Effects Of Downregulation Of DJ-1 Gene On Angiogenesis Induced By Multiple Myeloma Cell

Objectives:To investigate whether DJ-1 gene plays an important role in the angiogenesis mediated by myeloma cells.Methods:1.Human myeloma RPMI8226 cell line was infected with control shRNA lentiviral particles-A(shControl)and DJ-1 shRNA(h)lentiviral particles(shDJ-1),respectively.Next,the stably transfected cells were screened in medium containing 2%purinomycin,and DJ-1 proteinwas detected by Western Blot to verify the effectiveness ofdownregulation.2.After downregulating the expression of DJ-1 in RPMI8226 cells by lentivirus transfection,the culture medium of Control group,shControl group and shDJ-1 group were respectively collected as conditioned medium.3.The effects of conditioned medium on the proliferation,migration and tubulization of human umbilical vein endothelial cells(HUVECs)in vitro were detected by CCK8 colorimetry,transwell migration experiment and matrix gluing tube test,respectively.4.The levels of Vascular endothelial growth factor A(VEGF-A),basic fibroblast growth factor(bFGF),Interleukin 6(IL-6)and angiogenin1(Ang-1)in the conditioned medium were detected by enzyme linked immunosorbent assay(ELISA).5.Western Blot was used to detect VEGF-A,bFGF and Ang-1protein in the RPMI8226 cellsControl group,shControl group and shDJ-1-1 group.Results:1.Multiple myeloma cell line RPMI8226 was transfected with DJ-1 shRNA lentivirus interference vector(shDJ-1),and the transfected cells were screened out by continuous culture in a complete medium containing 2%purinomycin.It was verified that the protein level of DJ-1 in the shDJ-1 group was significantly lower than those of the Control group(P<0.001)and the shControl group(P<0.001),while there was no significant difference between the Control group and the shControl group(P=0.723),indicating that we successfully constructed a cell model of DJ-1 knockdownin RPMI8226.2.Compared with HUVECs treated in the conditioned medium from Control and shControl culture,the number of HUVECs treated in conditioned medium from shDJ-1 culture was significantly lowerat different time points(shDJ-1 vs Control:n=12601±3195 vs 14720±783 at 24h,P<0.001;n=23761±2366 vs 34802±3121 at 48h,P<0.001;n=38192±3097 vs 60151±1350 at 72h,P<0.001;n=62308±856 vs 74014±877 at 96h,P<0.001),(shDJ-1 vs shControl:n=12601±3195 vs 14240±501at 24h,P<0.001;n=23761±2366 vs 31984±1469 at 48h,P<0.001;n=38192±3097 vs 59249±873 at 72h,P<0.001n=62308±856 vs 72960± 2617 at 96h,P<0.001)3.After HUVECs were disposed with conditioned medium,the number of HUVECs passing through the membrane in the shDJ-1 group wassignifycantly reduced,compared with that in the Control group(n=27.33±2.08 vs 38.67±4.04,P=0.004)and theshControl group(n=27.33±2.08 vs36.33±2.52=P=0.01).4.HUVECs were treated with conditioned medium,after 8 and 12hours,the number of "tree bud" branches formed on matrix glue in shDJ-1 group was significantly lower than that in Control group(n=14.25±2.22 vs 33.00±3.92 at 8h,P<0.001;n=27.75±2.22 vs 58.00±3.37 at 12h,P<0.001)and shControl group(n=14.25±2.22vs 32.00±2.94 at 8h,P<0.001;n=27.75±2.22vs 53.50±4.04 at 12h,P<0.001).5.After lentivirus transfection of myeloma cell lines RPMI8226,the levels of VEGF-A,bFGFand IL-6in the supernatants ofshDJ-1 cultureweredistinctlylower than those in Control group(bFGF:2.01±0.03 vs 2.08±0.07pg/mL,P<0.001;VEGF-A:10.66±0.21 vs 12.19±0.92pg/mL,P<0.001;IL-6:1.24±0.03 vs 1.32±0.10pg/mL,P=0.001)and shControl group(bFGF:2.01±0.03 vs 2.060.03pg/mL,P=0.006;VEGF-A:10.66±0.21 vs 11.77±0.71pg/mL,P<0.001;IL-6:1.24±0.03 vs 1.29±0.07pg/mL,P=0.04),while the levels of Ang-1were not significant different(2.060.05 vs 2.120.16 vs 2.080.09ng/mL,P=0.072).6.VEGF-AandbFGFprotein levels in shDJ-1 RPMI8226 cellswere significantly lower than those in Control cells(VEGF-A:P<0.001,bFGF:P<0.001)and shControlcells(VEGF-A:P<0.001,bFGF:P<0.001),while there was no significant difference of Ang-1 protein among the three cell groups(P=0.153),Conclusion:Myeloma cells withDJ-1 knockdowninhibitthe proliferation,migration and angiogenesis of HUVECs in vitro,suggesting that DJ-1 may play an important role in the pro-angiogenic effect mediated by myeloma cells,and its mechanism may be related to the decreased secretion of pro-angiogenic factors such as VEGF-A,bFGF and IL-6.