Effect Of Thymic Mesenchymal Stem Cell Exosomes MiR-125a-5p On Macrophage Polarization

Background and ObjectiveMesenchymal stem cells(MSCs)are a kind of pluripotent stromal cells,which can be isolated from bone marrow,cord blood,fat,thymus and other tissues and expanded in vitro.The immunomodulatory effects of mesenchymal stem cells have been widely reported.Exosomes are vesicles secreted by cells,which are rich in protein,RNA,miRNAs and other biological small molecular substances.They affect the characteristics of receptor cells through transport,thereby affecting a variety of physiological and pathological processes.Therefore,they have been widely studied and appliied as biological carriers.Among the cell that can produce exosomes,human MSCs are the cells that produce the most.And MSCs-exosomes also show great potential for application in damage repair and immune regulation.The exosomes secreted by MSCs can play a mediating role in the tumor microenvironment,and participate in tumor genesis,metastasis and angiogenesis.Tumor-associated macrophages(TAMs)are the main immune cells in the tumor microenvironment and play an important role in the progression and regression of tumors.Studies have shown thatMSCs can induce M2 polarization of macrophages,and make macrophages show anti-inflammatory and immunosuppressive phenotypes.Thymic mesenchymal stem cells(tMSCs)are rarely studied and reported due to limited materials.However,their immunomodulatory function can not be ignored because they are derived from central immune organs.It has been reported that the expression of miR-125a-5p in thymoma is significantly increased.As an important thymoma-related miRNA,the effect of miR-125a-5p on the immunoregulation of tMSCs,especially on the M2 polarization of macrophages,is the focus of this study.In order to investigate whether miR-125a-5p overexpression can affect the polarization of macrophages through the exosomes of tMSCs,we constructed a lentiviral vector of miR-125a-5p to interfere with the expression of miR-125a-5p in the exosomes of tMSCs,and to explore the effect of miR-125a-5p on the polarization of macrophages.So as to provide experimental basis for clarifying the regulatory function of miR-125a-5p and the exosomes of tMSCs in anti-tumor immunity.Methods1.Isolation and identification of human tMSCs:human thymus tissue was collected,digested with collagenase IV and inoculated in culture dish to obtain adherent cells.The adherent cells were labeled with CD105,CD73,CD90,CD34,CD45 and Ep CAM,and the purity of tMSCs was determined by flow cytometry(FCM).2.Construction of lentiviral vector and intervention of miR-125a-5p expression in tMSCs:overexpression and inhibition of miR-125a-5p lentiviral vector,empty vector and packaging plasmid were co-transfected into HEK-293T cells respectively,and virus solution was collected.tMSCs were infected with overexpressed virus and empty vector virus respectively for 72 hours.The expression of miR-125a-5p was detected by q RT-PCR.3.Extraction and identification of tMSCs exosomes:the culture supernatants of tMSCs infected with virus were collected,and the exosomes were extracted with an exosome extraction kit.The morphology of exosomes was observed by TEM;the expressions of molecular markers CD81,CD63 and CD9 were detected by FCM;the expressions of exosomal marker proteins CD63 and CD9 were detected by WB;the expression of miR-125a-5p in tMSCs exosomes was detected by q RT-PCR.4.The effect of tMSCs exosomes loaded with miR-125a-5p on the polarization of THP-1 derived macrophages:PMA induces the differentiation of THP-1 cells into M0,which were induced by tMSCs derived exosomes(MEX)and exosomes overexpression miR-125a-5p(MEX^miR-125)for 48 hours.FCM and q RT-PCR were used to detect the phenotype and the cytokines secretion of macrophages.5.The effect of MEX^miR-125polarized macrophage supernatant on the growth of RAMOS and H9 cells:RAMOS and H9 cells were co-cultured with the MEX and MEX^miR-125polarized macrophage supernatants(CM^MEXand CM^MEX-miR-125)for 48h,collect cells and label cells with Annexin V and 7AAD,and use FCM to detect cell apoptosis.The effects of CM^MEXand CM^MEX-miR-125on the growth of RAMOS and H9 cells were detectd by CCK8.6.The effect of MEX^miR-125polarized macrophage supernatant on angiogenesis and wound healing of HUVEC:HUVEC was seeded on Matrigel,and then co-cultured with CM^MEXand CM^MEX-miR-125,and the angiogenesis of HUVEC was observed under microscope after 6 hours;scratches started when HUVEC confluence reached 100%,CM^MEXand CM^MEX-miR-125were added to co-culture with the cells.After 12 hours,the healing of HUVEC was observed under the microscope.Results1.Isolation and identification of human tMSCs:the adherent cells were observed under the microscope and showed a long spindle shape.The positive rates of CD105,CD73 and CD90 were(97.95±0.45)%?(97.25±0.55)%?(86.73±1.67)%,CD34,CD45 and Ep CAM were negative,which accorded with the basic characteristics of tMSCs.2.The expression of miR-125a-5p in tMSCs after lentivirus infection:the expression of miR-125a-5p in tMSCs increased significantly after lentiviral infection for 72 hours(P<0.001).3.Extraction and identification of exosomes derived from tMSCs:Transmission electron microscopy shows that the exosomes derived from tMSCs(MEX)have a double-layer membrane-like structure with a diameter of 50?120 nm.The extracted exosomes CD63 and CD9 are positive for protein expression.The expression of CD81,CD63 and CD9 on the surface of exosomes was positive.The expression of miR-125a-5p in tMSCs exosomes which overexpressing miR-125a-5p(MEX^miR-125)was significantly increased(P<0.001).4.The effect of MEX^miR-125on the polarization of THP-1 derived macrophages:The exosomes were labeled by PKH26 and co cultured with macrophages,which showed that macrophages could phagocytize exosomes.Compared with the control group,the expression of M1 molecule CD86 was significantly decreased in MEX and MEX^miR-125groups,while the expression of M2 molecule CD163 and CD206was significantly increased.In MEX^miR-125group,the down-regulation of CD86 and up-regulation of CD163 and CD206 were more significant.Biolegend multi-factor detection showed that compared with the control group,the expression of M2b cytokines IL-10 and IL-1?in the MEX group was significantly increased,and the expression of IL-6 and TNF-?were slightly increased,but there was no significant difference.IL-12 expression Lower but no significant difference,the expression of pro-inflammatory factors IL-8 and MCP-1 was significantly increased.Compared with the MEX group,the secretion of M2b cytokines IL-10,IL-1?,IL-6 and TNF-?in the MEX^miR-125group was significantly increased,and the expression of IL-12 was lower but there was no significant difference.The pro-inflammatory factor IL-8 and MCP-1 increased significantly(P<0.05).The m RNA results also confirmed that the expressions of IL-10,IL-1?,IL-6 and TNF-?in the MEX and MEX^miR-125groups were significantly increased,and the expression of the MEX^miR-125group was significantly higher than that of the MEX group(P<0.05).5.The effect of CM^MEX-miR-125on the growth of RAMOS and H9 cells:RAMOS and H9 cells were co-cultured with CM^MEXand CM^MEX-miR-125for 48 hours.Compared with the control group,the apoptosis rate of Ramos and H9 cells in CM^MEXand CM^MEX-miR-125groups decreased,and the cell viability increased.Compared with the CM^MEXgroup,the apoptotic rate of RAMOS and H9 cells in the CM^MEX-miR-125group was lower,and the activity of Ramos cells was increased(P<0.05).6.The effect of CM^MEX-miR-125on angiogenesis and wound healing of HUVEC:After co-cultured with CM^MEXand CM^MEX-miR-125respectively for 6 hours,the angiogenesis in CM^MEXand CM^MEX-miR-125groups increased compared with the control group,and CM^MEX-miR-125group had stronger effect on angiogenesis(P<0.05).After HUVEC was co-cultured with CM^MEXor CM^MEX-miR-125for 12 hours,tthe wound healing ability of CM^MEXand CM^MEX-miR-125groups was significantly increased;and the effect of CM^MEX-miR-125on promoting wound healing was stronger(P<0.001).Conclusion1.Human thymic mesenchymal exosomes can promote M2 polarization of THP-1-derived macrophages.2.miR-125a-5p can affect the regulation of tMSCs exosomes on macrophage polarization,promote the M2 like polarization of macrophages by MEX.3.Polarized macrophages can produce more inflammatory regulatory factors,improve the activity of Ramos and H9 cells and promote angiogenesis.