This article is mentioned in:

Introduction

Cardiovascular diseases (CVDs) are a series of heart and vascular diseases, which cause a huge social and economic burden worldwide. Acute myocardial infarction (AMI) is one important type of CVD (1). Mortality caused by CVD has been reported to account for 43.81 and 46.66% of total mortality in urban and rural areas respectively, and the mortality rate of AMI in China has risen (2). AMI is one common type of coronary heart disease, which is induced by acute and persistent ischemic hypoxia and ultimately causes myocardial necrosis (3). Traditionally, AMI has been diagnosed based on specific electrocardiogram (ECG) presentations and elevated cardiac serum markers, including lactate dehydrogenase, cardiac Troponin I (cTnI), cardiac muscle troponin T (cTnT) and creatine kinase MB (CK-MB) (4,5). However, the complexity of the condition of the patient, the onset of acute pericarditis, myocarditis, heart failure, hypertension and acute pulmonary embolism can also lead to elevated levels of existing markers, which makes distinguishing AMI from these conditions difficult (6,7). Only ST-segment elevation MI (STEMI) can be detected by electrocardiography, while the infarcted vessels cannot be predicted the first time. Although coronary angiography is the most effective method to diagnose and treat AMI, it may also be ineffective in certain cases where the infarcted vessels are complex. Furthermore, some areas do not have sufficient access to coronary angiography. This makes it difficult for a large proportion of patients with AMI to receive the optimal treatment (8). In summary, it is necessary to develop more comprehensive clinical indicators to aid the diagnosis and assessment of AMI as quickly as possible.

Exosomes (exos) are endogenously formed extracellular vesicles that were first identified in the late 1960s (9) and are considered micro-vesicles with a diameter of 30–150 nm. Exos can be produced by numerous types of cells and are widely present in the urine, blood and saliva. Exos have a phospholipid bilayer structure and the micro-vesicles are rich in biomolecules, such as proteins, lipids, mRNA and non-coding RNA; therefore, exos can act as messengers, which mediate intercellular communication and participate in the regulation of cellular functions (10). Moreover, certain proteins have been previously detected in exos, which demonstrate superiority in the timeliness and specificity of different diseases compared with traditional plasma markers, and these exos display great clinical value by serving as novel biomarkers and therapeutic agents for certain neoplastic diseases. For example, it has been shown that ABCG1 and PTEN can be proteins in exosomes that delay the progression of atherosclerosis (11,12). In the study of cardiovascular system diseases, increasing attention has been paid to the implications and functions of exosomal microRNAs (miRNAs) (13).

miRNAs are a class of highly conserved small non-coding RNAs, which widely exist in plasma or serum, either bound to protein complexes or present in microvesicles or lipoproteins (14–16). miRNAs mainly regulate genes by binding to the 3′-UTR of mRNAs and interfering with subsequent protein synthesis. Numerous miRNAs have been reported to have an association with the pathogenesis of CVD (17,18). It has been previously demonstrated that miRNA-133 (miR-133) widely exists in cardiomyocytes, that the inhibition of miR-133 increases cardiac hypertrophy and upregulation of miR-133 improves cardiac function (19). miR-199a and miR-590 have been reported to alleviate cardiac function after AMI by inducing mitosis (20). Furthermore, in terms of the therapeutic study of miRNAs, inhibition of miR-92a reduces endothelial inflammation and promotes angiogenesis and the functional recovery of ischemic myocardium (21). It has been reported that exosomal miRNA levels and corresponding disease scores, such as the SYNTAX score, can be used to predict the complexity of coronary lesions, which provides valuable references for the next step of treatment (22,23).

The Wnt/β-catenin signaling pathway has been reported to be reactivated after MI and is involved in the regulation of pathophysiological processes, such as myocardial apoptosis and myocardial fibrosis (24–26). Secretory frizzled-related protein 1 (SFRP1) is a protein structurally similar to the frizzled (FZ) receptor that inhibits the binding of Wnt or FZ receptor/β-catenin signaling pathway activity. It has been previously suggested that SFRP1 is closely associated with myocardial fibrosis and cardiac remodeling, and it is unclear whether its expression is affected by MI (27–33).

In conclusion, studies on AMI and exosomal miRNA are rare; however, it is necessary to explore their association to facilitate the clinical diagnosis and assessment of AMI. The aim of the present study was to identify top differential miRNAs from sequencing results, and to validate these miRNAs in AMI and healthy control groups. The aim was also to identify new markers of AMI by performing correlation analysis between exosomal miRNAs and existing clinical indicators in AMI. Our previous study reported the involvement of miRNAs carried by exosomes in the formation of frontal atherosclerosis (34). This study will investigate the value of miR-4516 and miR-203 in aiding the diagnosis of acute myocardial infarction and predicting the extent of vascular injury through sequencing analysis. These will help in the future diagnosis and treatment in the clinic.

Materials and methods

Ethics approval

The present study was performed at The Central Hospital Affiliated with Shandong First Medical University (Jinan, China). Blood samples from patients with AMI and healthy controls were collected from The Department of Cardiovascular Medicine and The Emergency and Health Examination Center between October 2020 and May 2021. The present study was performed according to the principles of the Declaration of Helsinki, and the study design was approved by The Ethical Committee of The Central Hospital Affiliated with Shandong First Medical University (approval no. 2018-039-01; Jinan, China).

Study participants

All participants in the present study were volunteers and signed an informed consent form. Basic information about the participants, including sex, age, disease situation and other relevant personnel information were recorded. A total of 62 patients with AMI and 31 healthy controls were enrolled in the present study. The diagnostic criteria for acute myocardial infarction were defined by The Joint European Society of Cardiology (35). The main AMI diagnostic basis includes typical AMI symptoms and signs, abnormal ECG, the level of serum biomarkers and coronary angiography results (36).

Inclusion criteria

The inclusion criteria for selecting participants were as follows: i) Aged 35–75 years old; ii) no history of smoking or had quit smoking >3 months before the study; iii) chest pain lasting >30 min with no relief. The time window was within 12 h from the start of chest pain, including both acute ST elevation and non-ST elevation MI, with samples collected from the AMI group within 12 h of onset; iv) cTnT or cTnI >upper limit of a normal level (99th percentile of the upper reference value) for at least one-time point; v) patients with ≥70% stenosis of the pathogenic vessel identified by coronary angiography, as this was required for percutaneous coronary intervention (PCI); vi) no anticoagulants administered; and vii) systolic blood pressure level was 90–140 mmHg, and diastolic blood pressure was <90 mmHg (patients with a history of well-controlled hypertension were also enrolled). All patients with acute myocardial infarction were given standard treatment. The appropriate medical records were retrieved for all participants.

Exclusion criteria

The exclusion criteria for selecting participants for the present study were as follows: i) Presence of infection, autoimmune disease, liver or kidney dysfunction, or history of tumor; ii) aged <35 or >75 years old; iii) a blood glucose level of >7 mmol/l; iv) systolic blood pressure <90 or >140 mmHg and/or diastolic blood pressure >90 mmHg; and v) psychiatric disorders (37).

SYNTAX score

SYNATX scores were assigned by assigning scores to individual coronary vessels. The greater the sum of the scores, the more severe the lesion. The complexity of coronary lesions was quantitatively evaluated according to the integration system by two or more experienced physicians according to anatomical characteristics, such as lesion location, severity, bifurcation and calcification of the left main coronary artery and/or three main vessels (38).

Extraction of exos from blood samples

A fasting blood sample (5 ml in a Becton, Dickinson and Company EDTA anticoagulant tube) was collected from all members of the healthy control group, and 5 ml of venous blood was collected before emergency PCI from participants in the AMI group. After centrifugation at 2,000 × g at 4°C for 10 min, plasma was collected in 1.5 ml RNase-free tubes and stored at −80°C. After 16,260 × g centrifugation at 4°C for 45 min, the supernatant of the plasma was collected and filtered using a 0.22 µm filter. The filtered supernatant was transferred into an ultracentrifuge tube and was centrifuged at 110,000 × g at 4°C for 120 min. At the end of the first ultracentrifugation step, the supernatant was aspirated, and the pellet was resuspended in 100 µl sterile PBS. The aforementioned centrifugation parameters were repeated for further centrifugation steps. After the second ultracentrifugation, the supernatant was removed, and the pellet was resuspended in 100 µl PBS, transferred to a new RNase-free tube and stored in a −80°C freezer.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) examination

A total of 10 µl exo suspension solution was dropped onto copper grids and subsequently transferred to 3% glutaraldehyde solution for fixation for 30 min, followed by the addition of 4% acetic acid oxygen dye solution and 1% methylcellulose solution, for visualization under TEM (ht-7700; Hitachi, Ltd.). Dilute exos obtained by ultracentrifugation were processed by NTA, to define the particle size distribution and exo concentration of all samples. Diluted exos obtained by ultracentrifugation were processed by NTA, to define the particle size distribution and exo concentration of all samples. The whole experiment was performed at 20–25°C.

Western blotting (WB)

RIPA lysis buffer (cat. no P0013B; Beyotime Institute of Biotechnology) was used to extract proteins from the exos previously isolated by ultracentrifugation. After ultrasonic cell disruptor processing, the lysate was centrifuged at 16,260 × g at 4°C for 5 min, and the supernatant was collected for concentration quantification using a BCA assay kit. Loading buffer was added to the samples, the samples were boiled for 10 min, and then stored at −80°C. A total of 20 µg protein sample per well was loaded on 10% sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis was completed, the separated proteins were subsequently transferred onto a 0.22 µm PVDF membrane for 90 min at 70 V. The membranes were blocked with 5% non-fat milk powder (diluted in TBST with 0.1% Tween 20) at room temperature. After blocking, membranes were incubated with primary antibodies against CD9 (1:1,000; cat. no. 20597-1-AP; Proteintech Group, Inc.), Alix (1:1,000; cat. no. sc-53540; Santa Cruz Biotechnology, Inc.) and calnexin (1:1,000; cat. no. 10427-2-AP; Proteintech Group, Inc.) as a negative control, overnight at 4°C. The membranes were washed three times using TBST and subsequently incubated with Anti-rabbit IgG (1:20,000; cat. no. 7074; Cell Signaling Technology, Inc.) and Anti-mouse IgG (1:20,000; cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies for 60 min at room temperature. The chemiluminescence reagent was Immobilon Western (MilliporeSigma) and Images were captured using a Tanon 5200 Multi Chemiluminescent Imaging System.

Extraction of total RNA

Exo RNA was isolated using a column-based isolation kit (cat. no. 217184; Qiagen China Co., Ltd.) according to the manufacturer's instructions. Briefly, 1 ml of RNA lysate was added to a 200 µl sample of exosomes, which was subsequently centrifuged at 16,260 × g 4°C for 90 min, resuspended and precipitated, centrifuged again and then isoacetone and anhydrous ethanol were added in that order. Finally, the RNA was obtained by adding enzyme-free water to resuspend the precipitate. The concentrations were measured using a spectrophotometer(ND-ONE-WA30221; Thermo Fisher Scientific, Inc.) at a wavelength of 260 nm.

Reverse transcription-quantitative (q)PCR

Synthesis of complementary cDNA was performed using an RT kit (cat. no. AG11717; Accurate Biology Inc.;) according to the manufacturer's protocol. The total reaction volume was 20 µl, which included 10 µl 2× miRNA RT Reaction Buffer, 2 µl miRNA RT Enzyme Mix and 8 µl RNA. Thermocycling was performed as follows: 42°C for 60 min and 95°C for 3 min. Real-time fluorescent quantitative PCR was performed on a QuanStudio 1 (Thermo Fish Scientific, Inc.) with thermocycling conditions as follows: 95°C for 30 sec for 1 cycle, and 35 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 30 sec. This process was repeated 3 times for each sample. qPCR was performed using the SYBR Green PCR kit (cat. no. AG11702; Accurate Biology Inc.) according to the manufacturer's protocol. The experiment was performed on a Light Cycle 480 machine (Roche Diagnostics). Each experiment was repeated three times and relative expression levels of miRNA were analyzed using the 2−ΔΔCq method (11). U6 was used for normalization. For RT-qPCR, a tailing method (addition of the poly(A) tail to the 3′ end of miRNA by a poly(A) polymerase to increase its length) was used. A specific primer was used upstream and a universal primer was used downstream. All miRNAs were purchased from Tiangen Biotech Co., Ltd. The sequences of the primers used were as follows: has-miR-203 forward (F), 5′-GUGAAAUGUUUAGGACCACUAG-3′; has-miR-4516 F, 5′-GGGAGAAGGGUCGGGGC-3′; and hsa-U6- F, 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′. The miRNA reverse primer was included in the RT-qPCR kit (cat. no. AG11717; Accurate Biology Inc.).

Bioinformatics analysis

Target genes of differentially expressed miRNAs were predicted using TargetScan 8.0 software (https://www.targetscan.org/vert_80/). Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/) enrichment analyses were performed using the David 6.8 online database (https://david.ncifcrf.gov). The ggplot2 package (version 3.3.5) (https://cran.r-project.org/web/packages/ggplot2/) in R (version 4.0.5) (https://www.r-project.org/) was used to plot bar and Venn diagrams for visualization.

ELISA

Plasma was obtained by centrifuging blood samples at 2,000 × g at 4°C for 10 min and was then stored at −80°C. A Human SFRP1 ELISA kit (cat. no. SEF880Hu; Cloud-Clone Corp.) was used to assess the SFRP1 secretion level in each individual, according to the manufacturer's protocols. The signal was measured at 450 nm excitation light using a SpectraMax® i3 multifunctional microplate reader. After the standard curve was established, the plasma expression levels of SFRP1 were calculated based on the absorbance values of each sample.

Statistical analysis

Mean ± standard deviation was used to present relative clinical characteristics. Ordinal variables were compared using one-way ANOVA. The statistical significance of normally distributed data was assessed with the unpaired Student's t-test. Pearson's correlation coefficient was used to validate the relationships between continuous variables. Independent factor risk analysis was performed using logistic regression. All experiments were repeated ≥3 times. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp.) and GraphPad Prism 8.0 (GraphPad Software; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Results

Baseline characteristics of the study population

The baseline characteristics of the 31 healthy control individuals and the 62 patients with AMI were summarized (Table I). Unpaired Student's t-test was used to compare the differences between the AMI and control groups. In parallel, drug use data was collected from both groups (Table II). There were no statistically significant differences in these characteristics between the two groups, except for total cholesterol).

Table I. Baseline data of the study participants.

Table I.

Baseline data of the study participants.

Baseline characteristic	Control, mean ± SD	AMI, mean ± SD	P-value
Age, years	52.81±7.6	62.02±9.46	0.058
HR, beats/min	74.06±11.6	76.76±13.02	0.379
SBP, mmHg	105.03±9.83	125.21±19.58	0.086
DBP, mmHg	79.19±9.77	75.73±14.34	0.277
Cr, µmol/l	75.1±15.73	77.97±15.44	0.289
Glu, mmol/l	5.19±0.74	5.59±1.14	0.054
TG, mmol/l	1.49±0.63	1.49±0.73	0.917
TC, mmol/l	3.96±1.04	4.51±1.03	0.022
LDL, mmol/l	2.69±1.08	2.71±0.81	0.985
HDL, mmol/l	1.21±0.4	1.18±0.47	0.676
Apolipoprotein a, mmol/l	231.83±291.8	330.56±304.14	0.144
Apolipoprotein B, mmol/l	1.01±0.25	1±0.17	0.671
Apolipoprotein E, mmol/l	46.63±12.61	51.59±16.35	0.185

[i] Unpaired Student's t-test was used to assess differences between the AMI and control groups. HR, Heart rate; SBP, Systolic blood pressure; DBP, Diastolic blood pressure; Cr, Creatinine; Glu, Blood glucose; TG, Triglycerides; TC, Total cholesterol; LDL, Low-density lipoprotein; HDL, High-density lipoprotein.

Table II. Summary of medication use.

Table II.

Summary of medication use.

Drugs/drug groups (Postoperative)	Control (n=31)	AMI (n=62)
Aspirin, n	12	39
Tegretol, n	NA	NA
Statins, n	4	10
Beta blockers, n	3	22
Proton pump inhibitors, n	NA	20
Isosorbide mononitrate, n	NA	2
ACEI/ARB, n	3	40
Trimetazidine, n	NA	NA
CCB, n	NA	30
Clopidogrel, n	NA	25
Diuretics, n	NA	20
Ivabradine, n	NA	2

[i] ACEI, angiotensin converting enzyme inhibitor; ARB, Angiotensin Receptor Blocker; CCB, calcium Channel Blockers.

Characterization of exos

Previously published reports and the standards of the International Society for Exosome Vesicles indicate that verification of exosomes can be performed using transmission electron microscopy, WB and particle size analysis, simultaneously (34,39). The size and morphology of exosomes can be objectively observed using transmission electron microscopy. Particle size analysis allows the size, diameter and concentration of exosomes to be assessed (40). The typical disc-like structure of exos was observed using TEM, which was a direct observation of plasma exosomes under TEM, whose shape and size met the criteria for exosomes (Fig. 1A). WB allowed verification of specific proteins on the surface of exosomes, such as CD9 and ALIX. Most common cell membranes contain Calnexin proteins; however, exos do not have Calnexin proteins on their membranes. Therefore, cell lysis was chosen as a control group. The identity of the exos was further confirmed by WB using antibodies against the exo-specific markers, Alix and CD9, and the negative control, calnexin (Fig. 1B). NTA results demonstrated that the average diameter of the isolated granule was 127.5 nm, with a median diameter of 117.7 nm (Fig. 1C), which was in-line with the standard size of exos. These results confirmed that exos were successfully extracted at a high quality and purity. The regulatory relationship between SFRP1 and the Wnt/β-Catenin signaling pathways, which serve a vital role in atherosclerosis or AMI was illustrated (Fig. 1D) (41,42).

Figure 1. Successful isolation of exos from plasma. (A) Ultrastructure of exos isolated from plasma was examined using transmission electron microscopy. Scale bar=200 nm. (B) The expression of exo markers, Alix and CD9, and the negative marker, calnexin, assessed using western blotting. (C) Size distribution of exos using particle size analysis. (D) SFRP1 serves an inhibitory role in the Wnt/β-Catenin signaling pathway. Exo, exosome; SFRP1, secretory frizzled-related protein 1.

Expression levels of exosomal miR-4516, miR-203 and plasma SFRP1 in each study group

Previous sequencing results were analyzed and the top 10 miRNAs with high variance ranking were selected for validation (Fig. 2A) (34). SFRP1 was predicted as the target protein for both miR-4516 and miR-203 using TargetScan software (Fig. 2B). Statistical analysis was performed using an unpaired Student's t-test. The levels of miR-4516 and miR-203 in plasma exos from the AMI group were significantly higher compared with those in the healthy control group. In addition, the SFRP1 protein expression level was significantly increased in the plasma of patients with AMI compared with the control group (Fig. 2C and Table III, Table IV, Table V). These data indicated that exosomal miR-4516, miR-203 and plasma SFRP1 were associated with AMI.

Figure 2. Indicators to diagnose AMI. (A) Previous sequencing results were analyzed and the top 10 miRNAs with high variance rankings were selected for validation. (B) SFRP1 was predicted as a target protein for both miRNAs using TargetScan software. There were 485 predicted downstream target proteins common to mir-4516 and miR-203. (C) Relative expression levels of miR-4516, miR-203 and SFRP1 in different subgroups. Unpaired Student's t-test was used for analysis. (D) Correlation analysis of miR-4516, miR-203 and SFRP1 in diagnosis of AMI. *P<0.05 and ****P<0.001. AMI, acute myocardial infarction; miR, microRNA; miRNA, microRNA; SFRP1, secretory frizzled-related protein 1.

Table III. SFRP1 expression levels in AMI and control groups.

Table III.

SFRP1 expression levels in AMI and control groups.

				95% Confidence interval
Group	Standard error	Mean deviation	P-value	Lower	Upper
Control (miR-4516)	4.284	0.272	0.001	0.176	0.368
AMI (miR-4516)	0.270	3.871	0.001	2.640	5.102
Control (miR-203)	3.578	2.387	0.001	1.359	3.415
AMI (miR-203)	1.171	1.050	0.001	0.659	1.440
Control (SFRP1)	1.802	4.657	0.001	4.196	5.119
AMI (SFRP1)	1.111	1.017	0.001	0.610	1.425

[i] AMI, acute myocardial infarction; miR, microRNA; SFRP1, secretory frizzled-related protein 1.

Table IV. MiR-203 expression levels in AMI and control groups.

Table IV.

MiR-203 expression levels in AMI and control groups.

				95% Confidence interval
Group	Standard error	Mean deviation	P-value	Lower	Upper
Control	3.578	2.387	0.001	1.359	3.415
AMI	1.171	1.050	0.001	0.659	1.440

[i] AMI, acute myocardial infarction.

Table V. Secretory frizzled-related protein 1expression levels in AMI and control groups.

Table V.

Secretory frizzled-related protein 1expression levels in AMI and control groups.

				95% Confidence interval
Group	Standard error	Mean deviation	P-value	Lower	Upper
Control	1.802	4.657	0.001	4.196	5.119
AMI	1.110	1.017	0.001	0.610	1.425

[i] AMI, acute myocardial infarction.

Exosomal miR-4516, miR-203 and plasma SFRP1 levels as diagnostic biomarkers for AMI

The area under the curve (AUC) of exosomal miR-4516 and miR-203 were 0.9809 (P<0.0001) and 0.6823 (P=0.006), respectively, and the AUC of plasma SFRP1 was 0.9603 (P<0.0001) in the AMI group, which indicated that exosomal miR-4516, miR-203 and plasma SFRP1 may be candidate diagnostic biomarkers for AMI (Fig. 2D).

Positive correlation between plasma exosomal miR-4516 and SYNTAX score of patients with AMI

After collecting coronary angiography images from each participant, the images were assessed using the SYNTAX scoring system (Fig. 3A). Plasma exosomal miR-4516 levels were significantly positively correlated with the SYNTAX scores of patients with AMI (r2=0.7875; P<0.0001) using Pearson's correlation analysis (Fig. 3B). However, there was no significant correlation between either the exosomal miR-203 or plasma SFRP1 levels and the SYNTAX score in the AMI group (r2=−0.0145, P=0.9461 and r2=−0.229, P=0.6572, respectively).

Figure 3. Correlation of plasma exosomal miR-4516, miR-203 and SFRP1 levels with SYNATX scores. (A) Representative images from a coronary angiography procedure. The three images show (left to right) occlusion of the gyral, anterior descending and right coronary branches of the left coronary artery, respectively. The degree of damage to the vessel was predicted based on the blockage and was assessed using the SYNATX score. (B) Correlation analysis of plasma exosomal miR-4516, miR-203 and SFRP1 levels with the SYNATX score of patients with AMI. Correlation of plasma exosomal miR-4516, miR-203 and SFRP1 levels with (C) cTnI and (D) LDL in patients with AMI. AMI, acute myocardial infarction; cTnI, cardiac troponin I; exo, exosome; LDL, low-density lipoprotein; miR, microRNA; SFRP1, secretory frizzled-related protein 1.

Plasma SFRP1 is positively correlated with plasma low-density lipoprotein (LDL) levels in patients with AMI

DBP and LDL of the participants correlated with plasma exosomal miR-4516, miR-203 and SFRP1 levels (Table VI). There was no correlation between the classical marker, cTnI, and plasma exosomal miR-4516, miR-203 and SFRP1 levels (Fig. 3C). Plasma SFRP1 was positively correlated with serum LDL levels (r2=0.667; P=0.0415); however, there was no correlation between plasma exosomal miR-4516 or miR-203 and serum LDL levels (Fig. 3D). A logistic regression analysis of common risk factors for coronary artery disease was performed.

Table VI. Correlations between serum exosomal miR-4516, miR-203 and SRFP1 levels and traditional risk factors of cardiovascular diseases in AMI patients.

Table VI.

Correlations between serum exosomal miR-4516, miR-203 and SRFP1 levels and traditional risk factors of cardiovascular diseases in AMI patients.

		Exosomal miR-4516		Exosomal miR-203		SRFP1
Characteristics	Value	Control	AMI	Control	AMI	Control	AMI
Age, years	P	0.802	0.918	0.746	0.994	0.691	0.175
	R2	−0.051	−0.015	0.160	0.007	0.573	−0.025
HR, beats/min	P	0.492	0.572	0.374	0.434	0.361	0.775
	R2	−0.189	−0.109	0.5876	−0.103	0.175	0.005
SBP, mmHg	P	0.590	0.222	0.081	0.076	0.003	0.984
	R2	0.206	0.342	−1.567	0.359	0.737	0.001
DBP, mmHg	P	0.420	0.842	0.842	0.014	0.616	0.804
	R2	0.193	0.042	0.115	0.376	−0.843	0.003
Cr, µmol/l	P	0.051	0.172	0.871	0.694	0.213	0.732
	R2	−0.706	0.313	−0.145	0.066	0.321	−0.004
Glu, mmol/l	P	0.223	0.051	0.365	0.128	0.986	0.663
	R2	0.021	−0.032	0.037	0.018	−0.002	0.091
TG, mmol/l	P	0.867	0.085	0.835	0.933	0.374	0.731
	R2	0.002	0.018	0.007	0.001	−0.091	0.118
TC, mmol/l	P	0.378	0.980	0.609	0.940	0.075	0.180
	R2	0.021	0.001	−0.029	0.001	0.296	0.319
LDL, mmol/l	P	0.939	0.954	0.446	0.991	0.934	0.041
	R2	−0.001	0.001	−0.046	0.091	−0.014	0.677
HDL, mmol/l	P	0.847	0.306	0.212	0.590	0.761	0.977
	R2	−0.001	−0.007	−0.028	0.003	−0.02	0.012
Apolipoprotein a	P	0.592	0.223	0.476	0.193	0.554	0.092
	R2	−3.757	0.005	0.119	−0.042	−0.29	0.001
Apolipoprotein B	P	0.817	0.855	0.533	0.902	0.747	0.111
	R2	0.001	−0.004	−0.008	0.002	−0.013	0.028
Apolipoprotein E	P	0.259	0.489	0.828	0.994	0.311	0.952
	R2	0.336	0.17	0.156	0.001	−0.211	0.001

[i] AMI, acute myocardial infarction; miR, microRNA; SFRP1, secretory frizzled-related protein; SBP, Systolic blood pressure; DBP, Diastolic blood pressure; Cr, Creatinine; Glu, Blood glucose; TG, Triglycerides; TC, Total cholesterol; LDL, Low-density lipoprotein; HDL, High-density lipoprotein.

miR-4516 and miR-203 are associated with adhesion

The KEGG enrichment analysis demonstrated that miR-4516 and miR-203 shared 748 enrichment pathways, and that miR-4516 and miR-203 were associated with adhesion function in the enriched pathways (Fig. 4A and B).

Figure 4. Bioinformatic analysis. (A) MiR-4516 and miR-203 shared 748 identical enrichment pathways. (B) Among the first 20 enrichment pathways, there was an association of miR-4516 and miR-203 with adhesion. KEGG, Kyoto Encyclopedia of Genes and Genomes; miR, microRNA.

Discussion

CVD remains one of the leading causes of death worldwide, and AMI is the most aggressive and problematic type of CVD (1). With health education and the continuous innovation of testing equipment, there is an increased awareness of early diagnosis and early treatment of AMI (43). Survival rates for AMI are gradually improving in developed cities, However, the relative lack of diagnostic capacity for AMI in less developed regions and the poor access to treatment increase the mortality rate of patients with AMI. Classical plasma markers of AMI, such as cTnI and CK-MB, are becoming less specific and have certain drawbacks in clinical diagnosis. This is due to multiple co-morbidities, such as myocarditis, heart failure and pulmonary heart disease, which also cause elevations in these markers (44). Exos are the products of cellular secretion and are found in large numbers in various bodily fluids and reflect intracellular status in real time. Previous studies have reported that the miRNAs transported in exos are of significant value in various CVDs. For example, miR-133, miR-146, miR-499 and miR-26a in plasma can be used as potential diagnostic markers for AMI (45).

In the present study, TargetScan predicted that SFRP1 may be a target protein for miR-4516 and miR-203. The results of the present study demonstrated that the expression levels of the three indicators were significantly elevated in the AMI group compared with the control, which prompted the hypothesis of a relationship between the three indicators and AMI. Pearson's correlation analysis further demonstrated that exosomal levels of miR-4516 and miR-203 and plasma SFRP1 levels were significantly correlated with AMI.

Coronary angiography can not only determine whether a coronary artery is blocked and the extent of obstruction, but also support the selection of a protocol for the next step of treatment. SYNTAX scores can be used to quantitatively evaluate the complexity of coronary lesions and provide a preliminary judgment, which can inform the choice of the surgical procedure according to a specific scoring system based on anatomical characteristics, such as coronary lesion location, severity, bifurcation and calcification. In addition, patients with MI often have multiple co-morbidities, such as heart failure, anemia or even uremia at an advanced age. Both ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction have corresponding blocked blood vessels, which can be identified on late coronary angiography. Where this occurs, it would be advantageous if the severity of the lesioned vessel could be determined as early as possible in conjunction with the SYNTAX score. Early assessment of the severity of the blockage in the vessel can predict the severity of damage to the heart muscle. For example, if the blockage is small, thrombolytic therapy may be an immediate option. However, if the blockage is severe and combined with chronic occlusion of multiple vessels, cardiac bypass surgery may be the immediate treatment of choice (46,47). In the present study, the relationship between the expression levels of exosomal miR-4516, miR-203 and SFRP1 in the AMI group with their SYNTAX scores were analyzed. Correlation analysis demonstrated that only miR-4516 was significantly positively correlated with the SYNTAX score of patients with AMI, and that neither miR-203 nor SFRP1 were correlated with SYNTAX score (48,49). These data indicated that exosomal miR-4516 may be used as a non-invasive means of predicting the lesion complexity of coronary vessels in patients with AMI, providing an initial basis for the next treatment step (PCI or coronary artery bypass grafting). The elevation of LDL in plasma is considered to be a major risk factor for atherosclerosis, and the most direct cause of AMI is the rupture of the atherosclerotic plate. Notably, in the present study, Pearson's correlation analysis demonstrated that SFRP1 protein expression levels were significantly correlated with LDL. After statistical analysis, no correlation was demonstrated between SFRP1 mRNA expression levels and SYNTAX scores, but the AMI group had significantly higher SFRP1 protein expression levels compared with the control group. Statistical analysis demonstrated that SFRP1 protein expression levels were significantly correlated with cTNl. As SFRP1 is a key protein in the WNT signaling pathway, it was hypothesized that SFRP1 may be positively correlated with the severity of cardiomyocyte injury and angiogenesis (42). In addition to this, based on the results of the present study, it was hypothesized that SFRP1 may be involved in lipid metabolism. However, for reasons of time, further validation was not performed.

Both miRNAs, miR-4516 and miR-203, were predicted by bioinformatic analysis to be closely associated with adhesion and endocytosis. The present study could not distinguish between STEMI and non-STEMI as the subsequent treatment regimens for these two types of AMI are different. Moreover, the detection of miRNA in plasma exos was quite time-consuming. The time must be decreased to facilitate use in a clinical setting. The main reason for this is that the process of exo extraction takes about one hour. SYNTAX 2 scores combine the clinical variables of the patient, such as sex, age, left ventricular ejection fraction, creatinine clearance and other information, which allow accurate evaluation of the condition of the patient (38). Therefore, combination of the difference in exosomal miRNA and SYNTAX 2 scores facilitates a more personalized treatment plan for patients.

The present study had limitations including that at the time of diagnosis of AMI, all patients had the necessary tests for cTnl and ECG. Obtaining the results of tests such as CK-MB and BNP was slow compared with cTnl, and we did not consider these as a necessary test considering that it would delay the patient's treatment; therefore, the data for these two indicators are relatively incomplete. The plasma samples collected were of human origin and as such the composition of the plasma was complex and, even if some of the more biased samples are excluded, the dispersion of some of the samples was still large, this was also a limitation of the present study. In addition to this, another limitation of the present study was the lack of data and survival information related to the later stages of recovery from myocardial infarction in patients. Therefore, no relevant prognostic and survival analysis were performed for this.

In conclusion, a combination of miR-4516, miR-203 and SFRP1 may help in the diagnosis of AMI and in the evaluation of the degree of coronary stenosis. Furthermore, the present study demonstrated a significant correlation between plasma SFRP1 protein expression levels and LDL levels. These results may provide novel diagnostic markers for AMI, which may contribute to the timely diagnosis and treatment of AMI.

Acknowledgements

The authors would like to thank Dr Yan Zheng (Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China) for their technical assistance.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (grant no. ZR202103050087).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

PL, KL and SW designed the present study, searched databases, extracted and assessed the literature and drafted the manuscript. PL, JT, YY, LW and YM statistically analyzed the data. YY and LW confirm the authenticity of all the raw data. PL, FD and GS conceived and designed the present study, provided general supervision and finalized the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the ethics committee of Jinan Central Hospital (approval no. 2018-039-01).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References