levels of RPL11 and p53 were detected by Western blot on the basis of the construction of a low expression vector for RPL11.Results:1.Western blot results showed that compared with Control group,M1 macrophage group showed high expression of i NOS and low expression of Arg-1,and M2 macrophage group showed high expression of Arg-1 and low expression of i NOS;ELISA results showed that M1 macrophage group showed high expression of IL-12 and low expression of IL-10,and M2 macrophage group showed The ELISA results showed that the M1 macrophage group showed high expression of IL-12 and low expression of IL-10,while the M2 macrophage group showed low expression of IL-12.2.Compared with the Control group,the tumor cell scratch healing rate was reduced in the M1 macrophage + non-small cell lung cancer cell co-culture(M1+NSCLC)group,but increased in the M2 macrophage + non-small cell lung cancer cell co-culture(M2+NSCLC)group.3.Compared with the Control group,the number of NSCLC cells passing through the film was significantly lower in the M1+NSCLC group,while in the M2+NSCLC group,the number of NSCLC cells passing through the film was increased.4.Compared with the Control group,the in vitro proliferation ability of nonsmall cell lung cancer cells was significantly decreased in the M1+NSCLC group;while in the M2+NSCLC group,the in vitro proliferation ability of non-small cell lung cancer cells was significantly increased.5.Compared with the Control group,the transition of G1/S of non-small cell lung cancer cells was inhibited after co-culture with M1-type macrophages,while the transition of G1/S of non-small cell lung cancer cells was promoted under co-culture with M2-type macrophages.6.Compared with the Control group,the expression of p53 and p21 increased in A549 cells after co-culture with M1-type macrophages,while the expression of p53 and p21 decreased after co-culture with M2 macrophages.7.Compared with the Control group,the expression of ribosomal RNA precursor(pre-r RNA)in A549 cells decreased after co-culture with M1-type macrophages,while the expression of ribosomal RNA precursor(pre-r RNA)in A549 cells increased after co-culture with M2-type macrophages.8.Compared with the Control group,the expression of RPL11 and MDM2 in the nucleus of A549 cells increased after co-culture with M1-type macrophages,while the expression of RPL11 and MDM2 in the nucleus of A549 cells decreased after coculture with M2 macrophages.9.Compared with the Control group,co-culture with M1-type macrophages induced a decrease in the distribution of RPL11 protein in the nucleolus and an increase in the nucleoplasm in A549 cells,while co-culture with M2-type macrophages induced an increase in the distribution of RPL11 protein in the nucleolus and a decrease in the nucleoplasm in A549 cells.10.Compared with the Control group,the interaction between MDM2 and RPL11 in the nucleus was enhanced after co-culture with M1-type macrophages.In contrast,the interaction between MDM2 and RPL11 in the nucleus was weakened after co-culture with M2 macrophages.11.Compared with the Control group,RPL11 expression in the nucleus of A549 cells was increased after the action of cytokine IL-12,and the effect of increased RPL11 expression in the nucleus of A549 cells by M1-type macrophages was decreased after IL-12 antibody intervention;compared with the Control group,the effect of cytokine IL-10 RPL11 expression was reduced and the effect of decreased RPL11 expression in the nucleus of A549 cells by M2-type macrophages was reduced after IL-10 antibody intervention;12.p53 protein expression was reduced in the si RPL11+M1 group compared to the si Ctrl+M1 group.Similarly compared to the si Ctrl+M2 group,p53 protein expression was increased in the si RPL11+M2 group.These results suggest that macrophage polarization influences p53 expression by affecting RPL11 in the nucleus position.Conclusion:M1/M2 macrophage polarization exerts regulatory effects on migration,invasion,and proliferation in NSCLC.Among them,M1 inhibits migration,invasion,and proliferation of NSCLC,and M2 promotes migration,invasion,and proliferation of NSCLC by a mechanism involving the RPL11-MDM2-p53 signaling axis in nucleolus accumbens stress. |
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