The VSMC cell line was obtained from the China Infrastructure of Cell Line Resources, Institute of Basic Medical Sciences (Chinese Academy of Sciences). The cells were cultured in DMEM (HyClone; Cytiva) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin (100 U/ml) and 1% streptomycin (100 mg/ml) (Beyotime Institute of Biotechnology) at 37˚C in a humidified incubator with 5% CO₂. VSMCs were cultured in 6-well plates for 12 h, and then transfected with indicated plasmids. ZFAS1-1 small interfering (si)RNA (5'-CTGGCTGAACCAGTTCCACAAGGTT-3'), ZFAS1-2 siRNA (5'-TACTTCTCCTAGTTGCAGTCAGG-3') and the scramble negative control siRNA (si-NC; 5'-ACGTGACACGTTCGGAGAATT-3') were obtained from Shanghai GenePharma Co., Ltd., and 100 nM of each siRNA was transfected into VSMCs for ZFAS1-knockdown using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. In addition, ZFAS1 transcript cDNA (5'-UGCGUGCCAAGCGCGACAUGGCGCGGAAGCCGAGAAGCCCCGGAGGCCC-3') was inserted into the pCDNA3.1 vector [Jiman Biotechnology (Shanghai) Co., Ltd.] and constructed by Biotech Integrated Solutions, and then 2 mg/l ZGAS1 pcDNA3.1 or empty pcDNA3.1 was transfected into VSMCs to achieve ZFAS1 overexpression (oe-ZFAS1) using Lipofectamine^® 2000. After transfection for 8 h at 37˚C, the VSMCs were exposed to ox-LDL (25, 50 and 100 mg/l; Beijing Solarbio Science & Technology Co., Ltd.) for 12, 24 and 48 h at 37˚C.

Total RNA from VSMCs was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RNA (1 µg) was reverse transcribed into first-strand cDNA using the Reverse Transcriptase kit according to the manufacturer's protocol (TransGen Biotech Co., Ltd.). qPCR was performed using SYBR Green Mixture (Takara Bio, Inc.) in the ABI Prism 7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were: Initial denaturation for 10 min at 94˚C, followed by 40 of cycles of denaturation for 30 sec at 94˚C, annealing for 30 sec at 55˚C and extension for 30 sec at 72˚C The 2^-ΔΔCq method (19) was applied to determine the relative target gene expression. The sequences for the qPCR primers were as follows: ZFAS1 forward, 5'-AGCGTTTGCTTTGTTCCC-3' and reverse, 5'-CTCCCTCGATGCCCTTCT-3'; GAPDH forward, 5'-GGTCTCCTCTGACTTCAACA-3' and reverse, 5'-AGCCAAATTCGTTGTCATAC-3'. GAPDH was employed as an internal control.

The cell viability of VSMCs was determined using the CCK-8 assay (Dojindo Molecular Technologies, Inc.). Briefly, VSMCs transfected with or without oe-ZFAS1 or si-ZFAS1 were seeded at the density of 2x10³ cells/well into 96-well plates and treated with ox-LDL. After transfection for 12, 24 and 48 h, CCK-8 reagent (10 µl) was added into each well and incubated with VSMCs for another 2 h. The absorbance at 450 nm was measured using an ELISA plate reader (Bio-Rad Laboratories, Inc.).

After treatment, proteins were isolated from VSMCs transfected with or without oe-ZFAS1 or si-ZFAS1 using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a BCA assay kit (Thermo Fisher Scientific, Inc.). Protein samples (25 µg/lane) were loaded and separated via 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with primary antibodies against Ki67 (cat. no. ab21700; 1:1,000; Abcam), proliferating cell nuclear antigen [PCNA; cat. no. 13110; 1:1,000; Cell Signaling Technology, Inc. (CST)], matrix metallopeptidase (MMP)2 (cat. no. 4022; 1:1,000; CST), MMP9 (cat. no. 3852; 1:1,000; CST) and GAPDH (cat. no. 8884; 1:2,000; CST) overnight at 4˚C. After washing with PBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (cat. no. ab97080; 1:10,000; Abcam) for 2 h at room temperature. ECL Reagent was used to develop color of protein bands (SuperSignal West Atto; Thermo Fisher Scientific, Inc). The gray values of the protein bands were determined using ImageJ software (version 1.48; National Institutes of Health). Wound healing assay. Wound healing assay was performed to assess the migration of VSMCs. Cells were plated in 6-well plates to generate a confluent monolayer. A scratch was created using a 200-µl sterile pipette tip, followed by washing with PBS three times. Subsequently, VSMCs were cultured with fresh serum-free DMEM for 24 h at 37˚C. Finally, the width of the scratch wound was observed, and images were captured at x100 magnification using a fluorescence microscope (Olympus IX53; Olympus Corporation). The recovered wound area (%) at the indicated time point (24 h) was calculated according to the following formula: [(wound width at 0 h) - (wound width at 24 h)] / wound width at 0 h.

The Transwell chamber assay was used to determine the invasive ability of VSMCs. Briefly, after transfection with or without oe-ZFAS1 or si-ZFAS1, the VSMCs were resuspended in 200 µl serum-free DMEM, and 4x10⁴ cells were loaded into the upper chambers of Transwell plates precoated with Matrigel Mix for 5 h at 37˚C (BD Biosciences), and the lower chambers were filled with 500 µl DMEM with 10% FBS as a chemoattractant. After incubation for 24 h at 37˚C, the membrane was fixed with 4% paraformaldehyde for 25 min at room temperature, and the cells on the lower surface of the membrane were stained with 0.1% crystal violet solution for 30 min at room temperature, and finally examined under a fluorescence microscope at x100 magnification (Olympus IX53; Olympus Corporation). The invasion rate was calculated according to the following formula: Number of cells in tested group / number of cells in control group.

Statistical analyses were performed using SPSS 19.0 (IBM Corp.), and data are presented as the mean ± SEM from at least three repeated experiments. Differences between multiple groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To verify the role of ZFAS1 in atherosclerosis, VSMCs were incubated with ox-LDL to simulate the high blood lipid environment, and ZFAS1 mRNA expression was detected by RT-qPCR. The results revealed that ZFAS1 mRNA expression was significantly higher with increasing doses of ox-LDL compared with the control group, and the increase was dose-dependent (Fig. 1A). Hence, 100 mg/l ox-LDL was selected for further experimentation. Moreover, VSMCs were stimulated with ox-LDL (100 mg/l) for 12, 24 and 48 h. The results of RT-qPCR revealed that ZFAS1 mRNA expression was significantly increased by ox-LDL stimulation at 24 and 48 h in a time-dependent manner (Fig. 1B). Thus, VSMCs treated with 100 mg/l ox-LDL for 48 h were selected for subsequent experiments.

To explore the effect of ZFAS1 expression on cellular behaviors, ZFAS1 expression was knocked down, and the proliferation of VSMCs was analyzed. First, si-ZFAS1 was used to achieve ZFAS1-knockdown. The RT-qPCR results demonstrated that ZFAS1 mRNA expression in the si-ZFAS1-1 and si-ZFAS1-2 groups was significantly lower compared with that in the control group, particularly in the si-ZFAS1-2 group (Fig. 2A). Hence, si-ZFAS1-2 was selected for further experiments. The CCK-8 assay results indicated that ox-LDL treatment significantly increased the viability of VSMCs at 24 and 48 h compared with the control group, while ZFAS1-knockdown significantly decreased VSMC viability when compared with the ox-LDL group (Fig. 2B). Finally, the expression levels of proteins associated with cell proliferation was quantified by western blotting. PCNA and Ki67 were identified as proliferation markers. As shown in Fig. 2C, ox-LDL stimulation caused significant upregulation of Ki67 and PCNA expression. Notably, ZFAS1-knockdown in ox-LDL-treated cells led to a significant decrease in Ki67 and PCNA expression (Fig. 2C). Hence, these results indicated that ZFAS1-knockdown inhibited the ox-LDL-induced excessive proliferation of VSMCs.

To explore the effect of ZFAS1 expression on cell proliferation, VSMC proliferation was determined following ZFAS1 overexpression. The RT-qPCR results revealed that ZFAS1 mRNA expression in VSMCs was significantly increased by oe-ZFAS1 transfection (Fig. 3A). The CCK-8 assay results revealed that ZFAS1 overexpression significantly increased the viability of VSMCs induced by ox-LDL (Fig. 3B). Finally, the western blotting results demonstrated that ZFAS1 overexpression significantly upregulated Ki67 and PCNA expression in ox-LDL-induced VSMCs (Fig. 3C). Thus, ZFAS1 overexpression promoted the proliferation of VSMCs stimulated by ox-LDL.

To investigate the effect of ZFAS1 expression on cellular behavior, ZFAS1 expression was inhibited, and the migration and invasion of VSMCs were analyzed. The results of the wound healing assay revealed that ox-LDL treatment significantly promoted the migration of VSMCs, while ZFAS1-knockdown significantly suppressed the migration of ox-LDL-induced VSMCs (Fig. 4A and C). Furthermore, the results of the Transwell chamber assay demonstrated that ox-LDL stimulation significantly promoted the invasion of VSMCs, whereas ZFAS1-knockdown significantly inhibited the invasion of ox-LDL-treated VSMCs (Fig. 4B and D). Additionally, the expression levels of proteins associated with migration and invasion were quantified. MMP2 and MMP9 are involved in cell migration and invasion. The western blotting results revealed that the expression levels of MMP2 and MMP9 in the ox-LDL and ox-LDL + si-NC groups were significantly higher compared with that in the control group (Fig. 5). Notably, ZFAS1-knockdown significantly inhibited the expression levels of MMP2 and MMP9 following ox-LDL stimulation (Fig. 5). These results indicated that ZFAS1-knockdown suppressed the excessive migration and invasion of VSMCs induced by ox-LDL.

To investigate the effect of ZFAS1 expression on cell migration and invasion, VSMC migration and invasion were determined following ZFAS1 overexpression. As shown in Fig. 6, ZFAS1 overexpression significantly promoted the migration and invasion of ox-LDL-induced VSMCs. Moreover, ZFAS1 overexpression significantly increased MMP2 and MMP9 expression (Fig. 7). These results suggested that ZFAS1 overexpression promoted the migration and invasion of VSMCs induced by ox-LDL.