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.
The incidence of diabetes mellitus (DM), a common chronic disease in clinical practice that affected over 100 million people in China, is increasing each year (
MicroRNA (miRNA or miR) is a class of non-coding small RNA molecules (18–22 nucleotides) that bind to the 3′-untranslated region of target gene mRNA to regulate their expression (
A total of 36 patients with T2DM (26 males and 10 females; mean age, 52.5±7.0 years) who received treatments at the Affiliated Hospital of Taishan Medical University (Taian, China) between January 2016 and January 2017 were included in the experimental group. In addition, 20 healthy subjects (10 males and 10 females; mean age, 55.6±4.5 years) who undertook physical examinations in the same time period were included in the control group. Fasting peripheral blood (5 ml) was obtained from all participants in the morning. According to the diagnostic standards for DM published by World Health Organization in 1999 (
Serum (250 µl) was separated from peripheral blood by centrifugation at 2,000 × g for 10 min at 4°C and mixed with 750 µl TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for lysis following the manufacturer's protocol. Following lysis, total RNA was extracted using the phenol chloroform method. The purity of RNA was determined by A260/A280 using ultraviolet spectrophotometry (Nanodrop ND2000; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). cDNA was obtained by RT at 37°C for 1 h using miScript II RT kit (Qiagen GmbH, Hilden, Germany) from 0.5 µg RNA according to the manufacturer's protocol and samples were stored at −20°C.
qPCR was performed using miScript SYBR®-Green PCR kit (Qiagen GmbH). The reaction mixture comprised 10 µl RT-qPCR-mix, 0.5 µl upstream primer (5′-ACAGTAGUCTGCACATTGGTTA-3′), 0.5 µl downstream primer (universal primer provided by the kit), 2 µl cDNA and 7 µl ddH2O. Thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 1 min and 60°C for 30 sec. The 2−ΔΔCq method (
Human umbilical vein endothelial cells (HUVECs; Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were seeded at a density of 1×105 cells/well in 24-well plates containing RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and cultured at 37°C in an atmosphere containing 5% CO2. Cells were divided into negative control (NC) and miR-199a-3p mimics groups. When cells reached 70% confluence, 1.25 µl miR-NC (universal sequence; Sangon Biotech Co., Ltd., Shanghai, China) or miR-199a-3p mimics (5′-ACAGTAGTCTGCACATTGGTTA-3′; Sangon Biotech Co., Ltd.) and 2 µl Lipofectamine® 3000 were added to individual vials containing 50 µl Opti Mem medium (both Thermo Fisher Scientific, Inc.) at room temperature for 5 min. Vials were mixed and incubated at room temperature for 15 min. Mixtures were added to the cells and incubated at 37°C for 6 h, following which the medium was replaced with RPMI-1640 medium supplemented with 10% FBS. Cells were cultured at 37°C in an atmosphere containing 5% CO2 for 48 h prior to experiments.
At 48 h following transfection, HUVECs were trypsinized, collected by centrifugation at 500 × g for 5 min at room temperature and inoculated into 96-well plates containing 200 µl RPMI-1640 medium at a density of 2,000 cells/well. At 0, 24, 48 and 72 h, 20 µl CCK-8 (5 g/l; Beyotime Institute of Biotechnology, Haimen, China) was added to the cells. Following incubation at 37°C for 2 h, the absorbance (490 nm) of each well was determined and cell proliferation curves were plotted. Each group was tested in triplicate and the values were averaged.
Matrigel chambers (Corning Incorporated, Corning, NY, USA) were used to determine the migration ability of cells. Matrigel was diluted with serum-free RPMI-1640 medium at a ratio of 1:2. A total of 50 µl diluted Matrigel was added to the upper chamber and incubated at 37°C for 1 h, following which 2×105 HUVECs and 200 µl serum-free RPMI-1640 medium were added. In the lower chamber, 500 µl RPMI-1640 medium supplemented with 10% FBS was added. Following incubation at 37°C and 5% CO2 for 24 h, cells in upper chamber were removed using a cotton swab. The chamber was fixed using 4% formaldehyde for 10 min at room temperature and subjected to 5% Giemsa's staining at room temperature for 1 min. Following washing for 3 times, migrated cells were counted using a light microscope (5 fields; magnification, ×200).
To simulate the high glucose environment in DM, HUVECs were cultured in RPMI-1640 medium supplemented with 40 mmol/l glucose following transfection in an atmosphere containing 5% CO2 at 37°C for 6 h. HUVECs were washed twice with PBS, trypsinized, collected by centrifugation at 500 × g for 5 min at room temperature and adjusted to a density of 1×106 cells/100 µl. Apoptosis was assessed using flow cytometry with an ANXN V FITC Apoptosis DTEC kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Using a flow cytometer and FlowJo software (version 7.6.1; BD Biosciences), cells with Annexin V-positive values were determined to be in early apoptosis, those with propidium iodide-positive values were necrotic and those with double positive values were in late apoptosis.
At 24 h following transfection, HUVECs were seeded onto culture plates at a density of 1×105 cells/well. When cells reached 70% confluence, they were infected following the manufacturer's instructions with Ad-GFP-LC3B adenovirus (Hanbio Biotechnology Co., Ltd., Shanghai, China) at a multiplicity of infection of 20 for 48 h at 37°C. Without fixing, cells were observed under a laser confocal microscope (SP8; Leica Microsystems GmbH, Wetzlar, Germany). Green vesicles represent autophagosomes. Autophagosome numbers in HUVECs were counted in five fields using a confocal microscope and averaged to evaluate autophagy activity.
HUVECs were trypsinized and collected by centrifugation at 500 × g for 5 min at room temperature. Cold radioimmunoprecipitation assay lysis buffer (500 µl; Beyotime Institute of Biotechnology) was mixed with the samples for 30 min on ice, followed by centrifugation at 12,000 × g at 4°C for 10 min. Extraction of nuclear proteins was performed using Cell nuclear protein and cytoplasmic protein extraction kit (P0027; Beyotime Institute of Biotechnology) A bicinchoninic acid protein concentration determination kit [RTP7102, Real-Times (Beijing) Biotechnology Co., Ltd., Beijing, China] was used to determine the protein concentration in the supernatant. Protein samples (10 µg) were mixed with 5X SDS loading buffer and the mixture was denatured by boiling in a water bath for 10 min. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were subsequently blocked with 5% skimmed milk at room temperature for 1 h. Membranes were incubated with phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110α (1:1,000; cat. no. 4249; Cell Signaling Technology, Inc., Danvers, MA, USA), PI3K regulatory subunit p85 (1:1,000; cat. no. AF1729; Beyotime Institute of Biotechnology) LC3BII (1:1,000; cat. no. AL221; Beyotime Institute of Biotechnology) and mouse anti-human GAPDH (1:5,000; cat. no. AF0006; Beyotime Institute of Biotechnology) primary antibodies at 4°C overnight. For nuclear proteins, membranes were incubated with rabbit anti-human polyclonal nuclear factor (NF)-κB (1:1,000; cat. no. AF0246; Beyotime Institute of Biotechnology), protein kinase B (AKT; 1:1,000; cat. no. AA326; Beyotime Institute of Biotechnology), phosphorylated-(p)AKT (1:1,000; cat. no. AA329; Beyotime Institute of Biotechnology) and histone H (internal reference for nuclear proteins; 1:5,000; cat. no. AF0009; Beyotime Institute of Biotechnology) primary antibodies at 4°C overnight. Membranes were washed with PBS with Tween-20 (PBST) five times for 5 min and incubated with goat anti-mouse (1:4,000; cat. no. A0216; Beyotime Institute of Biotechnology) and goat anti-rabbit (1:4,000; cat. no. A0208; Beyotime Institute of Biotechnology) horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Subsequently, membranes were washed five times with PBST for 5 min and developed using an enhanced chemiluminescence detection kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Image lab v3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to analyze imaging data. The relative expression of target proteins was normalized to GAPDH.
Data were analyzed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Data were tested for normality and multigroup measurement data were analyzed using one-way analysis of variance. Least Significant Difference and Student-Newman-Keuls post hoc tests were used for homogeneous data, while Tamhane's T2 or Dunnett's T3 tests were performed for heterogeneous data. Comparisons between two groups were made using Student's t-tests. P<0.05 was considered to indicate a statistically significant difference.
To measure the expression of miR-199a-3p in the peripheral blood of patients with T2DM, RT-qPCR was performed. The data illustrated that miR-199a-3p expression was significantly lower in patients with T2DM compared with healthy subjects (P<0.05;
To assess the effect of miR-199a-3p overexpression on HUVEC proliferation, cells were transfected with miR-NC or miR-199a-3p mimics and a CCK-8 assay was performed. The data revealed that the absorbance of HUVECs transfected with miR-199a-3p was significantly higher compared with the NC group at 48 h and 72 h (P<0.05;
To examine the effect of miR-199a-3p on HUVEC migration, a Transwell assay was performed. The number of migrated cells was significantly higher in the miR-199a-3p group compared with the NC group (P<0.05;
To investigate how miR-199a-3p overexpression affects apoptosis in HUVECs cultured under high glucose conditions, flow cytometry was performed. The results revealed that the apoptotic rate of HUVECs in the miR-199a-3p group was significantly lower compared with the NC group (P<0.05;
To investigate the effect of miR-199a-3p on autophagy, laser-scanning confocal microscopy and western blotting were performed. The results revealed that the number of autophagosomes in the miR-199a-3p group was significantly higher compared with the NC group (P<0.05;
To determine whether the biological functions of miR-199a-3p were associated with activity changes in the PI3K/AKT/NF-κB signaling pathway, western blotting was performed. The results demonstrated that protein levels of the PI3K catalytic subunit p110α and regulatory subunit p85 in HUVECs transfected with miR-199a-3p were significantly increased compared with the NC group (P<0.05;
Vascular injury is a basic pathological change in patients with DM and it has an important influence on the occurrence and development of DM. Sustained vascular injury aggravates local inflammatory responses, stimulates the proliferation and hypertrophy of smooth muscle cells, affects systolic and diastolic functions and eventually leads to vascular remodeling (
Vascular endothelial cells cover the surface of vascular intima and induce inflammatory signals, hormone levels, shear stress or pressure in the blood environment, as well as secreting a variety of vasoactive substances to regulate vascular functions (
Autophagy is a process in which cells encapsulate their own proteins or damaged organelles in vesicles that are then fused with lysosomes to form autophagy lysosomes and the contents are degraded (
In conclusion, the present study demonstrates that miR-199a-3p expression is downregulated in the peripheral blood of patients with T2DM and is associated with disease progression. Additionally, miR-199a-3p may activate the PI3K/AKT/NF-κB signaling pathway, promote the proliferation, migration and autophagy of vascular endothelial cells and suppress apoptosis, effectively inhibiting vascular injury.
The authors would like to thank Dr Xiange Tang from Endocrine Department of the Hospital.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HW and ZW collaborated to design the study. HW, ZW and QT were responsible for experiments. HW and ZW analyzed the data. All authors collaborated to interpret results and develop the manuscript. The final version of the manuscript has been read and approved by all authors, and each author believes that the manuscript represents honest work.
All procedures performed in the current study were approved by the Ethics Committee of Taishan Medical University. Written informed consent was obtained from all patients or their families.
Written informed consents for publication of any associated data and accompanying images were obtained from all patients or their parents, guardians or next of kin.
The authors declare that they have no competing interests.
(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.
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.
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.
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.
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.
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.