hsa_circ_0058092 protects against hyperglycemia‑induced endothelial progenitor cell damage via miR‑217/FOXO3

Abstract

Circular RNAs (circRNAs) regulate the expression of genes that are critical for various biological and pathological processes. Previous studies have reported that the expression of hsa_circ_0058092 is decreased in patients with diabetes mellitus (DM); however, the specific role of this circRNA in DM is unknown. In the present study, endothelial progenitor cells (EPCs) were isolated and a decreased hsa_circ_0058092 expression was found under conditions of hyperglycemia (HG). The overexpression of hsa_circ_0058092 protected the EPCs against HG‑induced damage by preserving cell survival, proliferation, migration and angiogenic differentiation. The overexpression of hsa_circ_0058092 also decreased the HG‑induced increase in NADPH‑oxidase proteins and inflammatory cytokines. Further investigation revealed that the overexpression of hsa_circ_0058092 enhanced FOXO3 expression, which was mediated through the interaction with miR‑217. Furthermore, the upregulation of miR‑217 or the downregulation of FOXO3 abolished the protective effects of hsa_circ_0058092 against HG‑induced EPC damage. On the whole, these data suggest that hsa_circ_0058092 acts via the miR‑217/FOXO3 pathway to protect against EPCs HG‑induced damage, and to preserve the migration and angiogenesis of EPCs.

Figure 1

HG decreases the expression of hsa_circ_0058092 in EPCs. (A) Flow cytometric analysis of cell surface markers in human peripheral blood-derived EPCs (CD34, KDR, CD45 and CD14). (B) RT-qPCR detection for the expression of hsa_circ_0058092 in EPCs following treatment with various glucose concentrations (0-40 mM). Data are presented as the means ± SD. ^*P<0.05, ^***P<0.001 vs. control. EPCs, endothelial progenitor cells.

Figure 2

Overexpression of hsa_circ_0058092 reverses EPC damage induced by 30 mM glucose (HG). (A) RT-qPCR detection for the expression of hsa_circ_0058092 in EPCs following transfection with hsa_circ_0058092 overexpression vector. Data are presented as the means ± SD. ^***P<0.001 vs. normal control (NC). (B) CCK-8 assay for the proliferation of EPCs. Data are presented as the means ± SD. ^**P<0.01, ^***P<0.001 vs. NC; ^##P<0.01 vs. HG. (C) Expression of the oxidative stress proteins, NOX1 and NOX4, measured by western blot analysis. GAPDH served as an internal control. (D and E) Apoptosis of EPCs determined by Annexin V/PI staining 24 h following HG induction. Data are presented as the means ± SD. ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. (F and G) Transwell assays for the migration of EPCs. Data are presented as the means ± SD. ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. Scale bar, 95 µm. (H and I) EPC tube formation capabilities were measured (magnification, 200). Data are presented as the means ± SD. ^*P<0.05, ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. (J-L) Levels of the inflammatory cytokines, IL-1β, IL-6 and TNF-α, were measured by ELISA. Data are presented as the means ± SD. ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. EPCs, endothelial progenitor cells; HG, high glucose.

Figure 3

miR-217 is a target of hsa_circ_0058092. (A) Predicted binding sites for miR-217 in hsa_circ_0058092. Mutated (Mut) version of hsa_circ_0058092 is also shown. (B) Relative luciferase activity was determined at 48 h following transfection with miR-217 mimic/NC or hsa_circ_0058092 wild-type/Mut in 293T cells. Data are presented as the means ± SD. ^***P<0.001. (C) RT-qPCR detection of miR-217 expression in EPCs transfected with or without hsa_circ_0058092 overexpression vector and treated with or without HG. Data are presented as the means ± SD. ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. (D) RT-qPCR detection of miR-217 expression following treatment with miR-217 inhibitor. Data are presented as the means ± SD. ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. (E) CCK-8 assay for the proliferation of EPCs. Data are presented as the means ± SD. ^**P<0.01, ^***P<0.001 vs. NC; ^##P<0.01 vs. HG. (F and G) Transwell assays for the migration of EPCs. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. Scale bar, 95 µm. (H and I) EPC tube formation capacity was measured (magnification, ×200). Data are presented as the means ± SD. ^**P<0.01, ^***P<0.001 vs. control; ^###P<0.001 vs. HG. (J) Expression of oxidative stress proteins NOX1 and NOX4 was measured by western blot analysis. GAPDH served as an internal control. (K-M) Levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were measured by ELISA. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. EPCs, endothelial progenitor cells; HG, high glucose.

Figure 4

FOXO3 is a target of miR-217. (A) Predicted binding sites for miR-217 in FOXO3 3′UTR. Mutated (Mut) version of 3′UTR-FOXO3 is also shown. (B) Relative luciferase activities were determined at 48 h following transfection with miR-217 mimic/NC or 3′UTR-FOXO3 wild-type/Mut in 293T cells. Data are presented as the means ± SD. ^***P<0.001. (C) RT-qPCR detection of FOXO3 expression in EPCs transfected with miR-217 mimic. Data are presented as the means ± SD. ^***P<0.001 vs. NC. (D) RT-qPCR detection shows miR-217 and FOXO3 expression in EPCs following transfection with or without FOXO3 over-expression vector under HG conditions. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. (E) CCK-8 assay for the proliferation of EPCs. Data are presented as the means ± SD. ^**P<0.01, ^***P<0.001 vs. control; ^#P<0.05 vs. HG. (F and G) Transwell experiments for the migration of EPCs. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. Scale bar, 95 µm. (H and I) EPC tube formation capacity was measured (magnification, ×200). Data are presented as the means ± SD. ^**P<0.01, ^***P<0.001 vs. NC; ^###P<0.001 vs. HG. (J) Expression of oxidative stress proteins NOX1 and NOX4 was measured by western blot analysis. GAPDH served as an internal control. (K-M) Levels of the inflammatory cytokines, IL-1β, IL-6 and TNF-α, were measured by ELISA. Data are presented as the means ± SD. ^***P<0.001 vs. control. ^###P<0.001 vs. HG. EPCs, endothelial progenitor cells; HG, high glucose.

Figure 5

Overexpression of miR-217 or downregulation of FOXO3 reverses the protective effects of hsa_circ_0058092 on EPC proliferation, migration and angiogenic differentiation. (A-C) RT-qPCR detection for the expression of (A) hsa_circ_0058092, (B) miR-217 and (C) FOXO3 in EPCs. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. (D) CCK-8 assay for the proliferation of EPCs. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^##P<0.01 vs. HG. (E and F) EPC apoptotic rate was determined by Annexin V/PI staining 24 h following induction with HG (30 mM). Data are presented as the means ± SD. ^***P<0.001 vs. control; ^##P<0.01 vs. HG. (G and H) Transwell assays for the migration of EPCs. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. Scale bar, 95 µm. (I and J) EPC tube formation capacity was measured (magnification, ×200). Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG. (K) Expression of the oxidative stress proteins, NOX1 and NOX4, was measured by western blot analysis. GAPDH served as n internal control. (L-N) Levels of the inflammatory cytokines, IL-1β IL-6 and TNF-α, were measured by ELISA. Data are presented as the means ± SD. ^***P<0.001 vs. control; ^###P<0.001 vs. HG.