| Objective:To investigate the effects of exosomes derived from two different human-derived gastric cancer cell lines(MKN-45 and AGS)on macrophages(RAW 264.7),and to explore the possible mechanism of gastric cancer-derived exosomes with promoting macrophage polarization,gastric cancer cell growth and metastasis.Methods:1.Bioinformatics technology was used to predict common potential targets and signaling pathways between gastric cancer and macrophages.2.Exosomes were extracted from supernatant culture of normoxic and hypoxic human-derived gastric cancer cell lines(MKN-45 and AGS).3.Transmission electron microscopy,nanoparticle tracking analysis(NTA)technology,western blotting(WB)and other methods were used to identify extracted gastric cancer-derived exosomes.4.The extracted gastric cancer-derived exosomes were labeled by PKH26 red dye,and co-culture of the macrophage nuclei labeled with DAPI blue dye,the results of exosome uptake were observed by inverted fluorescence microscopy.5.After co-culturing the extracted gastric cancer-derived exosomes with macrophages(RAW 264.7),the effects of gastric cancer-derived exosomes on the proliferated,migrated and invaded ability of RAW 264.7 cells were respectively detected by plate cloning experiment,cell scratch experiment and transwell chamber experiment.6.Flow cytometry was used to detect the expressions of M2 macrophage molecular marker protein CD206 and M1 macrophage molecular marker protein CD86.7.After co-culturing the extracted normoxic and hypoxic MKN-45 cell-derived exosomes with two human gastric cancer cells(MKN-45 and AGS),the effects of normoxic and hypoxic MKN-45 cell-derived exosomes on the growth and migration of gastric cancer cells were observed by the cell scratch experiment.8.WB technology was used to detect the effects of gastric cancer-derived exosomes on the expression level of MAPK(MEK/ERK)signaling pathway-related proteins in RAW 264.7cells.Results:1.Gastric cancer and macrophages were jointly enriched with 35 genes,10 potential molecules were screened from them,and gastric cancer may act on macrophages via the MAPK(MEK/ERK)signaling pathway.2.Both gastric cancer cell-derived exosomes can be successfully extracted under normal oxygen and hypoxia conditions.The morphology of exosomes detected by transmission electron microscopy was typical cup morphology.NTA technology detected exosome particle size between 30-150 nm.WB technology detected exosome characteristic markers,and all markers were positively expressed.These evidences were indicated that the substance extracted by differential centrifugation was exosomes.3.After co-culture of normoxic and hypoxic gastric cancer cell-derived exosomes and macrophages,the result of internalization experiments showed that PKH26-labeled exosomes could be up-taken by RAW 264.7 cells.4.Compared with the blank control group without exosomes,the proliferated and invaded ability of macrophages(RAW 264.7)were significantly enhanced in the experimental group adding gastric cancer-derived exosomes.Compared with normoxyic gastric cancer cell-derived exosomes,hypoxic gastric cancer cell-derived exosomes were more likely to promote the proliferation,migration and invasion of RAW 264.7 cells.In addition,whether it was incubated gastric cancer cells in normal oxygen or hypoxic environment,the experimental group adding MKN-45 cell-derived exosomes had stronger proliferated,migrated and invaded ability than the experimental group adding AGS cell-derived exosomes.5.Compared with the blank control group without exosomes,the polarization ability of RAW 264.7 cells was significantly enhanced in the experimental group adding gastric cancer-derived exosomes.Compared with normoxyic gastric cancer cell-derived exosomes,hypoxic gastric cancer cell-derived exosomes are more likely to promote the polarization of RAW 264.7 cells.In addition,whether it was incubated gastric cancer cells in normal oxygen or hypoxic environment,the experimental group adding MKN-45 cell-derived exosomes had stronger polarization and induction ability than the experimental group adding AGS cell-derived exosomes.6.Compared with the normoxic MKN-45 cell-derived exosomes,hypoxic MKN-45cell-derived exosomes were more likely to promote MKN-45 and AGS cell migration,and their cellular density of growth was also increased.7.After co-culture with extracted gastric cancer cell-derived exosomes and macrophages(RAW 264.7),the expression levels of p-MEK and p-ERK proteins in the MEK/ERK signaling pathway showed an upward trend,while the expression levels of MEK and ERK proteins did not change significantly.Compared with the experimental group adding normoxic gastric cancer cell-derived exosomes,the phosphorylated levels of MEK and ERK proteins were higher in the experimental group adding hypoxic gastric cancer cell-derived exosomes.In addition,whether it was incubated gastric cancer cells in normal oxygen or hypoxic environment,the experimental group adding MKN-45 cell-derived exosomes upregulated more p-MEK and p-ERK protein expression than the experimental group adding AGS cell-derived exosomes.Conclusion:Gastric cancer cell-derived exosomes can promote the polarization of macrophages,so as to enhance cell proliferation,migration and invasion,and hypoxic gastric cancer cell-derived exosomes are more likely to promote the above biological effects than normal oxygen gastric cancer cells-derived exosomes,and the experimental group adding MKN-45 cell-derived exosomes has stronger polarization inducting ability than the experimental group adding AGS cell-derived exosomes.In addition,gastric cancer cell-derived exosomes have a promoting effect on the growth and metastasis of gastric cancer cells.These phenomenons may be closely related to the regulation of the MEK/ERK signaling pathway. |
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