Exosomal MiR-382-5p From Helicobacter Pylori-induced Gastric Cancer Cells Promoted M2 Macrophage Polarization

Objective:This experiment was aimed to investigate the regulatory effect and mechanism of differentially expressed miR-382-5p in human gastric cancer SGC7901 cell-derived exosomes induced by H.pylori on M2-type polarization of macrophages,providing new clues for further elucidating the carcinogenic mechanism of H.pylori.Method:H.pylori was co-cultured with gastric cancer cell line SGC7901 for 24 hours,the exosomes were extracted by ultracentrifugation or kits,and the morphology of exosomes was observed by transmission electron microscope（TEM）.Nanoparticle tracking analysis（NTA）was used to detect the particle size of exosomes,and Western Blotting was employed to analyse exosomal marker proteins CD9,TSG101 and CD63 expression.Then,qRT-PCR was used to detect the expression of miR-382-5p in H.pylori-induced SGC7901 cell-derived exosomes.Human THP-1 leukemia cells were induced to differentiate into macrophages by PMA（Phorbol 12-myristate 13-acetate）,and qRT-PCR was used to detect the macrophage marker molecule CD68expression.PKH67-labeled exosomes were co-cultured with THP-1-derived macrophages,then the endocytosis of exosomes by macrophages was observed through the laser confocal fluorescent microscopy.Moreover,The Cy3 labeled miR-382-5p mimic was transfected into SGC7901 cells and then co-cultured with macrophages,laser confocal microscopy and qRT-PCR were used to verify the miR-382-5p could be delivered to macrophages through exosomes.The expressions of phenotype molecule CD206 and HLA-DR in miR-382-5p mimic-transfected macrophages were analyzed by flow cytometry,and the cytokines TNF-α,IL-6 and IL-10 levels were measured by qRT-PCR and ELISA.Furthermore,the dual luciferase reporter system was used to verify the targeting relationship between miR-382-5p and the 3’-UTR of the PTEN gene.Western Blotting was performed to detect the effect of miR-382-5p overexpression on the expression levels of PTEN protein and its downstream proteins PI3K,AKT,p-AKT.Finally,we investigated the effect of PI3K/Akt signaling pathway inhibitor LY294002 pretreatment on the expression of the miR-382-5p-induced cytokines secretion changes in macrophage.Results:1.TEM and NTA analysis showed that the extracted exosomes were consistent with exosome morphology,which were cup-like tiny vesicles with a average diameter of～115nm,and highly expressed the surface marker proteins CD9,TSG101 and CD63.2.qRT-PCR results demonstrated that the PMA-differentiated THP-1 cells express high level of CD68 molecule.After co-culturing with THP-1-derived macrophages for 24 hours,the exosomes could be internalized by macrophages as shown by fluorescence staining.3.The results of confocal laser microscope and qRT-PCR showed that miR-382-5p could be delivered to macrophages through exosomes.4.Flow cytometry results suggested that miR-382-5p mimic transfection increased the expression of M2-type phenotype molecule CD206 and the proportion of CD206^highHLA-DR^lowcells in macrophages.Furthermore,miR-382-5p mimic could also increase the expression of IL-10 and inhibit the expression of TNF-αand IL-6 in macrophages at both the mRNA and protein levels.5.The dual-luciferase reporter assay revealed that miR-382-5p can directly bind to the 3’-UTR of the target gene PTEN and inhibit its expression.Western Blotting results showed that the expression of PTEN protein in the miR-382-5p-transfected group decreased,while the expressions of PTEN/PI3K/AKT signaling pathway downstream proteins PI3K,AKT and p-AKT increased.6.The PI3K/Akt pathway inhibitor LY294002pretreatment reversed the changes of cytokine secretion induced by miR-382-5p.Conclusion:Exosomal miR-382-5p from H.pylori-induced gastric cancer cells promoted macrophage M2 polarization through the PTEN/PI3K/AKT signaling pathway.