Reduced expression of microRNA-199a-3p is associated with vascular endothelial cell injury induced by type 2 diabetes mellitus

Abstract

The aim of the present study was to investigate the function and mechanism of action of microRNA (miRNA or miR)-199a-3p in vascular endothelial cell injury induced by type 2 diabetes mellitus (T2DM). A total of 36 patients with T2DM (26 males and 10 females; mean age, 52.5±7.0 years) and 20 healthy subjects (10 males and 10 females; mean age, 55.6±4.5 years) were included in the present study. Peripheral blood samples were obtained from all participants and total RNA was extracted Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-199a-3p. Following the transfection of human umbilical vein endothelial cells (HUVECs) with a negative control (NC) miRNA or miR-199a-3p mimics, cell proliferation was assessed using a Cell Counting kit-8 assay. Cell migration was investigated using Transwell assays and flow cytometry was performed to detect the apoptosis of HUVECs. HUVECs were infected with Ad-GFP-LC3B and laser-scanning confocal microscopy was performed to observe autophagosomes in HUVECs. Western blotting was used to measure the expression of proteins associated with autophagy and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor (NF)-κB signaling pathway. MiR-199a-3p was downregulated in peripheral blood from patients with T2DM compared with healthy subjects. Transfection with miR-199a-3p mimics promoted the proliferation and migration of HUVECs. However, miR-199a-3p overexpression inhibited the apoptosis of HUVECs. MiR-199a-3p facilitated HUVEC autophagy by affecting autophagy-associated signaling pathways. Furthermore, miR-199a-3p regulated the biological functions of HUVECs via the PI3K/AKT/NF-κB signaling pathway. The results of the present study suggest that miR-199a-3p expression was reduced in patients with T2DM compared with healthy subjects and may be associated with vascular endothelial cell injury. In addition, miR-199a-3p promoted the proliferation, migration and autophagy of HUVECs, potentially by regulating the PI3K/AKT/NF-κB signaling pathway. Therefore, miR-199a-3p may function as protector of vascular endothelia.

Keywords: microRNA-199a-3p; type 2 diabetes mellitus; vascular endothelial cell injury.

Figure 1.

(A) Relative miR-199a-3p expression in control subjects and patients with T2DM. (B) Patients were further subdivided and the expression of miR-199a-3p in control subjects, subgroup 1, subgroup 2 and subgroup 3 was assessed. *P<0.05 vs. control. T2DM, type 2 diabetes mellitus; miR, microRNA; subgroup 1, T2DM without complications; subgroup 2, T2DM with macroangiopathy; subgroup 3, T2DM with macrovascular and microvascular lesions.

Figure 2.

Effect of miR-199a-3p overexpression on the proliferation of human umbilical vein endothelial cells. A Cell Counting kit-8 assay was performed to proliferation and the absorbance at 490 nm was measured at 24, 48 and 72 h. *P<0.05 vs. NC. NC, negative control; miR, microRNA.

Figure 3.

Effect of miR-199a-3p overexpression on the migration of human umbilical vein endothelial cells. A Transwell assay was used to assess migration and light microscopy was used to capture images following Giemsa's staining (magnification, ×100). *P<0.05 vs. NC. NC, negative control; miR, microRNA.

Figure 4.

Effect of miR-199a-3p on the apoptosis of human umbilical vein endothelial cells under high glucose conditions. Flow cytometry was performed to investigate apoptosis and to assess apoptosis in the NC and miR-199a-3p treated groups. *P<0.05 vs. NC. NC, negative control; miR, microRNA; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 5.

Effect of miR-199a-3p overexpression on the autophagy of HUVECs. (A) The number of autophagosomes in HUVECs was determined using laser-scanning confocal microscopy (magnification, ×200). (B) Western blotting was performed to assess the relative expression of autophagy-associated LC3BII protein in HUVECs *P<0.05 vs. NC. NC, negative control; miR, microRNA; HUVEC, human umbilical vein endothelial cell; LC3BII.

Figure 6.

Effect of miR-199a-3p overexpression on the expression of proteins in the phosphatidylinositol 3-kinase/AKT/NF-κB signaling pathway. Western blotting was used to measure p110α and p85 protein expression relative to GAPDH; and the ratio of p-AKT over AKT and NF-κB protein expression relative to histone H. *P<0.05 vs. NC. NC, negative control; miR, microRNA; AKT, protein kinase B; NF-κB, nuclear factor-κB; p110α, catalytic subunit; p85, regulatory subunit; p, phosphorylated.