Study on lung cancer cells derived exosomes promoting lung cancer cell me-tastasis through activation of neutrophils
Objective To investigate the activation of neutrophils by exosomes derived from lung cancer cells and the effect of activated neutrophils on the biological behavior of lung cancer cells.Methods In order to investigate the effect of lung cancer cell-derived exosomes on neu-trophils function,the experiment was divided into two groups:①NCM460-Exo group:neutro-phils were cultured with 200 μL(400 μg/μL)of NCM460-Exo in the medium for 24 h;②SW480-Exo group:200 μL(400 μg/μL)of SW480-Exo was added into the medium of neutro-phils and co-cultured for 24 h.In order to study the role of TGF-β1/Smad signaling pathway,SW480-Exo+SB-431542 group was added:1 h before adding SW480-Exo,the neutrophils were treated with TGFβR1 inhibitor SB-431542 in the medium,and then co-cultured with SW480-Exo for 24 h.In order to investigate the effect of neutrophils treated with lung cancer cell exosomes on the migration and invasion ability of lung cancer cells,the experiment was di-vided into two groups:①NCM460-Exo-Neu group:NCM460-EXO-activated neutrophils were co-cultured with SW480 cells;②SW480-EXO-NEU group:SW480-EXO-activated neutrophils were co-cultured with SW480 cells.The effects of SW480-Exo on neutrophil apoptosis and mR-NA expression of IL-8,TNF-α,OSM,MMP9 and VEGF were detected by flow cytometry and fluorescence quantitative PCR.Transwell and WB were used to detect the effects of SW480-EXO-activated neutrophils on SW480 cell migration,invasion and epithelial mesenchymal transformation related proteins expression.The differentially expressed genes of NCM460-Exo and SW480-Exo activated neutrophils were screened by transcriptome sequencing,and KEGG pathway enrichment analysis was performed in parallel.The effect of TGFβR1 inhibitor SB-431542 on SW480-Exo activated neutrophils was detected by Western blot and fluorescent quantitative PCR.Results Compared with neutrophils treated with NCM460-Exo,the apopto-sis rate of neutrophils treated with SW480-Exo was significantly decreased(P＜0.05),and the mRNA expression levels of IL-8,TNF-α,OSM,MMP9 and VEGF were significantly increased(P＜0.05).Compared with the control group,the migration and invasion ability of SW480 cells treated with SW480-Exo activated neutrophils were significantly increased(P＜0.05),and the expression levels of mesenchymal cell markers Vimentin and N-cadherin were signifi-cantly increased.The expression level of E-cadherin was significantly decreased(P＜0.05).Transcriptome sequencing showed that the TGF-β signaling pathway was significantly enhanced in SW480-Exo-activated neutrophils.For SW480-Exo-activated neutrophils,SB-431542 signifi-cantly inhibited the expression levels of p-Smad2/Smad2 and p-Smad3/Smad3(P＜0.05),as well as the mRNA expression levels of IL-8,TNF-α,OSM,MMP9 and VEGF(P＜0.05).Conclusion Exosomes derived from lung cancer cells can induce neutrophil activation,there-by enhancing the migration and invasion ability of lung cancer cells.
NETL
NSTL
万方数据
