Primary HUVECs were purchased from Lonza, Inc. (Allendale, NJ, USA). Sulfo-NHS-LC-biotin was purchased from Pierce; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). miRNA oligonucleotides (miR-126 mimic, 5′-CAGUACUUUUGUGUACAA-3′; miR-126 antagomir, 5′-CGCAUUAUUACUCACGGUACGA-3′; and negative control miRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′) and TransMessenger Transfection reagent were obtained from Qiagen China Co., Ltd. (Shanghai, China). Rhodamine600 (XRITC)-avidin was obtained from Pierce; Thermo Fisher Scientific, Inc. HUVECs were cultured in endothelial cell growth medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a 5% CO₂ atmosphere. The cells at passage 3–4 were used for experiments. All experiments were repeated at least three times.

All of the experiments performed in the present study were approved by the Ethics Committee of The First Affiliated Hospital of Anhui Medical University (Hefei, China). A total of 60 male apolipoprotein E (apoE)^−/− mice (age, 4 weeks; weight, 22±5 g) were obtained from the Institute of Basic Medical Sciences of Peking Union Medical College (Beijing, China). Mice were housed individually in screen-bottomed plastic cages in a temperature-controlled room (25°C) under a 12-h light/dark cycle with free access to food and water. Mice were divided into two groups: Standard diet (n=16) and standard diet plus 5% lard oil and 2% cholesterol (n=44); diets were maintained for 16 weeks. At 12 weeks, 8 control and 8 high-fat diet mice were euthanized for detection of miR-126 expression levels, and the remaining control and high-fat diet mice were randomly divided into the following three groups: Control miRNA oligonucleotides (n=8; 4 control mice and 4 high-fat diet mice); miR-126 antagomir oligonucleotides (n=18; 9 control mice and 9 high-fat diet mice); and miR-126 mimic oligonucleotides (n=18; 9 control mice and 9 high-fat diet mice). Control miRNA, or miR-126 antagomir or mimic was injected subcutaneously at a dose of 10 mg/kg twice in the first week, followed by once a week for 4 weeks. During the study period, 1 mouse died in the control miRNA oligonucleotide group, 2 died in the miR-126 antagomir oligonucleotide group and 2 died in the miR-126 mimic oligonucleotide group. At the end of the experiment, the aorta was removed from mice as previously described (8). Part of the aorta was embedded in optimal cutting temperature compound and snap frozen. The remaining aorta was stored at −80°C.

Total RNA was extracted from the aorta using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). miR-126 expression was determined using a miRNA Plate Assay kit (cat no. MA-0101; Signosis, Inc., Santa Clara, CA, USA) and an oligo mix specific for miR-126 (cat no. MO-0040; Signosis, Inc.), according to the manufacturer's protocol. The RNA content was normalized to U6 small nuclear (sn)RNA. miR-126 expression was also determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with the following primers: miR-126, forward 5′-UCGUACCGUGAGUAAUAAUGCG-3′, reverse 5′-CAUUAUUACUCACGGUACGAUU-3′; and U6 snRNA, forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′. Amplification conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, and at 60°C for 60 sec. The reaction mixture contained the following: 2X Power SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), cDNA template, 10 pmol/µl of each primer and sterile H₂O. Relative gene expression was quantified according to the comparative Cq method (32) using GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).

The permeability assay was performed as previously described (33). Briefly, frozen aorta sections (10-µm thick) were stained at 37°C with Sulfo-NHS-LC-biotin for 30 min, and blocked with 5% non-fat milk at 4°C overnight and subsequently submerged into blocking buffer containing XRITC-avidin (1:500) at room temperature for 1 h. The slides were washed three times with PBS and dried. Images were captured using an Olympus Provis AX70 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Caspase-3 activity was detected in aortic samples using a Caspase-3 Fluorometric assay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA), as previously described (34). Briefly, aortic samples were isolated from anesthetized mice and stored in liquid nitrogen. Samples were homogenized with lysis buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 2% SDS for 3 min on ice, maintained on ice for 10 min, centrifuged at 21,130 × g for 10 min at 4°C, and supernatants were collected and cryopreserved at −70°C until further use. Protein concentration was determined using a bicinchoninic acid (BCA) assay with the Micro BCA™ Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of extracted protein samples (50 µg) were incubated at 37°C overnight with N-acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). The quantity of pNA that was released was estimated at 405 nm using a microplate ELISA reader. Caspase-3 relative activity was calculated as follows: Caspase-3 activity=(mean experimental absorbance/mean control absorbance)x100%.

Aortic samples were homogenized, lysed for 3 min on ice in 1X SDS lysis buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 2% SDS and boiled for 10 min, followed by centrifugation at 16,000 × g for 10 min at room temperature. Protein concentration was determined using a Micro BCA™ Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of extracted protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Chalfont, UK). Membranes were blocked using 5% (w/v) bovine serum albumin (BSA; Amresco, LLC, Solon, OH, USA) for 2 h at room temperature, followed by incubation with the following primary antibodies: Anti-TGFβ (cat no. 3711; 1:1,000), anti-B-cell lymphoma (Bcl)-2 (cat no. 2872; 1:1,000), purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and anti-GAPDH (cat no. TA505454; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) diluted in TBS containing 0.05% Tween-20 (TBST) at 4°C overnight. Following 3 washes with TBST, horseradish peroxidase (HRP)-conjugated secondary antibodies (cat nos. ZB-2305 and ZB-2301; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) were added to membranes and incubated at room temperature for 2 h. Protein bands were visualized by enhanced chemiluminescence using ECL reagents (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was used as the loading control. Blots were semi-quantified by densitometric analysis using the Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Experiments were repeated 3 times.

Immunohistochemistry was performed as previously described (8). Sections (10 µm) of the frozen aorta tissue samples were blocked using PBS containing 0.05% Tween-20 and 1% BSA at room temperature for 30 min, and incubated at 4°C overnight with an anti-TGFβ (cat no. 3711; 1:1,000) primary antibody, purchased from Cell Signaling Technology, Inc. Subsequently, sections were incubated with a HRP-conjugated goat secondary antibody (cat no. ZB-2301; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 60 min. A Metal Enhanced 3,3′-diaminobenzidine Substrate kit (Pierce; Thermo Fisher Scientific, Inc.) was used for 3 min to develop the colour, and sections were counterstained with hematoxylin (10%) at room temperature for 30 sec. The integral absorbance was examined under a light microscope and results were quantified using the Image-Pro Plus software version 6.0 (Media Cybernetics, Inc.).

The region of the TGFβ 3′-UTR containing the potential binding site of miR-126 (source: NCBI GenBank; https://www.ncbi.nlm.nih.gov/nuccore/NM_011577.2) was predicted using TargetScan version 7.1 (http://www.targetscan.org/vert_71/). The sequence was inserted into the 3′ region of the luciferase gene in a luciferase vector (wt-Luc-TGFβ; Shanghai GeneChem Co., Ltd., Shanghai, China), and a mutated version of this sequence was inserted into the vector (mu-Luc-TGFβ). Plasmid DNA (300 ng) and miR-126 mimic, antagomir or control oligonucleotide (80 nmol/l) were co-transfected using TransMessenger Transfection reagent into HUVECs seeded into 6-well plates at a density of 1–2×10⁵ cells/well (confluence, 60–70%) for 24 h at 37°C. The pRL-TK vector (Promega Corporation, Madison, WI, USA) expressing Renilla luciferase served as a control. The luciferase assay was performed using a Dual-Luciferase^® Reporter assay system (Promega Corporation) 24 h following transfection, according to the manufacturer's protocol.

Data are presented as the mean ± standard deviation of 3 independent experiments. The statistical significance of the differences between groups was assessed using one-way analysis of variance followed by a Neuman-Keuls post hoc test for multiple comparisons. Comparisons between two groups were based on least significant differences. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS software version 21.0 (IBM Corp., Armonk, NY, USA).

To evaluate the impact of miR-126 on an established AS model, apoE^−/− mice were fed a high-fat diet for 12 weeks, followed by subcutaneous injection of 10 mg/kg control miRNA, miR-126 antagomir or miR-126 mimic twice for one week, and once a week for 4 weeks. Subsequently, the expression of miR-126 in the aorta was determined using a miRNA Plate assay kit. miR-126 levels were reduced in AS mice compared with control mice (P<0.05; Fig. 1A). Following 4 weeks of treatment, the miR-126 mimic significantly increased miR-126 expression and the miR-126 antagomir significantly reduced the expression of miR-126 compared with AS mice injected with control miRNA (P<0.05; Fig. 1B). In addition, caspase-3 activity was measured following 4 weeks of treatment with the miRNAs. Compared with the control group, caspase-3 activity was greater in AS mice (P<0.05; Fig. 1C). Treatment with miR-126 antagomir and mimic exhibited no effect on caspase-3 activity in mice fed a standard diet (Fig. 1D). Thus, the standard diet group was not analysed in subsequent experiments.

To investigate the effect of miR-126 on endothelial permeability, NHS-LC-biotin and XRITC-avidin were used to detect the endothelial permeability of aortic intima. Results presented in Fig. 2 demonstrated that the aortic intima from AS mice exhibited greater staining with NHS-LC-biotin compared with control mice, in which only the endothelial surface was stained. When AS mice were treated with an miR-126 antagomir (Fig. 2Ac), an increased quantity of NHS-LC-biotin leaked into the aortic intima compared with AS mice treated with control miRNA (Fig. 2Ab). By contrast, miR-126 mimic (Fig. 2Bc) treatment reduced endothelial permeability compared with control miRNA-treated AS mice (Fig. 2Bb).

As apoptosis is involved in the development of AS, the effect of miR-126 on apoptosis was investigated in the current study. Caspase-3 activity was significantly increased in AS model mice treated with control miRNA compared with control mice treated with control miRNA, and miR-126 antagomir treatment further increased caspase-3 activity (P<0.05; Fig. 3A). Treatment with an miR-126 mimic partially reversed the increased activity of caspase-3 induced by a high-fat diet (P<0.05; Fig. 3B). The opposite results were observed when the expression levels of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) were analysed (Fig. 3C and D).

Previous studies have demonstrated that TGFβ promotes apoptosis in endothelial cells (22–24). Therefore, the present study investigated the effect of miR-126 on the expression of TGFβ. TGFβ protein expression levels were increased in AS model mice treated with control miRNA compared with control mice treated with control miRNA, as determined by western blot analysis (Fig. 4A and B) and immunohistochemistry (Fig. 4C and D). This result indicated that TGFβ may be a target gene of miR-126. Results in Fig. 4 demonstrated that miR-126 antagomir treatment significantly increased levels of TGFβ in AS model mice (Fig. 4A and C), whereas an miR-126 mimic had the opposite effect (Fig. 4B and D).

miRNAs specifically bind to the 3′-UTR of target mRNAs and induce transcript degradation or translational repression. An miR-126 binding site in the TGFβ 3′-UTR was identified and the alignment between miR-126 and the wild-type and mutant TGFβ 3′-UTR are presented in Fig. 5A and B, respectively. This indicated that miR-126 may cause translation inhibition of TGFβ. To validate this result of bioinformatics analysis, wt-Luc-TGFβ and mu-Luc-TGFβ were inserted into the luciferase vector, which was co-transfected into HUVECs with control miRNA, miR-126 antagomir or miR-126 mimic. Treatment with miR-126 mimic reduced luciferase activity in HUVECs co-transfected with wt-Luc-TGFβ compared with HUVECs co-transfected with wt-Luc-TGFβ and control miRNA (Fig. 5C). In HUVECs co-transfected with mu-Luc-TGFβ and an miR-126 mimic, no inhibition was observed (Fig. 5C). miR-126 antagomir significantly promoted luciferase activity in the wt-Luc-TGFβ-transfected HUVECs, but had no effect on mu-Luc-TGFβ-transfected HUVECs (Fig. 5D).