| Background and ObjectiveMultiple myeloma(MM)is a plasma cell dysplastic disease,which is the second most common malignant tumor in the blood system.Angiogenesis is the primary condition for the growth of MM.It is of great significance for the treatment and long-term survival of patients with MM to explore the mechanism of MM angiogenesis and inhibit its angiogenesis.Vascular endothelial growth factor A(VEGFA)is the most important vascular growth factor,which can specifically bind to vascular endothelial growth factor receptor(VEGFR),stimulate the proliferation and migration of vascular endothelial cell(VEC),and play an important role in tumor angiogenesis.Tumor-associated macrophages(TAMs)are important part of tumor microenvironment,which can secrete vascular endothelial growth factor,angiopoietin,platelet-derived factor and other cytokines that promote vascular growth,and then affect the occurrence and development of tumor vessels.It was found that after transfected VEGFA siRNA into MM cells,the expression of VEGFA in MM cells was down-regulated,but the angiogenesis of MM was not significantly decreased,so it is speculated that inhibiting the expression of VEGFA in MM cells alone can not inhibit MM angiogenesis.There may be tumor microenvironment factors including TAMs that also affect MM angiogenesis.In order to investigate the synergistic effect of MM cells and TAMs on angiogenesis of MM,human monocytic leukemia cells(THP-1)were induced to differentiate into M1 and M2 macrophages,and then co-cultured with VEC.M2 macrophages and myeloma RPMI 8226 cells were transfected with VEGFA siRNA,and VEC was cultured in conditioned medium before and after transfection.The proliferation,migration and tubule formation ability of VEC in each group were compared by CCK8 proliferation test,transwell migration test and tubule formation test.ELISA test was used to detect the concentration of VEGFA in each group of culture medium,and qRT-PCR and Western blot techniques were used to detect the expression of VEGFR1 mRNA and protein in VEC of each group.To explore the effect of M1 and M2 macrophages on MM angiogenesis and whether MM cells and TAMs can promote MM angiogenesis together will provide a new target for the treatment of MM.Materials and Methods1 Induction of Ml and M2 macrophages in vitroTHP-1 cells were cultured in the medium containing Phorbol-12-myristate-13-acetate(PMA)at the concentration of 100 ng/ml for 48 h,and M2 macrophages were obtained by changing the medium containing interleukin 4(IL-4)at the concentration of 20 ng/ml for 48 h,and replacing the medium containing interferon y(IFN-y)at the concentration of 50 ng/ml and lipopolysaccharide(LPS)at the concentration of 100 ng/ml for 48 h to obtain M1 macrophages.2 Effects of Ml and M2 macrophages on the ability of VEC tube formation2.1 Co-culture of Ml and M2 macrophages with VECTHP-1 cells were induced into M1 and M2 macrophages in the upper chamber of 0.4 ?m Transwell,and then co-cultured with VEC in the lower chamber for 48 h.The co-culture groups were as follows:NC group:VEC cultured in normal medium;co-M2 group:VEC co-cultured with M2 macrophages;co-M1 group:VEC co-cultured with M1 macrophages.2.2 Detect the related indexes of each group(1)Detect the concentration of VEGFA in the supernatant of culture medium in each group by ELISA method.(2)Detect the proliferative ability of VEC in each group by CCK8 method.(3)Detect the migration ability of VEC in each group by Transwell migration method.(4)Detect the tubule formation ability of VEC in each group by tubule formation test.(5)Detect the expression of VEGFR1 mRNA of VEC in each group by qRT-PCR method.(6)Detect the expression of VEGFR1 protein of VEC in each group by Western blot method.3 Effects of the ability of VEC tube formation before and after VEGFA siRNA transfect of myeloma RPMI 8226 cells and M2 macrophages3.1 Construction and transfection of VEGFA siRNAThree interference sequences were designed for human VEGFA siRNA.The three sequences were transfected into myeloma RPMI 8226 cells respectively.The expression of VEGFA mRNA after transfection was detected by qRT-PCR method,and the best interference sequence was selected by comparison.3.2 Collect conditioned medium and groupCollected the media before and after transfection of RPMI 8226 cells and M2 macrophages to culture VEC,and grouped as follows:(1)Before transfection:NC group,VEC cultured in normal medium;RP group,VEC cultured in RPMI 8226 cell medium;M2 group,VEC cultured in M2 medium;RP+M2 group,VEC cultured in the mixed medium of RPMI 8226 cell medium and M2 medium.(2)After transfection:RP+M2 group,VEC cultured in the mixed medium of RPMI 8226 cell medium and M2 medium;siRP+M2 group,VEC cultured in the mixed medium of post-transfection RPMI 8226 cell medium and M2 medium;RP+siM2 group,VEC cultured in the mixed medium of RPMI 8226 cell medium and post-transfection M2 medium;siRP+siM2 group,VEC cultured in the mixed medium of post-transfection RPMI8226 cell medium and post-transfection M2 medium.3.3 Detect the related indexes in each group before and after transfectionThe detection method of related indexes is the same as 2.2.4 Statistical analysisThe experimental data were analyzed by SPSS 21.0 software and expressed as mean ± standard deviation(x ± S).The differences among groups were analyzed by one-way analysis of variance(ANOVA).The test level is ?=0.05.All the experiments were carried out independently for 3 times.Results1 Effects of Ml and M2 on the proliferation,migration and tube formation ability of VEC in vitro1.1 The concentration of VEGFA in the supernatant of VEC medium in each groupThe concentration of VEGFA in the supernatant in each group was detected by ELISA method.The concentration of VEGFA in co-M2 group was significantly higher than that of NC group(P<0.05),while the concentration of VEGFA in co-Ml group was significantly lower than that of NC group(P<0.05).1.2 The proliferation ability of VEC in each groupThe proliferation ability of VEC in each group was detected by CCK8 method.The proliferation ability of VEC in co-M2 group was significantly higher than that in NC group(P<0.05),while the proliferation ability of VEC in co-M1 group was significantly lower than that in NC group(P<0.05).1.3 The migration ability of VEC in each groupThe migration ability of VEC in each group was detected by Transwell migration method.The ability of VEC migration in co-M2 group was significantly higher than that in NC group(P<0.05),while the ability of VEC migration in co-M1 group was significantly lower than that in NC group(P<0.05).1.4 The tube formation ability of VEC in each groupThe tube formation ability of VEC in each group was detected by matrix glue tubule formation test.The ability of VEC tube formation in co-M2 group was significantly higher than that in NC group(P<0.05),while the ability of VEC tube formation in co-M1 group was significantly lower than that in NC group(P<0.05).1.5 The expression of VEGFR1 mRNA in VEC of each groupThe expression of VEGFR1 mRNA in VEC of each group was detected by qRT-PCR experiment.The expression of VEGFR1 mRNA in VEC of co-M2 group was 5 times higher than that of NC group and significantly higher than that of NC group(P<0.05),while the expression of VEGFR1 mRNA in VEC of co-M1 group was significantly lower than that of NC group(P<0.05).1.6 The expression of VEGFR1 protein in VEC of each groupThe expression of VEGFR1 protein in VEC of each group was detected by Western blot method.The expression of VEGFR1 protein in VEC of co-M2 group was significantly higher than that of NC group(P<0.05),and the expression of VEGFR1 protein in VEC of co-M1 group was significantly lower than that of NC group(P<0.05).2 Synergistic effects of myeloma RPMI8226 cells and M2 macrophages on the proliferation,migration and tube formation ability of VEC in vitro2.1 The concentration of VEGFA in the supernatant of VEC medium in each groupThe concentration of VEGFA in the supernatant in each group was detected by ELISA method.Compared with NC group,the concentration of VEGFA in culture medium of RP group,M2 group and RP+M2 group were higher(P<0.05),and the concentration of VEGFA in RP+M2 group was the highest(P<0.05).2.2 The proliferation ability of VEC in each groupThe proliferation ability of VEC in each group was detected by CCK8 method.Compared with NC group,the proliferation ability of VEC in RP group,M2 group and RP+M2 group were stronger(P<0.05),and the proliferation ability of VEC in RP+M2 group was the strongest(P<0.05).2.3 The migration ability of VEC in each groupThe migration ability of VEC in each group was detected by Transwell migration method.Compared with NC group,the migration ability of VEC in RP group,M2 group and RP+M2 group were stronger(P<0.05),and the migration ability of VEC in RP+M2 group was the strongest(P<0.05).2.4 The tube formation ability of VEC in each groupThe tube formation ability of VEC in each group was detected by matrix glue tubule formation test.Compared with NC group,the tube formation ability of VEC in RP group,M2 group and RP+M2 group were stronger(P<0.05),and the tube formation ability of VEC in RP+M2 group was the strongest(P<0.05).2.5 The expression of VEGFR1 mRNA in VEC of each groupThe expression of VEGFR1 mRNA in VEC of each group was detected by qRT-PCR experiment.Compared with NC group,the expression of VEGFR1 mRNA of VEC in RP group,M2 group and RP+M2 group were higher(P<0.05),and the expression of VEGFR1 mRNA of VEC in RP+M2 group was the highest(P<0.05).2.6 The expression of VEGFR1 protein in VEC of each groupThe expression of VEGFR1 protein in VEC of each group was detected by Western blot method.Compared with NC group,the expression of VEGFR1 protein of VEC in RP group,M2 group and RP+M2 group were higher(P<0.05),and the expression of VEGFR1 protein of VEC in RP+M2 group was the highest(P<0.05).3 Effects of VEC proliferation,migration and tube formation ability after VEGFA siRNA transfect of myeloma RPMI 8226 cells and M2 macrophages3.1 Select the best interference sequenceThree designed and synthesized VEGFA siRNA sequences,S1,S2,S3 and NC sequences,were transfected into RPMI 8226 cells in logarithmic phase respectively.The expression of VEGFA mRNA in each group was detected by qRT-PCR after 48 h.Compared with the NC sequence,the expression of VEGFA mRNA in each group was significantly decreased after transfection of S1,S2,S3 sequence(P<0.05),and the expression of VEGFA mRNA in S1 sequence group was the lowest,so this sequence was used as the transfection sequence in the follow-up experiment.3.2 The concentration of VEGFA in the supernatant of VEC medium in each group after transfectionThe concentration of VEGFA in the supernatant in each group after transfection was detected by ELISA method.Compared with RP+M2 group,the concentration of VEGFA in culture medium of siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the concentration of VEGFA in siRP+siM2 group was the lowest(P<0.05).3.3 The proliferation ability of VEC in each group after transfectionThe proliferation ability of VEC in each group after transfection was detected by CCK8 method.Compared with RP+M2 group,the proliferation ability of VEC in siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the proliferation ability of VEC in siRP+siM2 group was the lowest(P<0.05).3.4 The migration ability of VEC in each group after transfectionThe migration ability of VEC in each group after transfection was detected by Transwell migration method.Compared with RP+M2 group,the migration ability of VEC in siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the migration ability of VEC in siRP+siM2 group was the lowest(P<0.05).3.5 The tube formation ability of VEC in each group after transfectionThe tube formation ability of VEC in each group after transfection was detected by matrix glue tubule formation test.Compared with RP+M2 group,the tube formation ability of VEC in siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the tube formation ability of VEC in siRP+siM2 group was the lowest(P<0.05).3.6 The expression of VEGFR1 mRNA in VEC of each group after transfectionThe expression of VEGFR1 mRNA in VEC of each group after transfection was detected by qRT-PCR experiment.Compared with RP+M2 group,the expression of VEGFR1 mRNA of VEC in siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the expression of VEGFR1 mRNA of VEC in siRP+siM2 group was the lowest(P<0.05).3.7 The expression of VEGFR1 protein in VEC of each group after transfectionThe expression of VEGFR1 protein in VEC of each group after transfection was detected by Western blot method.Compared with RP+M2 group,the expression of VEGFR1 protein of VEC in siRP+M2 group,siM2+RP group and siRP+siM2 group were decreased(P<0.05),and the expression of VEGFR1 protein of VEC in siRP+siM2 group was the lowest(P<0.05).Conclusions1 M2 macrophages can promote the proliferation,migration and tube formation of VEC,while M1 macrophages can inhibit the proliferation,migration and tube formation of VEC.2 RPMI 8226 cells and M2 macrophages can synergistic promote the proliferation,migration and tube formation of VEC,inhibit the expression of VEGFA in RPMI 8226 cells and M2 macrophages can synergistic restrain the proliferation,migration and tube formation of VEC.3 VEGFA produced by RPMI 8226 cells and M2 macrophages may play a role in VEC through the receptor VEGFR1. |
PDF Full Text Request