2.1 Clinical specimens collection

A total of 30 patients with AS (age range, 41-59 years; male/ female, 15/15) and 30 healthy subjects (age range, 43-58 years; male/female: 15/15) were enrolled in this study. The atherosclerotic plaque tissues and corresponding normal plaque tissues were collected from atherosclerotic patients. Peripheral blood was received from 30 patients and 30 healthy subjects, and then centrifuged at 1,2000 g at 4°C for 8 min to separate serum. All specimens were separated and immediately snap-frozen in liquid nitrogen until analysis. A protocol for this study was approved by the Ethics Committee of the Guang’anmen Hospital, Chinese Academy of Chinese Medical Science and informed consents were obtained from all participants.

2.2 Cell culture and transfection

The Human VSMCs were obtained from American Tissue Culture Collection (ATCC, USA). The hVSMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, SiJi-Qing, Hangzhou, China) and 1% penicillin-streptomycin. Then, the cells were kept in a humidified incubator of 5% CO₂ at 37°C. The miR-204-5p inhibitor, miR-204-5p mimic, inhibitor control, and mimic control were obtained from GenePharma (Shanghai, China). MMP-9-plasmid, control-plasmid, MMP9-siRNA and control-siRNA were obtained form Santa Cruz. Then, the miRNAs and plasmids were transfected into VSMCs using Lipofectamine^TM 2000 transfection reagent (Invitrogen) in line with the manufacturer’s protocol.

2.3 MiRNA target analysis and dual-luciferase reporter assay

TargetScan 7.2 (http://www.targetscan.org/vert_72/) was applied to predict the potential target of miR-204-5p and MMP-9 was identified as a potential target gene of miR-204-5p. According to the bioinformatics results, the wild-type (WT) and mutant (MUT) seed regions of miR-204-5p in the 3’-untranslated region (UTR) of MMP-9 gene were synthesized in vitro and then inserted into a pmirGLO vector (Promega Corporation, Madison, WI, USA). Then, hVSMC cells were seeded in triplicate in 24-well plates and transfected with luciferase reporter plasmids and mimic control or miR-204-5p mimic using Lipofectamine 2000 reagent (Invitrogen). After transfection for 48 h, the luciferase signals were examined using the Dual Luciferase Reporter Assay System (Promega) and the results were normalized to the Renilla luciferase activity.

2.4 qRT-PCR

Total RNA was extracted from hVSMCs, tissue lysates or blood samples using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). The purity and concentrations of RNA were determined by Nanodrop ND1000 instrument. cDNA was obtained using HiScript^TM II Q RT SuperMix for qPCR (Vazyme Biozyme Co., Ltd) from 1 μg RNA and the temperature protocol was as follows: 42˚C for 2 min, 50˚C for 15 min and 85˚C for 5 sec. Amplification was performed using qRT-PCR in a Step one plus system (Roche Molecular Diagnostics, Pleasanton, CA, USA) using ChamQ^TM Universal SYBR qPCR Master Mix (Vazyme). The reaction protocol was as follows: 30 cycles of denaturing at 95˚C for 60 sec, annealing at 60°C for 60 sec, and chain extension at 72˚C for 1 min, followed by a final extension step at 72˚C for 10 min. The primer sequences were as follows:

GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′;

reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′;

U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′;

reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′;

miR-204-5p forward, 5′-ACACTCCAGCTGGGTTCCCTTTGT-CATCCTAT-3′;

reverse, 5′-CTCAACTGGTGTCGTGGA-3′;

MMP-9 forward, 5′-AGACCTGGGCAGATTCCAAAC-3′;

MMP-9 reverse, 5′-CGGCAAGTCTTCCGAGTAGT-3′.

GAPDH or U6 were applied as an internal control and relative quantification method (2^−δδCq) [18] was carried out to quantify relative expression levels.

2.5 Western blot analysis

The hVSMCs, tissue lysates and blood samples were treated with radioimmunoprecipitation lysis buffer (Invitrogen, CA, USA), and centrifuged at 12,000 rpm and 4˚C for 15 min to obtain the total proteins. The supernatant was applied to detect protein concentration using the Protein Assay Kit (Beyotime, Haimen, China). Protein samples were separated by 10% Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and then transferred onto polyvinylidene fluoride (PVDF) membranes. After transfer, the membranes were blocked with 5 g/L skimmed milk at room temperature for 1 h and subsequently incubated with MMP-9 (cat. no. 13667; dilution rate: 1:1000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; dilution rate: 1:1000; Cell Signaling Technology, Inc.) overnight. Following washing with phosphate buffered solution-tween-20 (PBST) four times of 10 min, the membranes were incubated with corresponding horseradish peroxidase (HRP)-linked secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Finally, membranes were washed four times with PBST for 10 min and developed using the chemiluminescence (ECL) plus Kit (Pierce, USA).

2.6 MTT assay

The proliferation of hVSMCs was determined by the MTT assay according to the manufacturer’s protocol. Briefly, the cells were seeded into 96-well plates at a density of 10,000 cells per well 24 h prior to transfection. After transfection for 48 h, MTT solution (25 μL) was added to each well and cultivated at 37°C for 4 h. After discarding the supernatant, 200 μL dimethyl sulfoxide (DMSO) was added and slightly shaken for 10 min to dissolve the formazan crystals. Lastly, the 96-well plates were placed at a micro-plate reader (Molecular Devices, Sunny- vale, CA, USA) and the extinction was measured at 490 nm.

2.7 Flow cytometry analysis

After transfected for 48 h, hVSMCs were trypsinized and then centrifuged at 1000 rpm for 5 min at room temperature. Furthermore, the sedimentary cells were re-suspended and adjusted at a density of 1×10⁶ cells/ml. Subsequently the cells were stained with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in dark. After staining, the cell apoptosis was analyzed by flow cytometry (Calibur; BD, USA), and data on cell apoptosis was collected analyzed using FlowJo software (version 10.0).

2.8 Transwell assay

The hVSMCs were starved through culturing in serum-free medium for 12 h. Then, cells were transfected with inhibitor control, miR-204-5p inhibitor, mimic control, miR-204-5p mimic, miR-204-5p inhibitor+control-siRNA, miR-204-5p inhibitor+MMP-9-siRNA, miR-204-5p mimic+control-plasmid or miR-204-5p mimic+MMP-9-plasmid. After 48 hours of transfection, cells were harvested and the migratory ability was detected using a Transwell chamber. In brief, 1×10⁵ hVSMCs were seeded into the upper chamber pre-coated with or without Matrigel (BD Biosciences). The lower chambers were filled with 400 μL medium containing 10% FBS. After incubation with 5% CO₂ at 37°C for 16 hours, non-migrated or no-invaded cells were removed from the on the topside of the filter by cotton bud. Then, the migrated or invaded cells were washed with PBS and fixed with 4% paraformaldehyde solution and stained with 0.1% crystal violet for half an hour at room temperature, imaged, and counted. Each sample was performed in triplicate.

2.9 Statistical analysis

Statistical analysis was performed with SPSS v16.0 (SPSS, Chicago, IL, USA). Measurement data were expressed as the mean ± standard deviation (SD) of 3 independent experiments. Student’s t-test was used to comparing the data between two groups. The one-way analysis of variance with Tukey’s post hoc test was used for comparisons of multiple samples. p<0.05 was considered to indicate a statistically significant difference.

3.1 miR-204-5p was downregulated in atherosclerotic tissues and blood

In order to detect the activity of miR-204-5p in the progression of AS, qRT-PCR assay was conducted to investigate the miR-204-5p expression level in atherosclerotic tissues and blood. Generally, the miR-204-5p expression in atherosclerotic blood samples was decreased in comparison to those the in healthy individuals (Figure 1A). In the same way, the atherosclerotic plaque tissues had lower miR-204-5p expression than adjacent tissues (Figure 1B).

Figure 1

The low expression of miR-204-5p in atherosclerotic tissues and blood.

(A) The expression levels of miR-204-5p in blood from 30 atherosclerotic patients and 30 healthy subjects were determined by qRT-PCR assay. (B) The expression levels of miR-204-5p in 30 atherosclerotic plaque tissues and 30 normal control tissues were detected by qRT-PCR assay. Data were exhibited as mean ± SD.**p < 0.01.

3.2 MMP-9 was a direct target of miR-204-5p

In order to understand the molecular mechanisms of miR-204-5p in AS, prediction program TargetScan was used to foresee the potential targets of miR-204-5p. As shown in Figure 2A, we found that MMP-9 was a candidate target of miR-204-5p. To further verify the predicted binding sites between MMP-9 and miR-204-5p, luciferase reporter constructs were performed. In Figure 2B, miR-204-5p markedly reduced the luciferase activity of MMP-9 wild-type 3’-UTR compared with the mimic control group, while no obvious effect on MMP-9 3’-UTR-mut reporter. Taken together, we concluded that MMP-9 was a direct target of miR-204-5p.

Figure 2

miR-204-5p directly targets MMP-9.

(A) The position of miR-204-5p target sites in 3′-UTR of MMP-9. (B) Dual-luciferase assay was used to confirm the binding sites between miR-204-5p and MMP9. Each value represents as means ± SD. **p < 0.01.

3.3 miR-204-5p negatively regulated the expression of MMP-9 in hVSMCs

To determine the effect of miR-204-5p on the expression of MMP-9 in hVSMCs, hVSMCs were transfected with inhibitor control, miR-204-5p inhibitor, miR-204-5p mimic, mimic control, MMP-9-plasmid, control-plasmid, MMP9-siRNA and control-siRNA. After transfected for 48 h, qRT-PCR analysis was performed to detect the transfection efficiency. The results showed that the expression level of miR-204-5p was lower in miR-204-5p inhibitor transfected group compared to the control group in hVSMCs (Figure 3A). In addition, the miR-204-5p expression was higher in miR-204-5p mimic group than that in the control group (Figure 3B). Furthermore, the expression of MMP-9 was significantly upregulated in MMP-9-plasmid transfected hVSMCs compared with the control group (Figure 3C). Meanwhile, the MMP-9 expression was downregulated after transfected with MMP9-siRNA compared with the control group (Figure 3D). Additionally, the expression of MMP-9 was reduced by miR-204-5p mimic in hVSMCs compared to the control at both mRNA and protein levels, while the effect was reversed by MMP-9-plasmid (Figure 3E and F). Consistently, the expression of MMP-9 was promoted by miR-204-5p inhibitor and compared to the control and the effect was also reversed by MMP-9-siRNA (Figure 3G and H). Collectively, the above results demonstrated that miR-204-5p could negatively adjust the expression of MMP-9 in hVSMCs.

Figure 3

miR-204-5p regulated the expression of MMP-9 in hVSMCs.

(A) After transfected with miR-204-5p inhibitor and inhibitor control, the expression of miR-204-5p in hVSMCs was detected by qRT-PCR assay. (B) After transfected with miR-204-5p mimic and mimic control, the expression level of miR-204-5p in hVSMCs was determined by qRT-PCR assay. (C) After transfected with MMP-9-plasmid and control-plasmid, the mRNA expression level of MMP-9 in hVSMCs was determined by qRT-PCR assay. (D) After transfected with MMP9-siRNA and control-siRNA, the mRNA expression level of MMP-9 in hVSMCs was determined by qRT-PCR assay. (E and F) The expressions of MMP-9 were detected by qRT-PCR and Western blot assay in miR-204-5p mimic accompanied by control-plasmid or MMP-9-plasmid groups at both mRNA and protein levels. (G and H) The expressions of MMP-9 were detected by qRT-PCR and Western blot assay in miR-204-5p inhibitor accompanied by control- siRNA or MMP-9-siRNA groups at both mRNA and protein levels. Data presented were the mean ± SD. **p < 0.01 vs. Control group; ## p < 0.01 vs. miR-204-5p mimic/inhibitor group.

3.4 Downregulation of MMP-9 abrogated the promotion of miR-204-5p inhibitor on hVSMC growth and migration

MMP-9 was considered as to be regulated by miR-204-5p in hVSMCs, we further investigated whether MMP-9 was involved in miR-204-5p - mediated regulations in hVSMCs. Firstly, hVSMCs were transfected with inhibitor control, miR-204-5p inhibitor alone or accompanied with control-siRNA or MMP-9-siRNA for 48 h. After transfection, MTT assay, flow cytometry assay and transwell analysis were conducted to detect cell viability, apoptosis and migration respectively. As shown in Figure 4A, downregulation of miR-204-5p resulted in increased cell viability in hVSMCs. In Figure 4B, transwell analysis demonstrated that downregulation of miR-204-5p promoted the migratory activity of hVSMCs. Results from flow cytometry assay showed that downregulation of miR-204-5p inhibited the cell apoptosis of hVSMCs (Figure 4C and D). However, all these functions were reversed by MMP-9- siRNA.

Figure 4

Downexpression of MMP-9 abrogated the promotion of miR-204-5p inhibitor on hVSMC growth and migration.

hVSMCs were respectively transfected with inhibitor control, miR-204-5p inhibitor alone or accompanied with control-siRNA or MMP-9-siRNA for 48 h. (A) MTT assay was used to detect cell viability of hVSMCs. (B) The transwell assay was performed to check cell migration. (C and D) hVSMC apoptosis was detected by flow cytometric assay. Results were presented as mean ± SD. *, **p < 0.05, 0.01 vs. Control group; #, ## p < 0.05, 0.01 vs. miR-204-5p inhibitor group.

3.5 Overexpression of MMP-9 abrogated the inhibition of miR-204-5p upregulation on hVSMC growth and migration

Based on the previous experiment, we further explored the crucial role of overexpressed MMP-9 on miR-204-5p-mediated regulations of cell growth and migration in hVSMCs. In brief, mimic control, miR-204-5p mimic alone or accompanied with control-plasmid or MMP-9-plasmid were transfected into hVSMCs for 48 h. The results demonstrated that miR-204-5p upregulation evidently inhibit the viability (Figure 5 A) and migration (Figure 5 B) in hVSMCs, while promoted apoptosis in hVSMCs (Figure 5 C and D). However, all the above effects were inverted by MMP-9-plasmid. Together, the results indicated that miR-204-5p targeted MMP-9 and regulated the proliferation, apoptosis and migration of hVSMCs.

Figure 5

Overexpression of MMP-9 abrogated the inhibition of miR-204-5p mimic on hVSMC growth and migration.

hVSMCs were respectively transfected with mimic control, miR-204-5p mimic alone or accompanied with control-plasmid or MMP-9-plasmid for 48 h. (A) MTT assay was used to detect cell viability of hVSMCs. (B) The transwell assay was performed to check cell migration. (C and D) hVSMC apoptosis was detected by flow cytometric assay. Results were presented as mean ± SD. **p < 0.01 vs. Control group; ## p < 0.01 vs. miR-204-5p mimic group.