Regulation Of Human Umbilical Vein Endothelial Cell Senescence By DCAF13 Through PI3K Signaling Pathway

Objective:The purpose of this study was to investigate the effects of CRL4 E3 ubiquitin ligase complexes(Cullin4 Ring E3 ligases)on the aging of human umbilical vein endothelial cells(HUVEC),as well as the effects of CRL4 E3 ubiquitin ligase complex substrate recognition protein DCAF13(DDB1-CUL4 associated factors,DCAF13)on the aging,proliferation,migration,and tubular formation of human umbilical vein endothelial cells.Methods: The effect of knocking down CRL4 E3 ubiquitin ligase complex and DCAF13 on HUVEC aging was detected by β-gal staining;The inhibitory effect of knocking down DCAF13 on HUVEC activity was determined by CCK-8 method;Scratch and transwell test were used to observe the effect of knocking down DCAF13 on HUVEC migration ability;The effect of knocking down DCAF13 on HUVEC tube forming ability was measured by tube forming test;Western blot was used to detect the expression of aging related proteins Lamin B1,FOXO4,PAI-1,IGFBP7 and cyclic-related proteins p21 and p53.Results: In this study,we used β-gal staining found that after knocking down the key proteins DDB1,ROC1,CUL4 B and DCAF13 in HUVEC,compared with NC group,the number of cells stained blue in si DDB1,si ROC1,si CUL4 B and si DCAF13 groups increased significantly(P<0 01);Western blot results showed a significant increase in the expression of aging related proteins PAI-1 and IGFBP7 in the si DDB1,si ROC1,si CUL4 B,and si DCAF13 groups;At the same time,Western blot,q-PCR and immunofluorescence results showed that the expression of cycle related proteins such as p21,p53,and cyclin B1 increased in the si DCAF13 group.Flow cytometry results showed that the cell cycle was blocked in G1 phase phase after knocking down DCAF13.In addition,experiments such as CCK-8,Transwell,and tube formation showed a decrease in the proliferation and migration ability of HUVEC cells after DCAF13 knockout,and a decrease in the tube formation ability of HUVEC.Meanwhile,mechanism studies have found that knocking down DCAF13 reduces the expression of PI3 K and AKT in HUVEC.Conclusion: CRL4 ubiquitin ligase complex promotes HUVEC cell aging by affecting DCAF13;Knocking down DCAF13 can promote the aging of HUVEC,affect the cell cycle of HUVEC,inhibit the proliferation and migration of HUVEC,and the ability to form tubules.Moreover,knocking down DCAF13 may promote HUVEC cell aging,inhibit HUVEC proliferation,migration,and tubule formation through the PI3K-AKT signaling pathway.