Long non‑coding RNA NORAD regulates angiogenesis of human umbilical vein endothelial cells via miR‑590‑3p under hypoxic conditions

Abstract

Dysregulation of angiogenesis can be caused by hypoxia, which may result in severe diseases of the heart, including coronary artery disease. Hypoxia‑inducible factor 1 (HIF‑1) modulates angiogenesis via the regulation of several angiogenic factors. However, the underlying mechanism of hypoxia‑induced angiogenesis remains unknown. In the present study, it was hypothesized that long non‑coding RNA (lncRNA) non‑coding RNA activated by DNA damage (NORAD) may serve a role in the process of angiogenesis via the regulation of microRNA(miR)‑590‑3p under hypoxic conditions. The effect of NORAD and miR‑590‑3p on cell viability and properties associated with angiogenesis, including cell migration and tube formation in human umbilical vein endothelial cells (HUVECs) under hypoxic conditions, were assessed. Potential downstream angiogenic factors of miR‑590‑3p were also determined by molecular experiments. It was identified that NORAD expression was upregulated and miR‑590‑3p expression was downregulated in hypoxia‑exposed HUVECs, and also in myocardial infarction (MI) left ventricle tissues in mice. Moreover, downregulation of NORAD expression resulted in decreased cell viability and angiogenic capacity, but further knocking down miR‑590‑3p expression reversed these alterations, resulting in increased cell migration and tube formation in HUVECs under hypoxic conditions for 24 h. It was demonstrated that NORAD overexpression also increased cell vitality and tube‑formation capacity. Furthermore, NORAD was identified to bind with miR‑590‑3p directly, and miR‑590‑3p was shown to target certain proangiogenic agents, such as vascular endothelial growth factor (VEGF)A, fibroblast growth factor (FGF)1 and FGF2 directly. Therefore, the present results suggested that lncRNA NORAD may bind with miR‑590‑3p to regulate the angiogenic ability of HUVECs via the regulation of several downstream proangiogenic factors under hypoxia. Thus, the lncRNA NORAD/miR‑590‑3p axis may be a novel regulatory pathway in the angiogenic mechanisms in HUVECs, which highlights a potentially novel perspective for treating ischemia/hypoxia‑induced angiogenic diseases.

Keywords: hypoxia; non-coding rna activated by dna damage; transcriptional activation; microrna-590-3p.

Figure 1.

HUVEC tube-forming ability is increased under hypoxic conditions. (A) mRNA expression levels of HIF-1α and its target, VEGFA, under normoxic and hypoxic conditions. (B) Protein expression of HIF-1α following hypoxic culturing for 24 h. (C) Cell viability of HUVECs following normoxia and hypoxia treatment for 12 or 24 h, respectively. (D) Transwell migration assay was performed to assess the migratory capacity of cells under normoxic and hypoxic conditions. Representative images of migration in both cells. Magnification, ×200. (E) Tube formation ability of HUVECs under normoxia and hypoxia. Representative images and quantification of tube count and length. Magnification, ×100. **P<0.01 and ***P<0.001 vs. normoxia. HIF-1α, hypoxia-inducible factor-1α; VEGFA, vascular endothelial growth factor A; OD, optical density.

Figure 2.

lncRNA NORAD expression is increased and miR-590-3p expression is decreased in hypoxic HUVECs in vitro and MI left ventricular tissues in vivo. mRNA expression levels of lncRNA NORAD and miR-590-3p in (A) HUVECs under normoxic and hypoxic conditions, and (B) mice left ventricle tissues after 24 h of MI modelling. **P<0.01 and ***P<0.001 vs. normoxia or control. MI, myocardial infarction; lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA.

Figure 3.

lncRNA NORAD knockdown decreases tube formation in HUVECs under hypoxia. (A) Relative expression of lncRNA NORAD in HUVECs following transfection with shNORAD and shNC1. ***P<0.001 vs. shNC1. (B) mRNA expression levels of NORAD and miR-590-3p in HUVECs transfected with shNORAD under hypoxic conditions. ***P<0.001 vs. hypoxia + shNC1. (C) Cell viability of HUVECs transfected with shNORAD under normoxia or hypoxia. *P<0.05 and ***P<0.001 vs. hypoxia + shNC1. (D) Representative images and quantification of migration in HUVECs transfected with shNORAD under hypoxia. Magnification, ×200. ***P<0.001 vs. hypoxia + shNC1. (E) Representative images and quantification of tube formation of HUVECs transfected with shNORAD under hypoxic conditions. Magnification, ×100. *P<0.05 and ***P<0.001 vs. hypoxia + shNC1. lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA; sh, short hairpin RNA; NC, negative control; OD, optical density.

Figure 4.

lncRNA NORAD overexpression increases tube formation in HUVECs under hypoxia. (A) Relative expression of lncRNA NORAD in HUVECs after transfection with NORAD_1-2000-OE plasmid and empty vector. ***P<0.001 vs. vector. (B) mRNA expression levels of NORAD and miR-590-3p following overexpression of NORAD under hypoxic conditions. (C) Cell viability, (D) cell migration (magnification, ×200) and (E) tube formation (magnification, ×100) were assessed 24 h after hypoxia treatment in the NORAD-overexpressing HUVECs. *P<0.05, **P<0.01 and ***P<0.001 vs. Hypoxia + shNORAD + vector. sh, short hairpin RNA; lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA; OD, optical density.

Figure 5.

Overexpression of miR-590-3p suppresses tube-formation in HUVECs under hypoxia. (A) HUVECs were transfected with miR-590-3p mimics and mimics-NC. Relative expression of miR-590-3p following transfection was assessed prior and subsequent to hypoxia treatment. ***P<0.001 vs. respective NC controls. (B) Cell viability, (C) cell migration (magnification, ×200) and (D) tube formation (magnification 100×) were examined in cells transfected with miR-590-3p mimics under hypoxia. *P<0.05, **P<0.01 and ***P<0.001 vs. Hypoxia + mimics-NC group. (E) Relative luciferase activity was measured 48 h after co-transfection of miR-590-3p mimics with NORAD-wt or NORAD-mut in 293T cells. The binding sequences of the wt and mut NORAD are shown. **P<0.01 vs. NORAD-mut + miR-590-3p mimics group. lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA; NC, negative control; mut, mutant; wt, wild-type; OD, optical density.

Figure 6.

lncRNA NORAD regulates angiogenesis of HUVECs under hypoxia via miR-590-3p. (A) Relative expression of miR-590-3p in HUVECs transfected with miR-590-3p inhibitor and inhibitor-NC. ***P<0.001 vs. inhibitor-NC. (B) HUVECs were co-transfected with shNORAD and miR-590-3p inhibitor or shNORAD and inhibitor-NC, and expression levels of lncRNA NORAD and miR-590-3p were determined under atmospheric conditions. (C) Expression levels of lncRNA NORAD and miR-590-3p were again assessed under hypoxic condition after the same HUVEC co-transfection. (D) Cell viability, (E) cell migration (magnification, ×200) and (F) tube formation (magnification, ×100) were measured in HUVECs transfected with both shNORAD and miR-590-3p inhibitor under hypoxia. *P<0.05, **P<0.01 and ***P<0.001 vs. respective inhibitor-NC group. lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA; NC, negative control; sh, short hairpin RNA; OD, optical density.

Figure 7.

Downstream target genes of the lncRNA NORAD/miR-590-3p axis in HUVECs under hypoxia. (A) Relative mRNA expression levels of VEGFA, FGF1 and FGF2 were identified in HUVECs transfected with shNORAD or shNC1, and co-transfected with shNORAD and miR-590-3p inhibitor, or shNORAD and inhibitor-NC under hypoxia for 24 h. (B) ELISA was performed to measure the concentration of VEGFA, FGF1 and FGF2 in transfected HUVECs under hypoxic condition. ***P<0.001 in hypoxia + shNORAD vs. hypoxia + shNC1. ^##P<0.01, ^###P<0.001 in hypoxia + shNORAD + miR-590-3p inhibitor vs. hypoxia + shNORAD + inhibitor-NC. (C) Dual luciferase reporter assay was used to assess binding of miR-590-3p with the proangiogenic factors VEGFA, FGF1 and FGF2. Luciferase activity was assessed in 293T cells following co-transfection of miR-590-3p mimics with the 3′UTR of VEGFA, FGF1 and FGF2 or mutant 3′UTRs. **P<0.01 vs. targeted mutants + miR-590-3p mimics. lncRNA, long non-coding RNA; lncRNA NORAD, lncRNA non-coding RNA activated by DNA damage; miR, microRNA; VEGFA, vascular endothelial growth factor A; FGF, fibroblast growth factor; sh, short hairpin RNA; NC, negative control; 3′UTR, 3′ untranslated region; wt, wild-type; mut, mutant.