Prostate Cancer-derived Exosomes Promote Macrophages To Differentiate Into M2 Phenotype Through The LncRNA MIR22HG/MYC Axis

Objective: To explore the effects of exosomes from prostate cancer cells on M2-type differentiation of macrophages,analyze the molecular mechanism of lnc RNA MIR22 HG mediating M2-type differentiation in macrophages,and clarify the role of lnc RNA MIR22 HG in macrophages.Methods: 1.Isolation of PC-3 exosomes(PC-3 exos)and RWPE-1(RWPE-1 exos)exosomes by Millipore ultrafiltration and identification of exosomes by transmission electron microscopy,nanoparticle size analysis and flow cytometry;2.Macrophages treated with PC-3 exos: THP-1 cells were induced into macrophages with PMA(10ng/m L),and then cultured with RPMI 1640 for 48 h,which was defined as M0 group.IL-4(25 ng/ m L)was added to stimulate M0 for 48 h,which was the M2 group.The extracted exosomes(100 ug/ m L)were added to M0 for 48 h,which were the PC-3-exo-Mφ group and the RWPE-1-exo-Mφ group separately.After PC-3 cells were treated with GW4869,the conditioned medium was collected,concentrated and washed.Then,the macrophages treated with above CM for 48 h were GW4869-Mφ group;3.The expression of CD206 in macrophages in each group was detected by immunofluorescence,and the expression of IL-10,IL-6,TNF-αand TGF-β in macrophages in each group was detected by ELISA method;4.The expression of lnc RNA MIR22 HG in M0,M2 and PC-3-exo-Mφ was detected by q PCR;5.ASOlnc RNA MIR22 HG was transfected into macrophages and the lnc RNA MIR22 HG in macrophages was knocked down.The ability of macrophages phagocytosing CY3-zymosan was observed by fluorescence microscope,and the effect of lnc RNA MIR22 HG on pro-angiogenic ability of macrophages was assessed using the tube formation assay in vitro;6.The distribution of lnc RNA MIR22 HG in PC-3-exo-Mφwas analyzed by RNA fluorescence in situ hybridization;7.RNA coimmunoprecipitation(RIP)assay was used to detect the binding of lnc RNA MIR22 HG to c-MYC in the PC-3-exo-Mφ cells;8.Chromatin immunoprecipitation(Ch IP)assay was used to detect the binding of c-MYC to CD206,TGF-β and SIRP-α promoters in PC-3-exo-Mφ;9.After knocking down lnc RNA MIR22 HG in macrophages,immunohistochemical assay was used to detect the expression of CD206 in macrophages in tumor tissues of nude mice.Results: 1.Exosomes enriched and isolated by Millipore ultrafiltration method.Electron microscopy and nanoparticle size analysis showed that exosomes were mostly circular in shape,with a diameter of about 40-160 nm.After the exosomes were sorted by CD63 magnetic beads,the surface expression of exosome-specific molecular marker CD9 was detected by flow cytometry;2.Macrophages were induced by cytokines or exosomes,and they were divided into five groups: M0 group,M2 group,PC-3-exo-Mφ group,RWPE-1-exo-Mφ group and GW4869-Mφ group;3.The positive rate of CD206 green fluorescence in the PC-3-exo-Mφ group was higher than M0,which was no difference from the M2 group,and the number of positive cells accounted for 70% of the total cells.The expressions of IL-10 and TGF-β in the supernatants of M2 and PC-3-exo-Mφ groups increased,while the expressions of IL-6 and TNF-αdecreased;4.The expression level of lnc RNA MIR22 HG in M2 and PC-3-exo-Mφ was higher than in M0,but there was no difference between PC-3-exo-Mφ and M2;5.After macrophages were transfected with ASO-lnc RNA MIR22 HG,the lnc RNA MIR22 HG was significantly down-regulated.After knockdown of lnc RNA MIR22 HG,the phagocytic ability of macrophages was significantly increased and the ability of the tube formation in vitro was decreased compared to untreated PC-3-exo-Mφ;6.The lnc RNA MIR22 HG in PC-3-exo-Mφ was distributed in the cytoplasm and nucleus;7.The results of RIP showed that the lnc RNA MIR22 HG was bound to c-MYC in the nucleus of PC-3-exo-Mφ cells;8.The results of Ch IP showed that c-MYC was bound to the promoters of CD206,TGF-β and SIRP-α;9.Knockdown of lnc RNA MIR22 HG,the expression of CD206 in nude mouse macrophages decreased significantly compared with the blank group and the NC group.Conclusion: PC-3 exos induced a significant increase in the expression of lnc RNA MIR22 HG in macrophages,promotes the expression of CD206,TGF-β and SIRP-α and leaded to M2 differentiation of macrophages,and the decreased macrophage phagocytic ability.This may be because PC-3 exos induced upregulation of the lnc RNA MIR22 HG in macrophages and promoted the downstream target genes of c-MYC,thus leading to the M2 differentiation of macrophages.