Human vascular smooth muscle cells (hVSMCs) were obtained from the Cell Bank of the Shanghai Institute of Cell Biology (Shanghai, China). Cells were grown in 75 cm² flasks with Dulbecco's Modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China). Cells were then incubated at 37°C with 5% CO₂.

100 nM Mimic controls (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; anti-sense: 5-′ACGUGACACGUUCGGAGAATT-3′), 100 nM miR-15b mimics (sense: 5′-UAGCAGCACAUCAUGGUUUACA-3′; anti-sense: 5′-UAAACCAUGAUGUGCUGCUAUU-3′), 100 nM miR-15b inhibitors (5′-UGUAAACCAUGAUGUGCUGCUA-3′), 100 nM inhibitor controls (5′-CAGUACUUUUGUGUAGUACAA-3′; all Shanghai GenePharma Co., Ltd., Shanghai, China), 2 µl control-plasmids (cat. no. sc-108083), 2 µl insulin growth factor 1 receptor (IGF1R)-plasmids (cat. no. sc-421057-ACT; both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and 100 nM miR-15b mimics+2 µl IGF1R-plasmids were transfected into hVSMCs using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Cells without any treatment were used as the control group. Following 48 h, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to assess transfection efficiency.

A MTT assay was performed to detect cell viability. 48 h following transfection, cells were seeded in 96-well plates (2×10⁴ cells/ml). Subsequently, 10 µl MTT reagent (Beyotime Institute of Biotechnology, Haimen, China) was added to each well and incubated for 4 h at 37°C. DMSO (100 µl; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was added to dissolve the formazan crystals. Thermo Scientific™ Multiskan™ FC (Thermo Fisher Scientific, Inc.) was then used to measure absorbance at a wavelength of 490 nm. Each experiment was performed in triplicate.

Following transfection for 48 h, hVSMCs were digested using 0.2% trypsin. Following a wash with PBS, cells were fixed with 70% ethanol overnight at 4°C. The apoptosis of cells was then assessed using the Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit [cat. no. 70-AP101-100; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] following the manufacturer's protocol. A FACS Calibur flow cytometer with Cell Quest software (version 5.1; BD Biosciences, San Jose, CA, USA) was utilized for the detection of cell apoptosis rate. Each experiment was performed in triplicate.

To assess cell migration, un-coated transwell chambers (pore size, 8 µm; Costar; Corning Inc., Corning, NY, USA) were utilized in the present study. Cells (2×10⁴) were seeded into the upper chamber with serum-free DMEM and 600 µl DMEM containing 30% FBS was added to the lower chamber. Following 48 h of incubation at 37°C, the migratory cells on the lower chamber were fixed with 4% paraformaldehyde at room temperature for 30 min and then stained with 0.5 ml 0.1% crystal violet at room temperature for 15 min. At the end of the experiment, migrated cells were counted under a light microscope at a magnification of ×200 using five random fields of view. Each experiment was performed in triplicate.

TargetScan bioinformatics software (www.targetscan.org/vert_71) was utilized to predict the targets of miR-15b and the binding sites between IGF1R and miR-15b. To confirm the binding sites between miR-15b and the 3′-untranslated region (3′-UTR) of IGF1R, a dual luciferase reporter assay was performed. The wild type (WT-IGF1R) and mutant (MUT-IGF1R) 3′-UTRs of IGF1R were cloned into a pmiR-RB-ReportTM dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). hVSMCs were then co-transfected with WT-IGF1R or MUT-IGF1R with miR-15b mimics or mimic controls using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. After cell transfection for 48 h, the dual-luciferase assay system (Promega Corporation, Madison, WI, USA) was utilized to detect luciferase activity. Luciferase activity was normalized to that of renilla luciferase in the current study.

The TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for hVSMC RNA extraction and the miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) was used for reverse transcription. The QuantiFast SYBR Green PCR kit (Qiagen GmbH) was used to perform RT-qPCR analysis under a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH and U6 were utilized as mRNA and miRNA controls, respectively. Primer sequences as shown in Table I. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 10 min, 35 cycles of 95°C for 15 sec and 55°C for 40 sec. Relative gene expression was calculated using the 2^−ΔΔCq method (16).

Protein was extracted from cells using radioimmunoprecipitate lysate buffer containing PMSF (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (70 mg of each extract) were separated using 12% SDS-PAGE, electrotansfered to polyvinylidene difluoride membranes and then blocked with 5% skimmed milk at room temperature for 1.5 h. Subsequently, membranes were incubated with the following primary antibodies overnight at 4°C: anti-IGF1R (1:1,000; cat. no. sc-81464; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-protein kinase B (AKT; 1:1,000; cat. no. 4691; Cell Signaling Technology Inc., Danvers, MA, USA), anti-phosphorylated (p)-AKT (1:1,000; cat. no. 4060; Cell Signaling Technology Inc.), anti-mechanistic target of rapamycin (mTOR; 1:1,000; cat. no. 2983; Cell Signaling Technology Inc.), anti-p-mTOR (1:1,000; cat. no. 5536; Cell Signaling Technology Inc.), anti-ribosomal protein S6 kinase beta-1 (p70S6K; 1:1,000; cat. no. 2708; Cell Signaling Technology Inc.), anti-p-P70S6K (1:1,000; cat. no. 9234; Cell Signaling Technology Inc.) and anti-β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology Inc.). Samples were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (1:1,000; cat. no. 7074; Cell Signaling Technology Inc.) at room temperature for 2 h. To visualize immunoreactive proteins, the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.) was utilized. Gel-Pro Analyzer densitometry software (Version 6.3, Media Cybernetics, Inc., Rockville, MD, USA) was used for band density quantification.

Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation. Comparisons between two groups were made using Student's t-test and comparisons between multiple groups were analyzed by one-way analysis of variance with a Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To assess the role of miR-15b in the viability and migration of hVSMCs, miR-15b mimics, mimic controls, miR-15b inhibitors or inhibitor controls were transfected into hVSMCs. A total of 48 h following cell transfection, RT-qPCR was performed to detect transfection efficiency. The results revealed that, compared with the control group, the miR-15b mimic significantly increased the level of miR-15b, while the miR-15b inhibitor significantly decreased miR-15b (Fig. 1A). Subsequently, MTT and transwell assays were performed to determine whether miR-15b effected viability and migration of hVSMC cells. It was demonstrated that, compared with the control group, the miR-15b mimic significantly reduced the viability and migration of hVSMCs, while the miR-15b inhibitor exhibited the opposite effect (Fig. 1B and C).

The current study further assessed whether miR-15b affected the proliferation of hVSMCs by inducing cell apoptosis. A total of 48 h following transfection, the apoptosis of hVSMCs was analyzed via flow cytometry. The results revealed that, compared with the control group, the miR-15b mimic significantly promoted the apoptosis of hVSMCs, while the miR-15b inhibitor reduced hVSMCs apoptosis (Fig. 2).

To assess the molecular mechanism of miR-15b on hVSMCs, bioinformatics software (TargetScan) was used to predict the potential targets of miR-15b (Fig. 3A). A dual luciferase reporter assay was then performed to confirm predictions. The results indicated that, compared with cells co-transfected with WT-IGF1R and mimic control, the luciferase activity in cells co-transfected with WT-IGF1R and miR-15b mimic were significantly reduced (Fig. 3B). The results demonstrated that IGF1R is a direct target of miR-15b.

Furthermore, the current study revealed that, compared with the control group, the miR-15b mimic significantly inhibited the expression of IGF1R in hVSMCs at the mRNA and protein level, while the miR-15b inhibitor exhibited the opposite effects (Fig. 3C and D).

The PI3K/AKT signaling pathway, an IGF1R-mediated downstream signaling pathway, was assessed in the current study. The results indicated that, compared with the control group, the mRNA levels of key components of the PI3K/AKT pathway, including AKT, mTOR and P70S6K, were significantly decreased in hVSMCs transfected with miR-15b mimics Additionally, treatment with the miR-15b inhibitor exhibited the opposite effect (Fig. 4A-C). Compared with the control group, the protein levels of AKT/p-AKT, mTOR/p-mTOR and P70S6K/p-P70S6K in hVSMCs were significantly inhibited by miR-15b mimics and significantly enhanced by miR-15b inhibitors (Fig. 4D-G).

To assess whether miR-15b affects hVSMCs by directly acting on IGF1R, a rescue experiment was performed. Mimic controls, miR-15b mimics, control-plasmids, IGF1R-plasmids or miR-15b mimic+IGF1R-plasmids were transfected into hVSMCs. A total of 48 h following, transfection efficiency was detected via RT-qPCR and western blotting. As presented in Fig. 5A and B, compared with the control group, the IGF1R-plasmid significantly promoted the protein and mRNA expression of IGF1R in hVSMCs. Furthermore, compared with the mimic control group, miR-15b mimics markedly reduced the protein and mRNA expression of IGF1R in hVSMCs, while the IGF1R-plasmid reversed this effect (Fig. 5C and D).

hVSMC viability, migration and apoptosis were measured using MTT, transwell and flow cytometry assays, respectively. The results revealed that, compared with the mimic group, the reduced viability and migration, and the increased apoptosis of hVSMCs induced by the miR-15b mimic was markedly reversed following transfection with the IGF1R-plasmid (Fig. 6A-D).