VSMCs were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences (Shanghai, China) and maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; both Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator (Thermo Fisher Scientific, Inc.), in an atmosphere containing 5% CO₂. Ethyl 3,4-dihydroxybenzoate (EDHB) was purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany) and dissolved in ethanol. VSMCs were exposed to EDHB (50 µg/ml) for 24 h.

VSMC lysates were prepared and measured using a caspase-3 ELISA Kit (no. KGA203, Nanjing KeyGEN BioTECH, Co., Ltd., Nanjing China). In brief, 50 µl supernatant was mixed with 2X reaction buffer (50 µl) and dithiothreitol (0.5 µl). Immunocomplexes were incubated with 5 µl peptide substrate in assay buffer for 2 h at 37°C. Release of p-nitroaniline was measured at 405 nm using an ELISA reader (SpectraMax M5; Molecular Devices, LLC., Sunnyvale, CA, USA) according to the manufacturer's instructions.

Quantitative assessment of apoptotic cells was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, examining DNA-strand breaks during apoptosis with the ApoAlert DNA Fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were incubated at 37°C in hypoxic conditions for 48 h. Cells were trypsinized, fixed with 4% paraformaldehyde at room temperature for 24 h and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. Cells were washed and incubated with the reaction mixture for 60 min at 37°C, and immediately analyzed using FACScan and the Cellquest program ver. 5.1 (BD Biosciences).

Lentiviral vectors containing miR-17-5p were constructed to transfect VSMCs. Briefly, VSMCs were cultured in McCoy's 5α medium containing 10% FBS (MP, Biomedicals, Santa Ana, CA, USA) and when the exponential growth phase was reached, 1.0×10⁵ cells/well were seeded in 96-well plates. A total of 300 µl complete culture medium, containing recombinant lentiviruses, control lentiviruses or McCoy's 5α medium (all of which contained 6 µg/ml polybrene; Sigma-Aldrich; Merck Millipore) was added to the plates when the cells reached 50–60% confluence. The virus-containing medium was replaced with fresh complete medium two days later.

The siRNAs or antagomirs for miR-17-5p, were obtained from GE Healthcare Dharmacon, Inc. (Pittsburgh, PA, USA), and were designed with the following sequences: miR-17-5p, 5′-CUGAGGUCCAGGACACACA-3′; scramble, 5′-AGAGAUGACUCACUGUCAC-3′. VSMCs were transfected with siRNA oligonucleotides using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

The HIF-1α-siRNA oligonucleotide fragment was designed and synthesized according to the human HIF-1α gene sequence (GenBank no. NM001530). The sequence was as follows: Forward, 5′-GATCCCGAGGAAGAACTATGAACATAATTCAAGAGATTATGTTCATAGTTCTTCCTCTTTTTGGAT-3′ and reverse, 5′-AGCTATCCAAAAAGAGGAAGAACTATGAACATAATCTCTTGAATTATGTTCATAGTTCTTCCTCGG-3′. Scramble and HIF-1α-siRNA oligonucleotide were cloned into the pSIREN-RetroQ plasmid (Addgene, Inc., Cambridge, MA, USA) for retrovirus production. After 48 h, infected cells were selected with puromycin (2 mg/ml) and the clones were selected and cultured for further experiment.

The three prime untranslated region (3′UTR) of the STAT3 gene containing the predicated target sites for miR-17-5p was obtained by polymerase chain reaction (PCR) amplification. The fragment was inserted into the multiple cloning sites of the pMIR-REPORT luciferase miR expression reporter vector (Ambion; Thermo Fisher Scientific, Inc.). Cells from the human embryonic kidney cell line HEK-293 (Cell Resource Center, Shanghai Institutes for Biological Sciences) were co-transfected with 0.1 µg luciferase reporters containing STAT3 3′UTR and microR-17-5p mimics using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cell lysates were harvested 48 h post-transfection and luciferase activity was measured with a dual luciferase reporter assay kit (no. RG028. Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol.

RNA extraction was performed using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Synthesis of cDNA was performed by RT reactions with 2 µg total RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) with oligo dT (15) primers (Fermentas; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-17-5p level was measured using qPCR with the mirVana RT-qPCR miR detection kit (Ambion; Thermo Fisher Scientific, Inc.) in conjunction with SYBR Green (Invitrogen; Thermo Fisher Scientific, Inc.). Following the circle reaction, the threshold cycle (Cq) was determined and the relative miR-17-5p level was calculated based on the Cq values and normalized to U6 level in each sample. PCR was performed with the following primers: miR-17-5p, forward 5′-TCTAGATCCCGAGGACTG-3′ and reverse, 5′-ATCGTGACCTGAACC-3′; U6, forward 5′-CTCGCTTTGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′.

Autophagy in VSMCs was detected via western blotting and fluorescence microscopy, and cultured VSMCs were prepared for observation under a transmission electron microscope (TEM) to investigate the formation of autophagosomes. Cells were washed with phosphate-buffered saline, fixed with 4% formaldehyde/1% glutaraldehyde at room temperature overnight, and then rinsed three times with 0.1 M sodium cacodylate/0.2 M sucrose buffer. Following fixation, cells were incubated in 1% osmium tetroxide for 1 h, dehydrated in a series of ethanol washes, and then embedded in epoxy resin. When the epoxy resin had polymerized at 65°C for 24 h, sections 100-nm thick were then cut onto a carbon-coated copper grid. Cells were stained with uranyl acetate and lead citrate (SPI Supplies, West Chester, PA, USA) and examined under a TEM (H-800; Hitachi, Ltd., Tokyo, Japan).

VSMCs were homogenized and extracted in NP-40 buffer (Thermo Fisher Scientific, Inc.), followed by 5–10 min boiling and centrifugation at 7,500 × g, for 15 min at 4°C to obtain the supernatant. Protein samples were quantified using the bicinchoninic Acid kit for Protein Determination, (no. BCA1-1KT; Sigma-Aldrich; Merck Millipore).

Samples containing 30 µg protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Following blocking for 2 h at room temperature with 5% (w/v) non-fat dry milk in tris-buffered saline and 0.1% (w/v) Tween 20 (TBST), membranes were incubated with primary antibodies for LC3 (sc-292,354, dilution, 1:1,000), P62 (sc-48389, dilution, 1:1,000), β-actin (sc-81178, dilution, 1:2,000), Bax (sc-6236, dilution, 1:1,000), p-caspase3 (sc-22171-R, dilution, 1,000), caspase3 (sc-7272, dilution, 1:1,000), STAT2 (sc-483, dilution, 1:1,000) at 4°C overnight. All primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The membranes were washed three times with TBST and incubated with secondary antibodies donkey anti-mouse immunoglobulin (Ig) G (sc-2096, dilution, 1:10,000) and goat anti-rabbit IgG (sc-2004, dilution, 1:10,000; both Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and visualized with an Amersham ECL Western blotting Detection reagent (GE Healthcare Life Sciences, Chalfont, UK). Signals were densitometrically assessed using Quantity One^® software ver. 4.5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each experiment was performed four times.

The data from these experiments were presented as the mean ± standard deviation for each group. Statistical analyses were performed using PRISM version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Inter-group differences were analyzed via one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Significant upregulation of miR-17-5p expression was observed in VSMCs subjected to hypoxic conditions (P<0.05; Fig. 1A). HIF-1 is stabilized under hypoxic conditions as a cellular response to low oxygen concentration. Furthermore, EDHB has been shown to have a cytoprotective effect against oxidative stress in various types of cells, including VSMCs (17). In the present study, lower miR-17-5p levels were observed in EDHB-treated and HIF-1α loss-of-function cells (Fig. 1B). These results indicate that miR-17-5p may be a novel hypoxia-responsive miR that has not been reported in previous miR profiling data.

The ultrastructure of VSMCs was observed under a TEM to assess levels of autophagy in response to hypoxia and miR-17-5p loss-of-function. An increase in typical autophagic vacuoles containing extensively degraded organelles, including mitochondria and endoplasmic reticulum, was observed in the cytoplasm of VSMCs following treatment with hypoxia as compared with the control group (Fig. 2). However, this effect was reversed by miR-17-5p antagomir in hypoxic conditions (Fig. 2). To assess autophagosome formation, LC3-II expression was measured via immunofluorescent staining and western blotting was used to measure the LC3-II/LC3-I ratio and p62 levels. The results demonstrated that the LC3-II intensity of green fluorescence in response to hypoxia was greater than that of the control group following incubation for 24 h in RPMI-1640 (Fig. 3A). Consistent with the fluorescence imaging results, the LC3-II/LC3-I ratio increased in response to hypoxia (Fig. 3B) and the expression of p62 was markedly decreased following exposure of cells to hypoxic conditions. However, the results of LC3-II degeneration and p62 accumulation demonstrated that miR-17-5p loss-of-function inhibited hypoxia-induced autophagy (Fig. 3A and B). Furthermore, it was investigated whether hypoxia induced apoptosis in VSMCs via an apoptotic mechanism using a caspase-3 activity assay and TUNEL staining following the exposure of VSMCs for 24 h. The results indicate that caspase-3 activity in the hypoxia-treated group was significantly lower than that of the control group (P<0.05; Fig. 3C), and cell apoptosis was significantly inhibited in response to hypoxia (P<0.05; Fig. 3D); however, these reductions were significantly reversed by miR-17-5p antagomir transfection (both P<0.05). The apoptotic response was further investigated by measuring the expression of apoptosis-related proteins. Western blot analysis demonstrated that treatment of VSMCs with hypoxia markedly decreased pro-apoptotic proteins BAX and p-caspase levels (Fig. 3E). These results suggest that hypoxia induced protective autophagy in VSMCs via inhibition of cell apoptosis as a response to an anaerobic environment, which is consistent with the literature (18).

To determine whether miR-17-5p regulated STAT3 through the predicted binding sites in its 3′-UTR (Fig. 4A), a luciferase construct was designed by incorporating wild-type or mutant 3′-UTR of STAT3, which expressed luciferase unless repressed by the incorporated 3′-UTR. No significant difference was observed between the control group and the group that underwent cotransfection of VSMCs with the pMIR-REPORT construct containing mutant STAT3 3′-UTR and PLemiR-17-5p. Cotransfection with the luciferase construct containing wild-type STAT3 3′-UTR and PLemiR-17-5p resulted in significantly lower luciferase activity than the control group, leading to a ~70% decline in luciferase activity compared with control group (P<0.05; Fig. 4B). Western blotting results indicated that the expression of STAT3 was significantly decreased following cotransfection with the luciferase construct containing wild-type STAT3 3′-UTR and overexpressing miR-17-5p compared with that of the wild-type control group (P<0.05; Fig. 4C). Furthermore, the combination of miR-17-5p loss-of-function and hypoxia was demonstrated to significantly upregulate the protein expression of STAT3 compared with hypoxia single treatment in VSMCs (P<0.05), which was confirmed by immunofluorescent staining (Fig. 5A) and western blotting (Fig. 5B).