miR-28-5p improved carotid artery stenosis by regulating vascular smooth muscle cell proliferation and migration

Abstract

Objectives

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in carotid artery stenosis. The purpose of this study was to investigate the diagnostic value of serum miR-28-5p in asymptomatic carotid artery stenosis and its regulation on the proliferation and migration of VSMCs.

Methods

Serum miR-28-5p levels in 65 healthy controls and 68 asymptomatic carotid artery stenosis patients were detected by qRT–PCR. The receiver-operating characteristic curve was applied to elucidate the diagnostic value of serum miR-28-5p for carotid artery stenosis patients. The specificity of miRNA targets was detected by luciferase reporter assay. CCK-8 and Transwell assay were applied to detect proliferation and migration of cells. Pearson correlation test was used to investigate the correlation between Forkhead box subclass O 1 (FOXO1) and serum miR-28-5p.

Results

Serum miR-28-5p was significantly reduced in asymptomatic carotid artery stenosis patients. Moreover, miR-28-5p could distinguish asymptomatic carotid artery stenosis patients from healthy controls, with sensitivity and specificity of 86.8% and 81.5%, respectively, indicating its high diagnostic value. The overexpression of miR-28-5p inhibited the proliferation and migration of VSMCs, while inhibition of miR-28-5p resulted in the opposite effect. What is more, FOXO1, a direct target of miR-28-5p, was significantly increased in asymptomatic carotid artery stenosis patients. Inhibition of miR-28-5p in VSMCs reversed the reduction of FOXO1 levels in patients.

Conclusions

miR-28-5p is a valuable diagnostic biomarker for asymptomatic carotid artery stenosis and can affect the proliferation and migration of VSMCs by regulating FOXO1.

Keywords

miR-28-5p asymptomatic CAS diagnostic VSMCs

Introduction

Carotid artery stenosis (CAS) is one of the key risk factors for ischemic neurological events, accounting for 10% of ischemic stroke.¹ Asymptomatic CAS occurs quietly, and its non-negligible disability and mortality rates remain a worldwide concern.² Initially, there is the only thickening of the intima-media, and atherosclerotic plaques accumulating, blocking blood flow, and then symptomatic stenosis occurs.³ Nevertheless, the proliferation and migration of vascular smooth muscle cells (VSMCs) promoted the thickening of the local intima and accelerated the progression of asymptomatic CAS.⁴ Therefore, diagnosis of patients at high risk of asymptomatic CAS in the early stage can contribute to the development of reasonable and effective treatment plans and reduce the risk of stroke. Although digital subtraction angiography is the gold standard for diagnosis of CAS, there is a risk of complications.⁵ Minimally invasive angiographies such as carotid ultrasound and CT angiography (CTA) are widely used in asymptomatic CAS. However, the consistency between the two assessments of carotid stenosis is 79.1%, so a second confirmation is needed.⁶ What is more, the preferred treatment for asymptomatic CAS patients is still controversial. Carotid endarterectomy and carotid artery stenting are good strategies for the prevention of stroke in patients with symptomatic CAS. However, they are only applicable in patients with moderate and severe stenosis, and their efficacy in asymptomatic CAS patients remains uncertain.⁷ Therefore, it is particularly important to find new and valuable biomarkers and targets for the early diagnosis and prevention of asymptomatic CAS.

As a small, highly conserved non-coding RNA molecule, microRNAs (miRNAs) can regulate the physiological and pathological processes of disease by inducing target mRNA degradation or blocking translation.⁸ In recent years, many studies have shown that a variety of miRNAs are dysregulated in CAS, such as miR-330-5p,⁹ miR-186-5p,¹⁰ and miR-214,¹¹ suggesting that miRNAs have a greater potential regulatory role in CAS. Among the reported miRNAs, miRNA-28-5p attracted our attention. CircCBFB promotes the occurrence of abdominal aortic aneurysms by influencing the apoptosis of VSMCs through miRNA-28-5p.¹⁰ Besides, miR-28-5p affects the upregulation of the ABCA1-signaling pathway by regulating ERK2, thus affecting atherosclerosis.¹² Although miR-28-5p can affect common vascular disease, including abdominal aortic aneurysm and atherosclerosis, its effect on the pathogenesis of CAS and its VSMCs in CAS has not been reported. Based on the above, we speculate that miR-28-5p may affect CAS.

The present study explored the expression pattern of serum miR-28-5p in asymptomatic CAS patients. The diagnostic value of serum miR-28-5p level in differentiating asymptomatic CAS patients from healthy controls was verified for the first time. Besides, considering the crucial role of VSMCs in the pathogenesis of CAS, we further examined the effect of miR-28-5p level on VSMCs proliferation and migration and attempted to validate their potential targets to seek new therapeutic approaches for ameliorating asymptomatic CAS.

Materials and methods

Research subjects and clinical data

This study was in accordance with Helsinki Declaration, and all subjects signed the informed consent. Moreover, this protocol was approved by the Medical Ethics Committee of Weifang Yidu Central Hospital.

A total of 68 asymptomatic patients with CAS and 65 age-matched healthy individuals admitted to Weifang Yidu Central Hospital from June 2017 to December 2019 were included in this study. All subjects underwent carotid artery ultrasound and angiography in other ongoing studies to examine bilateral carotid arteries. The degree of CAS was defined according to the previously published criteria.¹³ Patients with asymptomatic CAS were identified based on their clinical data and the National Institutes of Health Stroke Scale.¹⁴ Patients diagnosed with ischemic attack acute coronary syndrome, cerebrovascular disease, malignancy, and taking antiplatelet therapy or oral anticoagulant therapy were excluded. The inclusion criteria for the control group were carotid artery stenosis < 20%, and no history of cardiovascular disease, cancer, or mental illness. The basic clinical data of the subjects were collected in Table 1. The process flow chart of this study was shown in the Supplementary figure.

Table 1.

Clinical data of the study population.

Paraments	Total (n = 133)		P value
Paraments	Healthy individuals (n = 65)	CAS patients (n = 68)	P value
Gender (male and female)	(26/39)	(36/32)	0.165
Age 0 (years)	61.0 (12.0)	64.43 ± 6.61	0.095
BMI (kg/m²)	23.3 (3.6)	23.42 ± 3.16	0.488
FBG (mg/dL)	86.7 (31.7)	91.77 ± 16.57	0.179
SBP (mm Hg)	123.0 (11.5)	130.15 ±12.23	0.001
DBP (mm Hg)	73.0 (10.5)	84.03 ± 8.54	<0.001
HDL (mg/dL)	49.59 ± 3.39	48.95 ±3.90	0.399
LDL (mg/dL)	111.75 ± 6.46	113.5 (12.4)	0.396
TG (mg/dL)	121.60 ± 12.71	120.5 (21.2)	0.704
TC (mg/dL)	192.55 ± 4.40	192.8 (6.9)	0.946
Degree of carotid stenosis	11.36 ± 4.86	61.04 ± 7.91	<0.001

CAS: carotid stenosis; BMI: body mass index; FBG: fasting blood glucose; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; TC: total cholesterol.

Normally distributed data are expressed by mean ± standard deviation and non-normal distributed data are expressed by median (interquartile interval).

Specimen collection and serum separation

After the subjects fasted for 12 h, 5 ml of upper limb vein blood was taken. The blood samples were then placed at room temperature for 20 min and centrifuged at 3000 r/min for 30 min to collect the supernatant serum. Finally, routine clinical biochemical tests and laboratory mRNA extraction were performed respectively.

Cell culture and transfection

VSMCs were purchased from the Institute of Biochemistry Cell Biology (Shanghai, China). The cells were cultured in a Dulbecco’s modified eagle medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidity incubator at 37°C and 5% CO₂. When the cell density reached 60–80%, miR-28-5p mimic or inhibitor was transferred into the cells through the transfection reagent lipofectamine 2000 (Invitrogen, USA). According to the manufacture’s instruction, transfected cells were replaced with a new culture medium 6–8 h later.

Real-time quantitative PCR (RT-qPCR)

Validation of miRNAs level in serum and cells by RT–qPCR assay. Total RNA was extracted from serum and cells using Trizol reagent, and the quality and concentration of RNA were measured by Nanodrop. Then, the extracted mRNA was reverse-transcribed into complementary DNA (cDNA) according to the miR-X miRNA First-Strand Synthesis Kit (TaKaRa, Japan). Finally, qRT–PCR was performed using the mir-X miRNA qRT-PCR SYBR Kit (TaKaRa, Japan) in an ABI 7300 Sequencing Detection System (Applied Biosystems, Foster City, USA). The expression levels were normalized to the reference gene U6 or GAPDH. Relative miRNA and Forkhead box subclass O 1 (FOXO1) levels were calculated as 2^−ΔΔCt. Each experiment was repeated at least three times. The primer sequence was as follows: has-miR-28-5p forward 5’-AAGGAGCUCACAGUCUAUUGAG-3’ and reverse 5′-CTCGTTCGGCAGCACA-3′; U6 forward 5′-AACGCTTCACGAATTTGCGT-3′ and reverse 5′-CTCGTTCGGCAGCACA-3′; FOXO1 5′-GCGGGCTGGAAGAATTCAAT-3′ and reverse 5′-TCCAGTTCCTTCATTCTGCA-3′; GAPDH 5′-TGCACCACCAACTGCTTAGC-3′ and reverse 5′-GGCATGGACTGTGGTCATGAG-3′.

VSMCs proliferation assay

Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of VSMCs. Transfected cells were seeded with 5 × 10⁴ cells/well into 96-well plates and placed in an incubator for culture. CCK-8 reagent (10 μl) was added into cells at the 0, 24, 48, and 72 h, respectively, and incubated in an incubator for another 1 h. Then the change of absorption value at 450 nm was detected by a microplate reader (BioTek, Winooski, USA).

Cell migration assay

Transwell assay was used to detect cell migration. After transfection with miR-28-5p mimic or inhibitor, 5 × 10⁴ cells were seeded into the upper chamber. DMEM medium (500 μl) containing 10% FBS was added to the lower chamber. After cultured in the incubator for 24 h, the cells that unmigrated in the upper chamber were wiped with cotton swabs. Then, the migrated cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 10 min. The number of cells in the five fields was counted under the microscope.

Luciferase reporter assays

The specificity of miRNA targets was detected by luciferase reporter assay. FOXO1 3’UTR wild-type (WT) and mutant-type (MUT) sequences were synthesized and cloned into pMIR-report luciferase vector (Biosystems, Shanghai, China). Cells were seeded into 24-well plates until the density reached 60–80%, then miR-28-5p mimic or inhibitor, Renilla luciferase plasmid, and recombinant pMIR report plasmid were transfected into VSMCs, respectively. Luciferase activity was measured 48 h after transfection by lipofectamine 2000 using the Dual-luciferase reporter assay system (Promega, USA). Renilla luciferase activity was used for normalization.

Statistical analysis

All statistical analyses were performed with GraphPad Prism 6.0 and SPSS 23.0. Kolmogorov–Smirnov test was used for normal distribution test. In clinical data, normally distributed data are expressed by mean ± standard deviation, and non-normal distributed data are expressed by median (interquartile interval). The receiver-operating characteristic curve (ROC) was applied to elucidate the diagnostic value of miR-28-5p for CAS patients. Pearson correlation test was used to investigate the correlation between FOXO1 and serum miR-28-5p. Data were analyzed by the Mann–Whitney U test, nonpaired Student’s t-test, and one-way ANOVA followed by the post hoc Tukey’s test. P < 0.05 was considered statistically significant.

Results

Clinical characteristics of the subjects

The clinical characteristics of 68 asymptomatic CAS patients and 65 healthy individuals were analyzed. As shown in Table 1, there were no significant differences in gender, age, fasting blood glucose, low-density lipoprotein, high-density lipoprotein, total cholesterol, and triglycerides between the two groups (P > 0.05). However, compared with the healthy individuals group, asymptomatic CAS patients had higher diastolic blood pressure (DBP), systolic blood pressure (SBP, P < 0.05), and degree of carotid stenosis (P < 0.05).

Serum level of miR-28-5p in asymptomatic CAS patients

The level of miR-28-5p in the serum of the subjects was detected by qRT–PCR. Compared with healthy individuals, serum miR-28-5p was significantly reduced in asymptomatic CAS patients (P < 0.001, Figure 1). Therefore, we speculated that the dysregulated miR-28-5p may play a regulatory role in CAS.

Figure 1.

qRT–PCR was detected in the miR-28-5p expression in the subjects. Serum miR-28-5p expression reduced in asymptomatic CAS patients compared with the healthy control group. ***P < 0.001 by nonpaired Student’s t-test.

Figure 2.

ROC confirmed the high diagnostic value of miR-28-5p in asymptomatic patients with CAS. AUC was 0.889, and sensitivity and specificity were 86.8% and 81.5%, respectively. Serum miR-28-5p could significantly distinguish asymptomatic CAS patients from healthy controls.

High diagnostic value of miR-28-5p in asymptomatic CAS

To confirm our hypothesis, the diagnostic value of miR-28-5p in asymptomatic CAS patients was subsequently examined. ROC curve showed in Figure 2, miR-28-5p could significantly recognize asymptomatic CAS patients from healthy controls. The area under the curve (AUC) was 0.889, sensitivity was 86.8%, and specificity was 81.5%. The results suggest that miR-28-5p is a new potential biomarker for asymptomatic CAS.

miR-28-5p significantly reduced the proliferation and migration of VSMCs

Since the pathogenesis of CAS is involved in the proliferation and migration of VSMCs, we further analyzed the effect of miR-28-5p on cell function in vitro. miR-28-5p mimic and inhibitor were transfected into VSMCs. qRT–PCR confirmed that miR-28-5p mimic increased the level of miR-28-5p, while miR-28-5p inhibitor significantly inhibited the level (P < 0.05, Figure 3(a)). Besides, CCK-8 and Transwell assay confirmed that upregulation of miR-28-5p significantly inhibited cell proliferation and migration, while downregulation of miR-28-5p significantly promoted cell proliferation (P < 0.05, Figure 3(b) and (c)).

Figure 3.

miR-28-5p significantly reduced the proliferation and migration of VSMCs: (a) the level of miR-28-5p after transfection with miR-28-5p mimic or inhibitor was detected by qRT–PCR; (b) CCK-8 assay demonstrated that increasing miR-28-5p significantly reduced cell proliferation, whereas inhibiting miR-28-5p promoted cell proliferation; (c) Transwell assay confirmed that increasing miR-28-5p reduced cell migration while inhibiting miR-28-5p promoted cell proliferation. ***P < 0.001, compared with control group. Statistical significance in one-way ANOVA followed by the post hoc Tukey’s test.

FOXO1 is a direct target of miR-28-5p in VSMCs

Finally, we analyzed the specific targets of miR-28-5p in CAS. The TargetScan 7.0 online software demonstrated that miR-28-5p had binding sites with the 3’ UTR of FOXO1 (Figure 4(a)). Then, the luciferase reporter assay confirmed that miR-28-5p mimic significantly inhibited the activities of FOXO1 3’ UTR WT luciferase reporter, while miR-28-5p inhibitor increased luciferase activity. The results showed that FOXO1 was the direct target of miR-28-5p (P < 0.05, Figure 4(b)). Subsequent analysis showed that the level of FOXO1 in asymptomatic CAS serum was increased (P < 0.05, Figure 4(c)). Moreover, correlation analysis confirmed that the downregulation of serum miR-28-8p in patients was negatively correlated with the upregulation of FOXO1 (P < 0.05, Figure 4(d)). Finally, we also found that the increase of miR-28-5p downregulated the expression FOXO1 in cells, while the decrease of miR-28-5p had the opposite effect (P< 0.05, Figure 4(e)).

Figure 4.

miR-28-5p targeted FOXO1: (a) binding regions between miR-28-5p and FOXO1; (b) luciferase reporter assay of miR-28-5p and FOXO1. miR-28-5p mimic significantly inhibited the activities of FOXO1 3’ UTR WT luciferase reporter, while miR-28-5p inhibitor increased luciferase activity; (c) serum FOXO1 was significantly elevated in asymptomatic CAS patients compared with healthy controls; (d) the decreased of serum miR-28-5p in patients was negatively correlated with the increase of FOXO1; (e) the increase of miR-28-5p can decrease the expression of target FOXO1. **P < 0.01, ***P < 0.001, compared with control group. Statistical significance in one-way ANOVA followed by the post hoc Tukey’s test.

Discussion

CAS is a narrowing or contraction of the internal surface of the carotid artery.¹⁵ It is known to be an important risk factor for ischemic stroke. CAS gradually developed from simple thickening of intima medium to symptomatic stenosis. In asymptomatic patients, there is currently a lack of effective noninvasive subclinical biomarkers for the disease early diagnosis.¹ Besides, prophylactic surgery has certain side effects in asymptomatic patients with CAS. Therefore, it is very necessary to find new and valuable diagnostic biomarkers for the diagnosis of CAS. It is very important for the early intervention of CAS.

Evidence of miRNA dysregulation has emerged in a variety of cardiovascular and cerebrovascular diseases, including CAS and its common complications (atherosclerosis, abdominal aortic aneurysm).^16,17 It is worth noting that Ding et al.¹⁸ have analyzed miRNAs differentially expressed in endotoxin-induced myocardial injury in 2015, and the downregulation of miR-28-5p is identified. Besides, miR-28-5p participates in atherosclerosis by affecting the upregulation of the ANCA1 pathway through ERK2.¹² CircCBFb promotes the occurrence of abdominal aortic aneurysms by affecting miR-28-5p.¹⁹ Based on the above role of miR-28-5p in myocardial injury, abdominal aortic aneurysm, and atherosclerosis, we speculate that miR-28-5p may also have some influence on CAS. Therefore, we analyzed the expression of miR-28-5p in asymptomatic CAS patients and healthy controls included in the study. It was found that serum miR-28-5p in asymptomatic CAS patients was reduced. This is consistent with the expression pattern in atherosclerosis, abdominal aortic aneurysms, suggesting that miR-28-5p may be involved in the occurrence and development of CAS.

Previous studies have confirmed that the proliferation and migration of VSMCs are involved in the pathogenesis of CAS.²⁰ Moreover, various miRNAs have been reported to affect CAS by regulating the proliferation and migration of VSMCs.^21,22 What is more important, it has been confirmed that miR-28-5p affects the apoptosis of VSMCs in abdominal aortic aneurysms.¹⁹ In this study, we analyzed the effects of miR-28-5p on the proliferation and migration of VSMCs. The results confirmed that overexpression of miR-28-5p inhibited cell proliferation and migration, while inhibition of miR-28-5p promoted cell proliferation and migration. The results suggested that inhibition of miR-28-5p may significantly promote the CAS.

FOXO1 is a transcription factor and can be a potential target gene for many miRNAs. In the liver, miR-542-5p inhibited hyperglycemia and hyperlipidemia by targeting FOXO1.²³ What is more, FOXO1 has been shown to play a key role in the regulation of vascular remodeling and diabetic cardiomyopathy.^24,25 FOXO1 has been reported to regulate atherosclerosis as a complication of CAS, affects the proliferation and migration of VSMCs, and is associated with plaques in carotid artery stenosis.^24,26–28 In this study, we confirmed that FOXO1 is the target gene of miR-28-5p and was significantly increased in asymptomatic CAS. What is more, the decrease of serum miR-28-5p in patients was negatively correlated with the upregulation FOXO1. The increase of miR-28-5p can decrease the expression of target FOXO1. The results suggested that miR-28-5p may affect the proliferation and migration through the regulation of FOXO1 in CAS.

There are some limitations in this study. For example, the specific signaling pathway that miR-28-5p influences CAS through the regulation of FOXO1 has not been thoroughly studied, and the degree of asymptomatic carotid artery stenosis can be used to find its correlation with miRNA, which was not involved in this study and those will be focused on in the following studies. Besides, this study was conducted on other subjects in the ongoing study, so invasive carotid ultrasound and angiography was used for bilateral carotid artery examinations. At the same time, to rule out the influence of drugs on the expression level of miRNA in patients’ serum, and to maintain the rigor of the study, patients diagnosed with asymptomatic CAS for the first time were selected in this study. The influence of drugs on serum miR-28-5p will also be focused on in the following studies.

The research for the first time confirmed that miR-28-5p was significantly downregulated in asymptomatic CAS patients. And the downregulation of serum miR-28-5p is an effective diagnostic biomarker to distinguish asymptomatic patients with CAS from a healthy control. Besides, overexpression of miR-28-5p significantly inhibited the proliferation and migration of VSMCs, and this effect was realized through the targeted regulation of FOXO1. Therefore, promoting the expression of miR-28-5p to inhibit FOXO1 level may be a new way to improve CAS and prevent cerebral ischemic stroke.

Supplemental Material

sj-jpg-1-vas-10.1177_17085381211019510 - Supplemental material for miR-28-5p improved carotid artery stenosis by regulating vascular smooth muscle cell proliferation and migration

Supplemental material, sj-jpg-1-vas-10.1177_17085381211019510 for miR-28-5p improved carotid artery stenosis by regulating vascular smooth muscle cell proliferation and migration by Qiangrui Liu, Shibiao Yan, Yangyi Yuan, Shishun Ji and Long Guo in Vascular

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Shishun Ji

References

Dolz

Gorriz

Tembl

, et al. Circulating microRNAs as novel biomarkers of stenosis progression in asymptomatic carotid stenosis. Stroke 2017; 48: 10–16.

Bai

Feng

, et al. Treatment strategies for asymptomatic carotid artery stenosis in the era of lipid-lowering drugs: protocol for a systematic review and network meta-analysis. BMJ Open 2020; 10: e035094.

Fanelli

Boatta

Cannavale

, et al. Carotid artery stenting: analysis of a 12-year single-center experience. J Endovasc Ther 2012; 19: 749–756.

Wang

Ren

, et al. Association between ACE gene polymorphism and carotid stenosisand and construction of related gene regulatory networks. Saudi J Biol Sci 2019; 26: 2000–2005.

Huang

The role of miRNA-146a and proinflammatory cytokines in carotid atherosclerosis. Biomed Res Int 2020; 2020: 6657734.

Skagen

Skjelland

Zamani

, et al. Unstable carotid artery plaque: new insights and controversies in diagnostics and treatment. Croat Med J 2016; 57: 311–320.

Gaba

Ringleb

Halliday

Asymptomatic carotid stenosis: intervention or best medical therapy?

Curr Neurol Neurosci Rep 2018; 18: 80.

Zhu

, et al. MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation. Circ Res 2013; 113: 1117–1127.

Wei

Sun

Han

, et al. Upregulation of miR-330-5p is associated with carotid plaque’s stability by targeting Talin-1 in symptomatic carotid stenosis patients. BMC Cardiovasc Disord 2019; 19: 149.

10.

Zhang

Zhao

, et al. Diagnostic value of miR-186-5p for carotid artery stenosis and its predictive significance for future cerebral ischemic event. Diagn Pathol 2020; 15: 101.

11.

Chen

Sheu

Sun

, et al. MicroRNA-214 modulates the senescence of vascular smooth muscle cells in carotid artery stenosis. Mol Med 2020; 26: 46.

12.

Liu

, et al. MicroRNA 28-5p regulates ATP-binding cassette transporter A1 via inhibiting extracellular signal-regulated kinase 2. Mol Med Rep 2016; 13: 433–440.

13.

Aboyans

Ricco

Bartelink

MEL

, et al. ESC guidelines on the diagnosis and treatment of peripheral arterial diseases, in collaboration with the European Society for Vascular Surgery (ESVS). Rev Esp Cardiol (Engl Ed) 2017; 71: 111.

14.

Lal

Dux

Sikdar

, et al. Asymptomatic carotid stenosis is associated with cognitive impairment. J Vasc Surg 2017; 66: 1083–1092.

15.

Meissner

Meyer

FB.

Carotid stenosis and carotid endarterectomy. Cerebrovasc Brain Metab Rev 1994; 6: 163–179.

16.

Hartmann

Zhou

Natarelli

, et al. Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4. Nat Commun 2016; 7: 10521.

17.

Zhou

Wang

, et al. Overexpression of miR-431 attenuates hypoxia/reoxygenation-induced myocardial damage via autophagy-related 3. Acta Biochim Biophys Sin (Shanghai) 2020; 53: 140-148.

18.

Ding

Tang

, et al. [Differential expression of miRNAs in myocardial tissues of rats with lipopolysaccharide-induced endotoxemia]. Nan Fang Yi Ke Da Xue Xue Bao 2015; 35: 213–217.

19.

Yue

Zhu

Yang

, et al. CircCBFB-mediated miR-28-5p facilitates abdominal aortic aneurysm via LYPD3 and GRIA4. Life Sci 2020; 253: 117533.

20.

Yue

, et al. Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells. Biochem Biophys Res Commun 2018; 495: 2030–2037.

21.

Bras

Silva

Calin

, et al. miR-195 inhibits macrophages pro-inflammatory profile and impacts the crosstalk with smooth muscle cells. PLoS One 2017; 12: e0188530.

22.

Elia

Quintavalle

Zhang

, et al. The knockout of miR-143 and -145 alters smooth muscle cell maintenance and vascular homeostasis in mice: correlates with human disease. Cell Death Differ 2009; 16: 1590–1598.

23.

Tian

Ying

Wang

, et al. MiR-542-5p inhibits hyperglycemia and hyperlipoidemia by targeting FOXO1 in the liver. Yonsei Med J 2020; 61: 780–788.

24.

Yang

Zhao

, et al. Circular RNA circCHFR facilitates the proliferation and migration of vascular smooth muscle via miR-370/FOXO1/Cyclin D1 pathway. Mol Ther Nucleic Acids 2019; 16: 434–441.

25.

Zhu

, et al. Exogenous hydrogen sulfide attenuates the development of diabetic cardiomyopathy via the FoxO1 pathway. J Cell Physiol 2018; 233: 9786–9798.

26.

Jian

Wang

Jian

, et al. METTL14 aggravates endothelial inflammation and atherosclerosis by increasing FOXO1 N6-methyladeosine modifications. Theranostics 2020; 10: 8939–8956.

27.

Zou

, et al. Down-regulation of SENCR promotes smooth muscle cells proliferation and migration in db/db mice through up-regulation of FoxO1 and TRPC6. Biomed Pharmacother 2015; 74: 35–41.

28.

Jia

Aggarwal

Tyndall

, et al. Tumor necrosis factor-alpha regulates p27 kip expression and apoptosis in smooth muscle cells of human carotid plaques via forkhead transcription factor O1. Exp Mol Pathol 2011; 90: 1–8.

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