In the current study, a total of 45 male mice (age, 8–12 weeks) were purchased from the Animal Centre of the Second Military Medical University (Shanghai, China). All animals received care in compliance with the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council (Washington, DC, USA). Animals were housed at room temperature, with free access to food and water, and were maintained in a 12 h light/dark cycle. Prior to the experiments, mice were acclimated to laboratory conditions for at least 7 days. The current study was approved by the Medical Ethics Committee of Gongli Hospital (Shanghai, China).

Mice at 8–12 weeks of age were sacrificed by CO₂ overexposure. Using fine-tipped forceps and spring scissors, the sheath around the aorta was opened. The connective fascia and adventitia were then carefully removed, and the aorta (from the aortic arch to the iliac bifurication) was removed and placed in a 6-cm culture plate containing Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Fungizone (cat. no. SV30079.01; HyClone; GE Healthcare Life Sciences, Logan, UT, USA). The aorta was subsequently opened with spring scissors and the blood clots were removed. The aortic intima was then scraped with a scissor blade, before it was cut into sections (4-mm in length). Each section was subsequently placed under a plastic cell culture coverslip and maintained in DMEM supplemented with 20% fetal bovine serum (HyClone; GE Healthcare Life Sciences) at 37°C, with 5% CO₂. The coverslip was removed after tissue sections had adhered, and the culture medium was refreshed every 3–4 days. Once cells had reached ~80% confluence, they were dissociated by adding 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.). Following inactivation of trypsin, cells were subcultured at ratio of 1:3. Cells at passages 3–5 were used in downstream experiments. Each experiment was repeated at least 3 times with different cell preparations.

Cells were cultured to 90% confluence in 6 cm² culture dishes and then subcultured at a ratio of 1:2. VSMCs (5–10×10⁶ cells) were then subjected to normoxic or hypoxic conditions. For normoxic conditions, mouse VSMCs (mVSMCs) were incubated at 37°C with 5% CO₂ humidified atmosphere. For hypoxia, mVSMCs were cultured in normoxic conditions for ≥24 h until adherent, and then cultured at 37°C for 3 h in a humidified hypoxic chamber supplemented with 1% O₂, 94% N₂ and 5% CO₂.

The micrON™ miR-26b-5p agomir and micrON™miRNA agomir control (scramble) were purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China) and used to treat mVSMCs using riboFECT CP reagent (RiboBio Co., Ltd.) in accordance with the manufacturer's instructions.

According to the manufacturer's instructions, the total RNA was extracted using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following quantification of RNA samples using a NanoDrop spectrophotometer (Thermo Fisher Scientific. Inc., Wilmington, DE, USA), 1 µg RNA was used to generate cDNA by RT reaction using PrimeScript Reverse Transcriptase kit (Takara Bio. Inc., Otsu, Japan) according to the manufacturer's instructions. The PCR thermal cycling parameters consisted of 1 cycle for 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, and a final extension step at 72°C for 5 min. A dissociation curve was obtained for each PCR product. qPCR was performed on an ABI PRISM 7000 Sequence Detection System using SYBR Premix Ex Taq™ II kit (Takara Bio. Inc.) with specific primers for hypoxia inducible factor (HIF)-1α, desmin, H-caldesmon, smoothelin and β-actin. Each sample was analyzed in triplicate and target gene expression levels were normalized to β-actin mRNA levels. The fold change in target gene expression was calculated using the 2^−∆∆Cq method (14). The primer sequences were as follows: HIF-1α forward, 5′-GATGAGGCTTACCATCAGCT-3′, and reverse, 5′-ATGTCACCATCATCTGTGAG-3′; desmin forward, 5′-GTTTCAGACTTGACTCAGGCAG-3′, and reverse, 5′-TCTCGCAGGTGTAGGACTGG-3′; H-caldesmon forward, 5′-ATGGTAGAGGAGAAAACACCAGA-3′, and reverse, 5′-CCATCCCCTTCTATTTTGGACTC-3′; smoothelin forward, 5′-GAGCGGCAAGACAACAAGGA-3′, and reverse, 5′-CAGTCTCCCTGCCAATCGT-3′; β-actin forward 5′-CAACCGTGAAAAGATGACCC-3′, and reverse, 5′-GTCTCCGGAGTCCATCACAA-3′.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed at 42°C for 60 min followed by 70°C for 10 min. PCR was performed using a Bulge-Loop™ miRNA RT-qPCR kit (catalogue no. R11067.1; Ribobio, Co., Ltd.). Thermal cycling conditions consisted of 1 cycle for 10 min at 95°C followed by 40 cycles at 95°C for 2 sec, 60°C for 20 sec, and 70°C for 10 sec. A dissociation curve was obtained for each PCR product. The primers used for the detection of miR-26b-5p and control U6 small nuclear RNA were designed and produced by Guangzhou Ribobio Co., Ltd. Each sample was analyzed in triplicate and target gene expression levels were normalized to U6 mRNA levels. The fold change in target gene expression was calculated using the 2^−ΔΔCq method.

Cells were harvested and lysed with radioimmunoprecipitation assay lysis buffer to extract the total protein. Protein concentration was measured by the micro-bicinchoninic acid assay, and 100 µg protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel, then transferred onto polyvinylidene difluoride membranes (Beyotime Institute of Biotechnology, Haimen, China). Non-specific binding sites were blocked by immersing the membrane in tris-buffered saline (TBS) solution containing 5% non-fat milk for 1 h at room temperature and agitating at 50 rpm. The membranes were then washed twice for 2 min in TBS solution. Immune complexes were formed by incubating the membranes overnight at 4°C with primary antibodies against collagen Iα (polyclonal rabbit anti-mouse; dilution, 1:1,000; cat. no. ab34710; Abcam, Cambridge, UK), as well as β-actin (monoclonal rabbit anti-mouse; dilution, 1:1,000; cat. no. 8457), Smad2 (monoclonal rabbit anti-mouse; dilution, 1:100; cat. no. 5339) and p-Smad2 (polyclonal rabbit anti-mouse; dilution, 1:1,000; cat. no. 3101), Smad3 and p-Smad3 (monoclonal rabbit anti-mouse; dilution, 1:1,000; cat nos. 9523 and 9520, respectively), Smad4 (monoclonal rabbit anti-mouse; dilution, 1:1,000; cat. no. 38,454; all from Cell Signaling Technology, Inc., Danvers, MA, USA). Blots were then washed and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. SA00001-2; ProteinTech, Rosemont, IL, USA). Subsequently, immunoreactive protein bands were analyzed using the Pierce ECL Western Blotting Substrate (cat. no. 32106; Pierce Biotechnology, Inc., Rockford, IL, USA), and quantified using ImageJ software (version 1.44p; National Institutes of Health, Bethesda, MD, USA).

The culture medium was removed and the differently treated cells were fixed in 4% paraformaldehyde for 15 min at room temperature before they were washed three times in TBS. Subsequently, cells were blocked with 5% bovine serum albumin (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) diluted in TBS for 1 h at room temperature on a shaker. The blocking solution was then removed, and a primary antibody against Smad4 (polyclonal rabbit anti-mouse; dilution, 1:1,00; cat. no. ab208804; Abcam) diluted in TBS was added to appropriate wells and incubated at 4°C overnight. The cells were washed with TBS and then incubated with an Alexa Fluor^®-labeled polyclonal goat anti-rabbit secondary antibody (2 µg/ml; cat. no. A-11008; Invitrogen; Thermo Fisher Scientific, Inc.) for 3 h at room temperature. Slides were subsequently washed, air dried and mounted on coverslips with DAPI (1 mg/ml; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. D1306). The samples were mounted on glass slides with ProLong^® Gold Antifade Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and visualized using an inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). A rhodamine phalloidin (Cytoskeleton Inc., Denver, CO, USA) probe was used to label F-actin. The cell area was calculated as: Total area/nuclear number.

A potential miRNA-26b-5p target sequence in the Smad4 3′-untranslated region (3′-UTR) was analyzed using TargetScanMouse 7.1 software (www.targetscan.org/mmu_71). In brief, the mouse ‘Smad4’ gene and miRNA ‘miR-26b-5p’ sequence names were entered and submitted. The predicted Smad4 3′UTR-miR-26b-5p target binding regions were then shown.

The results presented are the average of at least three experiments and reported as the mean ± standard deviation. Statistical analyses were performed with one-way analysis of variance or Student's t-test using SPSS 11.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

As demonstrated in Fig. 1, compared with normoxia, mVSMCs exposed to low oxygen displayed a statistically significant upregulation in HIF-1α mRNA levels (P=0.0018; Fig. 1A), and a significant downregulation in miR-26b-5p expression (P=0.0275; Fig. 1B). Additionally, significant downregulation of desmin, H-caldesmon and smoothelin mRNA expression levels was detectable in response to hypoxia in mVSMCs compared with the levels in normoxic conditions (Desmin, P=0.0010; H-caldesmon, P=0.0052; smoothelin, P=0.0456; Fig. 1C).

To further investigate the importance of miR-26b-5p in hypoxia-induced phenotypic switching of mVSMCs, the miR-26b-5p agomir and scramble were transfected into mVSMCs cultured in normoxic and hypoxic conditions. The cell area analysis (Fig. 2A and B) demonstrated that mVSMCs transfected with scramble exhibited an area increase in hypoxic mVSMCs compared with those in normoxic conditions (P=0.0075). However, hypoxic mVSMCs transfected with miR-26b-5p agomir were smaller compared with hypoxic mVSMCs transfected with scramble (P=0.0051). Western blot analysis demonstrated a similar effect on collagen Iα expression (Fig. 2C). Hypoxia was associated with an upregulation in collagen Iα protein expression when compared with normoxia (P=0.0006). However, expression of collagen Iα in hypoxic mVSMCs transfected with miR-26b-5p agomir was significantly downregulated compared with that of hypoxic mVSMCs transfected with agomir (P=0.00009; Fig 2D).

Western blotting demonstrated that the expression levels of Smad2 and Smad3 proteins in hypoxic and normoxic mVSMCs transfected with agomir or scramble were comparable (Fig. 3A). However, hypoxia resulted in upregulation of p-Smad2 and p-Smad3 proteins in scramble-transfected mVSMCs, and the effect was not altered by miR-26b-5p agomir. Furthermore, the expression level of Smad4 in hypoxic mVSMCs transfected with scramble was increased compared with normoxic mVSMCs transfected with scramble. However, transfection with miR-26b-5p agomir resulted in reduced expression of Smad4 in hypoxic mVSMCs compared with normoxic mVSMCs. Furthermore, miRNA target analysis identified the miR-26b-5p target sequence in the Smad4 3′UTR as UACUUGA between 1,030–1,040 bp (Fig. 3B). Additionally, immunofluorescent staining demonstrated that immunoreactivity of Smad4 in the cytoplasm of normoxic mVSMCs transfected with scramble was more extensive and strongly distributed compared with hypoxic mVSMCs transfected with agomir (Fig. 3C).