Blood specimens, vascular plaque tissues and corresponding vascular tissues were obtained from 30 patients with AS (age range, 45–57 years; male/female, 15/15), and blood specimens were obtained from 30 healthy individuals (age range, 43–56 years; male/female: 15/15). The samples were obtained from the Affiliated Hospital of Qingdao University (Qingdao, China) between February 2016 and February 2018. Vascular plaque tissues and corresponding vascular tissues were obtained from patients with AS following coronary artery bypass surgery. Venous peripheral blood was drawn into heparin tubes from the 30 AS patients before surgery and from the 30 healthy individuals. Patients with the following conditions were excluded: Chronic or acute inflammatory disease, asthma, type I diabetes mellitus, autoimmune disease, cancer, severe heart failure and renal and hepatic dysfunction. The exclusion criteria for healthy individuals were: History of myocardial infarction, cerebrovascular accident, coronary bypass, coronary angiography with angioplasty or stenting or both or peripheral vascular disease were excluded. Informed consent was obtained from each patient and the study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Qingdao, China).

hVSMCs were purchased from Shanghai Jining Shiye Co., Ltd. (cat. no. JN-3270) and grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Cells were then incubated at 37°C with 5% CO₂.

The hVSMCs were first seeded into 6-well plates (5×10⁴ cells/well) and cultured at 37°C for 24 h. Then, the hVSMCs were transfected with 1 µg control-shRNA plasmid (cat. no. sc-108060; Santa Cruz Biotechnology, Inc.), 1 µg MMP-9-shRNA plasmid (cat. no. sc-29400-SH, Santa Cruz Biotechnology, Inc.), 100 nM mimic control (5′-CAGCUGGUUGAAGGGGACCAAA-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-133a-3p mimic (5′-UUUGGUCCCCUUCAACCAGCUG-3′; Shanghai GenePharma Co., Ltd.), or 100 nM miR-133a-3p mimic + 1 µg MMP-9-plasmid (cat. no. sc-400083-ACT, Santa Cruz Biotechnology, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A total of 48 h after cell transfection, RT-qPCR was performed to determine the transfection efficiency.

Total RNA was extracted from blood, tissues or cells using the TRIzol Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RT of extracted total RNA into cDNA was performed using the miScript Reverse Transcription kit (Qiagen GmbH), according to the manufacturer's instructions. The temperature protocol for the reverse transcription reaction was as follows: 25°C for 5 min, 42°C for 60 min and 80°C for 2 min. PCR was performed using the QuantiFast SYBR Green PCR kit (Qiagen GmbH). The amplification conditions were as follows: 10 min at 95°C, followed by 35 cycles of 15 sec at 95°C and 40 sec at 55°C. U6 or GAPDH was used as the internal reference. The primer sequences were as follows: U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′; U6 reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′; GAPDH reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′; miR-133a-3p forward, 5′-CTTTAACCATTCTAGCTTTTCCAGGTA-3′; miR-133a-3p reverse, 5′-GACTTCGGCTGTGGACAAGATTAG-3′; MMP-9 forward, 5′-AGACCTGGGCAGATTCCAAAC3′; MMP-9 reverse, 5′-CGGCAAGTCTTCCGAGTAGT-3′. The relative expression levels of genes were calculated using the 2^−ΔΔCq method (21). All experiments were performed in triplicate.

TargetScan version 7.2 (http://www.targetscan.org/vert_72/) was used to predict the potential targets of miR-133a-3p, and the binding sites between miR-133a and MMP-9. To confirm the association between miR-133a-3p and MMP-9, a dual-luciferase reporter assay was performed.

The wild-type and mutant 3′-UTR of MMP-9 (WT-MMP-9 and MUT-MMP-9, respectively) were cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's protocol. hVSMCs were first seeded into 24-well plates (5×10⁴ cells per well) and then co-transfected with 100 ng WT-MMP-9 or 100 ng MUT-MMP-9 and 100 nM miR-133a-3p mimic or 100 nM mimic control using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol, together with Renilla luciferase pRL-TK vector (Promega Corporation) as a control. Following transfection for 48 h, the relative luciferase activity was measured using the dual-luciferase reporter assay system (Promega Corporation), as per the manufacturer's protocol. All firefly luciferase activities were normalized to Renilla luciferase activity.

To determine cell proliferation, a CCK-8 assay was performed in accordance with the manufacturer's protocol (Sigma-Aldrich; Merck KGaA). hVSMCs were seeded into a 96-well plate (1×10⁴ cells/well) and transfected with 1 µg control-shRNA, 1 µg MMP-9-shRNA, 100 nM mimic control, 100 nM miR-133a-3p mimic, or 100 nM miR-133a-3p mimic + 1 µg MMP-9-plasmid for 48 h using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. Subsequently, 10 µl CCK-8 solution (Sigma-Aldrich; Merck KGaA) was added and the cells were incubated for an additional 2 h at 37°C with 5% CO₂. Absorbance was detected at a wavelength of 490 nm using a micro-plate reader.

Following cell transfection for 48 h, the apoptotic rate of hVSMCs was determined using the Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit [cat. no. 70-AP101-100; Hangzhou MultiSciences (Lianke) Biotech Co., Ltd.], according to the manufacturer's protocol. A flow cytometer was used to analyze cell apoptosis and data was analyzed using FlowJo software (version 7.6.1; FlowJo LLC).

Protein was extracted from blood, tissues or cells using radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd.), according to the manufacturer's protocol. Total protein was quantified using a Bicinchoninic Acid protein assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein (30 µg protein/lane) were separated via SDS-PAGE on a 10% gel and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1.5 h, followed by incubation with the following primary antibodies: MMP-9 (cat. no. 13667) and β-actin (cat. no. 4970; all 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Subsequently, membranes were incubated with an anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature for 2 h. Protein bands were detected using the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Inc.).

All experiments were performed at least three times. SPSS software version 17.0 (SPSS, Inc.,) was used for data analyses. Data were expressed as the mean ± standard deviation. Differences between two groups were analyzed using a paired or unpaired Student's t-test, and comparisons between multiple groups were analyzed using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the role of miR-133a-3p in AS, the level of miR-133a-3p expression was detected in the blood and vascular plaque tissue of patients with or without AS using RT-qPCR. The results revealed that the expression of miR-133a-3p was significantly reduced in the blood of patients with AS compared with healthy controls (Fig. 1A). Furthermore, the expression of miR-133a-3p was significantly reduced in atherosclerotic plaque tissue compared with corresponding vascular tissue (Fig. 1B). These data indicated that downregulation of miR-133a-3p expression may be involved in the development of AS.

A search was performed using TargetScan to predict the target genes of miR-133a-3p. The results revealed the binding sites between miR-133a-3p and the 3′UTR of MMP-9 mRNA (Fig. 2A). Subsequently, a dual-luciferase reporter assay was performed to confirm whether miR-133a-3p interacts directly with the target gene, MMP-9 (Fig. 2B). The relative luciferase activity of hVSMCs co-transfected with WT-MMP-9 and miR-133a-3p mimic was significantly decreased compared with cells co-transfected with WT-MMP-9 and mimic control (Fig. 2B). No significant differences in relative luciferase activities were observed in hVSMCs co-transfected with MUT-MMP-9 and miR-133a-3p mimic and cells co-transfected with MUT-MMP-9 and mimic control (Fig. 2B). The results revealed that MMP-9 was a direct target gene of miR-133a-3p.

The expression of MMP-9 in the blood and vascular plaque tissue of patients with or without AS were determined via RT-qPCR and western blotting. The results demonstrated a significant upregulation of the mRNA expression of MMP-9 in the blood of patients with AS compared with the healthy controls (Fig. 3A). Compared with healthy controls, the protein levels of MMP-9 in the blood of patients with AS markedly increased (Fig. 3B). Additionally, compared with the normal vascular tissue, the mRNA levels of MMP-9 were significantly upregulated in atherosclerotic plaque tissue (Fig. 3C), whilst the protein levels of MMP-9 in atherosclerotic plaque tissues were also markedly higher compared with that in the normal vascular tissue (Fig. 3D).

hVSMCs were transfected with MMP-9-shRNA or control-shRNA for 48 h. Transfection efficiency was then detected via RT-qPCR and western blot analysis. The results indicated a significantly decreased expression of MMP-9 in MMP-9-shRNA transfected hVSMCs at the mRNA level (Fig. 4A). Compared with the control group, MMP-9-shRNA transfection markedly reduced MMP-9 protein expression in hVSMCs (Fig. 4B). The role of MMP-9 in hVSMCs was determined using a CCK-8 assay (Fig. 4C) and flow cytometry (Fig. 4D). The results demonstrated the inhibition of proliferation and the induction of apoptosis upon MMP-9 downregulation.

hVSMCs were transfected with the mimic control, miR-133a-3p mimic or miR-133a-3p mimic+MMP-9-plasmid for 48 h. RT-qPCR revealed a significantly enhanced miR-133a-3p expression (Fig. 5A) in miR-133a-3p mimic-transfected hVSMCs. Compared with the control group, miR-133a-3p mimic transfection significantly reduced MMP-9 mRNA expression in hVSMCs, which was significantly reversed by MMP-9-plasmid (Fig. 5B). The protein levels of MMP-9 was also markedly reduced in VSMCs transfected with the miR-133a-3p mimic, which was reversed by transfection with the MMP-9-plasmid (Fig. 5C). The function of miR-133a-3p in hVSMC proliferation and apoptosis was subsequently investigated. The results of the CCK-8 assay demonstrated significant inhibition of proliferation (Fig. 5D) and induction of apoptosis (Fig. 5E) in miR-133a-3p mimic hVSMCs. These effects were reversed by the addition of the MMP-9-plasmid.