Inhibition of USP7 suppresses advanced glycation end-induced cell cycle arrest and senescence of human umbilical vein endothelial cells through ubiquitination of p53

Abstract

Diabetes mellitus is a n arising public health concern, and diabetic foot is one of the most common complications of diabetes. Current management for diabetic foot cannot reach optimal remission. In this study, we aim to explore the mechanism underlying the pathogenesis of diabetic foot and provide novel strategies for the treatment of diabetic foot. A total of 10 normal skin tissues and 20 diabetic foot ulcer specimens are collected. Cell proliferation is determined by CCK-8 assay. Cell cycle is determined by flow cytometry, and cell senescence is evaluated by β-galactosidase staining. Co-immunoprecipitation assay is used to explore the interaction between USP7 and p53. Advanced glycation end products (AGEs) are used to establish diabetic cell model, and streptozotocin (STZ) is used to establish diabetic rat model. Our results showed that USP7 expression is increased in diabetic foot ulcer and in human umbilical vein endothelial cells (HUVECs) after treatment with AGEs. Inhibition of USP7 can reduce cell cycle arrest and cell senescence in HUVECs. Moreover, USP7 can interact with p53 and promote its expression through mediating its deubiquitination. Knockdown of p53 can reverse USP7-mediated cell cycle arrest and cell senescence in HUVECs. In diabetic rats, HBX 41108, the specific inhibitor of USP7, can significantly accelerate wound healing. Our study reveals that the inhibition of USP7 can suppress AGEs-induced cell cycle arrest and cell senescence of HUVECs through promoting p53 ubiquitination. USP7 is a potential target for the treatment of diabetic foot ulcers.

Keywords: USP7; cell senescence; deubiquitination; diabetic foot; p53.

Figure1

USP7 expression is increased in diabetic foot ulcer and HUVECs after AGEs treatment(A) USP1, USP4, USP7, USP14, USP15, USP20, USP25 and USP47 mRNA expressions in normal skin tissues (n=10) and diabetic foot ulcers (n=20). (B–D) HUVECs were treated with AGEs. (B) Cell viability of HUVECs. The mRNA level (C) and protein level (D) of USP7 in HUVECs. *P<0.05, ***P<0.001 vs control.

Figure2

I nhibiting USP7 expression and its deubiquitinating activity reduces cell cycle arrest and cell senescence in HUVECs after AGEs treatment (A,B) USP7 expression in HUVECs after the transduction of shUSP7 lentivirus. (C) Cell viability, (D,E) cell cycle, (F) cell senescence (scale bar=100 μm), and (G,H) expressions of p53 and p21 in HUVECs transduced with shUSP7 lentivirus or treated with USP7 inhibitor HBX 41108 (5 μM), following treatment with 250 μg/mL AGEs. *P<0.05, ***P<0.001 vs control. ##P<0.01, ###P<0.001 vs AGEs+shNC+vehicle.

Figure3

USP7 interacts with p53 and mediates its deubiquitination(A–C) USP7 and p53 expressions in HUVECs transduced with oeUSP7 vector, blank vector, shUSP7, or shNC lentivirus vector. (D) Immunoprecipitation was performed using anti-USP7 or anti-p53 antibody. (E) p53 expression in HUVECs transduced with shUSP7 or shNC lentivirus and treated with 10 μM MG132 or vehicle. (F) The ubiquitination level of p53 in HUVECs transduced with shUSP7 lentivirus. (G) USP7 and p53 expressions in skin tissues of patients with diabetic foot ulcer (n=20) and normal skin tissues (n=10). (H) Pearson correlation plot of the expressions of p53 and USP7 in skin tissues (n=20). ***P<0.001 vs vector or control. ##P<0.01, ###P<0.001 vs shNC or DUSS.

Figure4

p53 silencing ameliorates USP7 overexpression-mediated cell cycle arrest and cell senescence in HUVECs(A,B) p53 expression in HUVECs after the transduction with shP53 lentivirus. (C) Cell viability, (D,E) cell cycle, (F) cell senescence (scale bar=100 μm), and (G,H) the expressions of p53 and p21 in HUVECs transduced with oeUSP7 vector and shP53 lentivirus. ***P<0.001 vs shNC or vector+shNC. ###P<0.001 vs oeUSP7+shNC.

Figure5

HBX 41108 facilitates wound healing in diabetic rats Rats were divided into control group, the STZ group, and HBX 41108 group(A) Blood glucose level (mM) changes in different groups of rats. (B) The images of skin wounds at day 0, 7, and 14 post-injury. (C) The wound healing rates at day 0, 7, and 14. (D) HE staining of wound tissues (scale bar=100 μm). (E) Expressions of USP7, p53 and p21 in tissues at day 7 and 14 post-injury in each group. ***P<0.001 vs control. #P<0.05, ###P<0.001 vs STZ+vehicle. S, scab; E, epidermis.