| BackgroundMultiple myeloma(MM)is a magligancy of terminally differentiated plasma cells,and is the second most common haemato logical malignancy which accounts for approximately 10%of the total.Most of MM patients are middle-aged and elderly people with diverse clinical symptoms.With the recent advancements of its detection,diagnosis and treatment methods,the prognosis of MM patients has been improved to a certain extent.However,it continues to remain a hard-to-cure disease due to the emergence of drug resistance and frequent relapses.Therefore,it is of great significance to further carry out the research on the mechanism of the occurrence and development of MM.And tumor angiogenesis is one of the important factors that promotes the survival,proliferation,and migration of myeloma cells,and is closely related to the prognosis of patients.The bone marrow microenvironment(BMM)is an internal environment that supports and regulates the settlement,proliferation,differentiation,development,and maturation of hematopoietic cells.And it is mainly composed of stromal cells,extracellular matrix,hematopoietic regulatory factors,and vascular nerves,which play important roles in the survival,proliferation,migration,and drug resistance of MM cells.Tumor-associated macrophages(TAMs)is the most important immune cell in the bone marrow microenvironment,which is derived from monocytes in the blood.The infiltration of TAMs is closely related to MM angiogenesis.Macrophages are polarized under the stimulation of different cytokines in the BMM and are classified into Ml and M2 type according to their phenotypes.M1 TAMs can exert anti-tumor effects and inhibit tumor angiogenesis,while M2 TAMs mainly promote the occurrence and development of tumors,and promote tumor angiogenesis.TAMs in BMM of MM are mainly M2 type macrophages.Exosomes are bilayer-lipid membrane nanovesicles formed by the endocytosis of cell membranes and multivesicular bodies(MVB),with 30-150 nm in diameter.They are secreted by various types of cells,with abundant proteins,mRNA and microRNA.Studies have shown that exosomes can transport their contents to target cells through a variety of pathways,thereby regulating the functions of target cells.Exosomes not only participate in inflammatory response through antigen presentation,immunosuppression,signal molecule transport,but also are closely related to tumor growth,metastasis,and angiogenesis.In addition,exosomes are also involved in the polarization of TAMs.MicroRNA(miRNA)is a type of endogenous non-coding single-stranded RNA that participates in post-transcriptional gene regulation and is about 20-24 nucleotides in length.Studies have shown that tumor cell-derived exosomes can synthesize miRNAs,and do not need to rely on their origin cells.The exosomes secreted by different tumor cells contain special miRNAs,which can induce tumor cell proliferation,extracellular matrix remodeling and MM cell migration,invasion,and tumor blood vessel formation.Bone marrow mesenchymal stem cells(BMMSCs)are important stromal cells in the tumor microenvironment.They can communicate with leukemia cells by miRNAs carried by BMMSCs-derived exosomes.Through such cell-cell communication,miRNA participates in regulating the proliferation,apoptosis,migration,and drug resistance of leukemia cells.Among them,miR-let-7c is highly expressed in exosomes secreted by BMMSCs.So far,there are few studies on the role of exosomes released by bone marrow mesenchymal stem cells in MM.From previous research and literature analysis,we found that miR-let-7c in BMMSCs-derived exosomes of MM patients plays an important role on regulating angiogenesis in the BMM,and proposed that miR-let-7c in BMMSCs-derived exosomes may enhance the angiogenesis of MM by promoting the polarization of macrophages into M2 TAMs on the one hand.On the other hand,hypoxia-inducible factor 1α(HIF-1α)/Let-7/argonautel(AGO1)/VEGF signaling pathway participates in the hypoxia-induced blood vessel formation.In this study,we will study the effect and possible mechanisms of miR-let-7c in BMMSCs-derived exosomes on macrophage polarization and MM angiogenesis and investigate the effects and possible mechanisms of inhibition and overexpression of miR-let-7c on macrophage polarization and MM angiogenesis in vivo and in vitro,which can provide a theoretical basis for further block the formation of MM blood vessels.Methods1.Isolation,culture,and identification of BMMSCs:we collected bone marrow aspirate samples of patients with MM under sterile conditions,and obtained mononuclear cells by density gradient centrifugation,then cultured them with αminimal essential medium in a six-well plate.After 2-4 passages of subculture,flow cytometry is used to detect BMMSCs specific surface markers STRO-1,CD 105 and CD90 to identify the cultured cells.2.Extraction and characterization of BMMSCs-derived exosomes:we both used ultracentrifugation and ExoQuick-TC kit to extract exosomes.We clarified the morphology of the extracted exosomes by transmission electron microscope,and analyzed the size and distribution using nanoparticle tracking analyzer.Exosomes were stained for their membrane markers using anti-CD63-FITC and anti-CD81-PE antibodies and analyzed by flow cytometry.3.BMMSCs-derived exosomes induce macrophage polarization:peripheral blood mononuclear cells(PBMCs)are separated from the peripheral blood of healthy donors by density gradient centrifugation,and macrophages in PBMCs are obtained by CD 14 magnetic beads positive sorting.The macrophages were divided into BMMSCs and macrophages co-culture group,exosomes and macrophages co-culture group,and control group where macrophages were cultured alone.Flow cytometry was used to detect the expression of CD206,the surface marker of M2 macrophages in each group;qRT-PCR was used to detect the expression of miR-let-7c in each group.According to the operating protocol of PKH26 staining solution,PKH26 fluorescently labeled exosomes were co-cultured with CD68-FITC macrophages,and the uptake of exosomes by macrophages was observed under a confocal microscope after DAPI staining.4.Regulating the expression of miR-let-7c in macrophages influences its polarization:we used LV3-miR-let-7c mimics and LV3-miR-let-7c inhibitor lentiviral vectors to infect macrophages,and selected stable cell lines with activation and inhibition of miR-let-7c expression,and LV3-NC was set as a control group.qRTPCR was used to verify the expression level of miR-let-7c in macrophages of each group.Flow cytometry was used to detected the changes in the level of M2 specific marker CD206 in each group.5.Isolation,culture,and identification of myeloma vascular endothelial cells(MVECs):mononuclear cells were obtained from the bone marrow aspirate of MM patients by density gradient centrifugation,and myeloma endothelial progenitor cells were extracted by the adherent sorting,and MVECs were further esatablished in EGM-2 by induction of growth factors like VEGF,hFGF.After 2-4 passages of subculture,MVECs were collected and the vascular endothelial cell specific surface markers CD31 and CD34 were analysed d by immunocytochemistry.6.The effect of M2 macrophages on MVECs:The cells were divided into MVECs+M2 macrophages co-culture group,MVECs+MΦs macrophages coculture group,and MVECs alone culture group(as a control group),We used CCK-8 kit to detect the proliferation ability of MVECs in each group,the scratch test and transwell migration test to analysed the migration ability,and tube formation assay to observe the tube formation ability of MVECs in vitro,and qRT-PCR was used to detects the expression of miR-let-7c in MVEC cells of each group.7.We established myeloma xenograft mouse models where mononuclear and macrophages system was remained or depleted in vivo.The experimental group was injected with exosomes derived from BMMSCs,and the control group was injected with the same dose of sterile deionized water.At the end of the experiment,the tumors were removed,photographed,and measured for their volume and weight.qRT-PCR was used to anaylse the expression of miR-let-7c in the xenograft tissues of each group,and immunohistochemical(IHC)were used to label vascular endothelial cells with CD31 and then measure the micro vessel density(MVD).CD 16 and CD 163 were marked by IHC to reveal the M1 and M2 macrophages respectively in each group,and we alse used IHC and Western blot to detect the protein expression of HIF-1α,AGO1 and VEGF,FISH and qRT-PCR to detect the mRNA expression of HIF-1α,AGO1 and VEGF in xenograft tissues of each group.8.The LV3 lentiviral vector of miR-let-7c mimics and miR-let-7c inhibitor was used to infect MVECs,and an NC groups was set,qRT-PCR was used to verify the expression level of miR-let-7c in MVECs of each group.We used CCK-8 kit to detect the proliferation ability of MVECs in each group,the scratch test and transwell migration test to analysed the migration ability,and tube formation assay to observe the tube formation ability of MVECs.9.The miRDB and Targetscan was used to predict and screen the target regulatory region of miR-let-7c to AGO1.Luciferase expression assay verified the targeted regulation of miR-let-7c on AGO1:The Mimics-control group and the miRlet-7c mimics group were transfected with AGO1 wild-type(AGO1 3’ UTR-WT)and AGO1 mutant-type(AGO1 3’ UTR-MUT)plasmids,respectively.The luciferase reporting system was used to verified the targeted relationship between miR-let-7c and AGO1.10.We established a myeloma xengraft mouse model where RPMI8226 cells transfect with LV3-miR-let-7c mimics,LV3-miR-let-7c inhibitor,and LV3-NC were injected subcutaneously into the back and shouders of mice.At the end of the experiment,the tumors were removed,photographed,and measured for their volume and weight.qRT-PCR was used to anaylse the expression of miR-let-7c in the xenograft tissues of each group,and IHC were used to label vascular endothelial cells with CD31 and then measure the MVD.CD 16 and CD 163 were marked by IHC to reveal the M1 and M2 macrophages respectively in each group,and we alse used IHC and Western blot to detect the protein expression of HIF-1α,AGO1 and VEGF,FISH and qRT-PCR to detect the mRNA expression of HIF-1α,AGO1 and VEGF in xenograft tissues of each group.11.Statistical analysis:Data were analyzed statistically using the GraphPad Prism 7.0 software and were described as mean ±SD.Between-group differences were analyzed using two-tailed unpaired Student t-tests and ANOVA was used to compare data of more than two groups.Results with α ≤0.05 were considered statistically significant.Results1.The BMMSCs successfully obtained by primary culture were positive for the MSC surface specific markers by flow cytometry,which were:STRO-1(98.8%),CD105(96.2%),and CD90(98.8%).These results meet the characteristics of BMMSCs.2.A classic cup-shaped morphology of exosome was revealed by TEM.Nanoparticle tracking revealed that 71.5%of the particles were in the range of 20200 nm,and the biomarkers,CD63 and CD81,were positively expressed in 81.1%and 91.7%,which meet the characteristics of exosomes.3.Flow cytometry showed that,the levels of M2 macrophages in the exosomes group were significantly higher(p<0.01),qRT-PCR results revealed:the expression level of miR-let-7c in exosomes group were significantly up-regulated.Under a confocal microscope,PKH26-labeled exosomes were found to accumulate in the cytoplasm of DAPI-stained macrophages.4.Stable cell lines with overexpression and inhibition of miR-let-7c-5p were successfully established.Flow cytometry showed that the proportion of M2 macrophages in the control group was significantly lower than that in the miR-let-7c mimics group(p<0.05),but higher in the miR-let-7c inhibitor group(p<0.01).5.MVECs successfully obtained by primary culture were strongly positive for the vascular endothelial cells specific surface markers CD31 and CD34 by immunocytochemistry.These results meet the characteristics of MVECs.6.The CCK-8 assays showed after MVECs and M2 macrophages were cocultured for 72 hours,the cell proliferation ability of MVECs was significantly enhanced(p<0.05),Scratch tests and Transwell migration assays revealed that MVECs migration ability was significantly enhanced after 72 hours of co-cultivation with M2 macrophages(p<0.01).The tube formation assays showed that after macrophages co-cultured with MVECs,the tube forming ability of MVECs was significantly enhanced(p<0.01).7.In the myeloma xenograft mouse model where mononuclear and macrophages system was remained and depleted in vivo,compared with the control group,the volume and weight of the xenograft in exosomes group increased significantly(p<0.05).The qRT-PCR showed that the expression level of miR-let-7c in the exosomes group was significantly higher than that in the control group(p<0.01).The immunohistochemical for marker CD31 showed that MVD of the exosomes group was significantly increased(p<0.05),suggesting that exosomes can promote the vascular formation of myeloma xenografts.M2 macrophages in exosomes group significantly increased compared with the control group(p<0.01),while M1 macrophages did not show any significant reduce compared with the control group(p=0.0698),compared with the control group.And mRNA and protein levels of HIF1α and VEGF in myeloma xenograft tumors increased significantly after exosomal injection(p<0.05),while the mRNA and protein levels of AGO1 decreased(p<0.01).8.The stable strains of MVECs screened by puromycin were infected with LV3miR-let-7c mimics,inhibitor and NC and then were identified by qRT-PCR,We found after overexpression of miR-let-7c,the cell proliferation,migration,and tube formation ability of MVECs were significantly increased(p<0.01).On the contrary,after miR-let-7c expression was inhibited,the proliferation ability,migration ability,and tube formation ability are weaker than the control group(p<0.01).9.The prediction results of miRDB and Targetscan showed that miR-let-7c has the regulatory sites binding that bind to the 3’UTR of targeted AGO1.Compared with the mimics-control+AGO1 WT group,the luciferase activity of the miR-let-7c mimics+AGO1 WT group was significantly reduced,and the difference was statistically significant(p<0.05),which suggested miR-let-7c can target to inhibit the expression of AGO 1.10.In the myeloma xenograft mouse model,he volume and weight of myeloma xengraft of the miR-let-7c mimics group increased significantly(p<0.05);the growth of tumors in the miR-let-7c inhibitor group was inhibited,and its volume and weight is significantly reduced(p<0.01).After up-regulating miR-let-7c level,the infiltration of M2 TAMs in the myeloma xengraft increased significantly(p<0.001),and the M1 TAMs decreased significantly(p<0.01),the MVD value increased significantly(p<0.05),and angiogenesis was promoted correspondingly,while inhibiting the expression of miR-let-7c,the infiltration of M2 TAMs in the myeloma xengraft decreased significantly(p<0.001),and the M1 TAMs show no significant increase(p<0.01),but the MVD value decreased(p<0.05),and angiogenesis was inhibited.Compared with the control group,the mRNA and protein levels of HIF-1α and VEGF in the miR-let-7c mimics group increased significantly(p<0.05),while the mRNA and protein levels of AGO 1 decreased(p<0.05).However,the mRNA and protein levels of HIF-1α and VEGF in the miR-let-7c inhibitor group were significantly reduced(p<0.05),while the mRNA and protein levels of AGO1 increased(p<0.05).Conclusions1.The miRNA-let-7c carried by BMMSCs-derived exosomes can increase the angiogenesis in MM by polarizing M2 macrophages in vitro and in vivo.2.miR-let-7c can participate in regulating the angiogenesis in MM through the HIF-1α/Let-7/AGO1/VEGF pathway.3.The miR-let-7c in exosomes has the potential to become a novel therapeutic target for multiple myeloma. |
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