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Serum miRNA-499 and miRNA-210: A potential role in early diagnosis of acute coronary syndrome

Corresponding Author

Sally M. Shalaby

Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Address correspondence to: Sally Shalaby, Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. Tel.: 00-201-0708-3153.E-mail: [email protected]Search for more papers by this author

Amal S. El-Shal,

Amal S. El-Shal

Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Amira Shoukry,

Amira Shoukry

Internal Medicine Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Mohamad H. Khedr,

Mohamad H. Khedr

Cardiology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Nader Abdelraheim,

Nader Abdelraheim

Cardiothoracic Surgery Department, Faculty of Medicine, Zagazig University Zagazig, Egypt

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Sally M. Shalaby,

Corresponding Author

Sally M. Shalaby

Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

Amal S. El-Shal,

Amal S. El-Shal

Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Amira Shoukry,

Amira Shoukry

Internal Medicine Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Mohamad H. Khedr,

Mohamad H. Khedr

Cardiology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Nader Abdelraheim,

Nader Abdelraheim

Cardiothoracic Surgery Department, Faculty of Medicine, Zagazig University Zagazig, Egypt

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First published: 27 June 2016

https://doi.org/10.1002/iub.1529

Citations: 39

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Abstract

In clinical practice, there is still a need for novel biomarkers, which can reliably rule in or rule out acute coronary syndrome (ACS) immediately on admission. This is of particular interest in patients with unstable angina (UA) and non-ST-segment elevation myocardial infarction (NSTEMI) in whom diagnostic uncertainty is high. The aim of the present study is to evaluate the potential role of miRNA-499 and miRNA-210 as novel molecular biomarkers for early diagnosis of UA and NSTEMI suspected patients presented at the emergency unit. A total of 110 patients presenting to the intensive care unit (ICU) within 24 h of onset of chest pain suggestive of ACS were enrolled in the study. They included 37 UA, 48 NSTEMI and 25 noncardiac chest pain (NCCP) patients. Immediately at enrollment, blood samples were taken for estimation of serum miRNA-499 and miRNA-210 expression levels by real time PCR. miRNA-499 and miRNA-210 expression levels were significantly increased in UA and NSTEMI patients compared with NCCP patients (P < 0.001). Receiver operating characteristic (ROC) curve analysis revealed that the area under curve (AUC) of miR-499 for the diagnosis of UA and NSTEMI was 0.98 and 0.97, respectively; while the AUC of miRNA-210 was 0.84 and 0.90, respectively. The important finding of our study was that the AUC of miRNA-499 for the diagnosis of ACS patients with symptoms onset <3 h was 0.89, while the AUC of miRNA-210 was 0.86. Interestingly, combining miRNA-499 and miRNA-210 significantly improved the diagnostic value by increasing the AUC to 0.96, P < 0.001. In conclusion, serum miRNA-499 and miRNA-210 are associated with UA and NSTEMI and with those presenting within 3 h of symptom onset. Both miRNAs might be potentially novel biomarkers for accelerating the diagnosis of ACS patients in emergency unit. © 2016 IUBMB Life, 68(8):673–682, 2016

Introduction

Acute coronary syndrome (ACS) is a term applied to any group of clinical symptoms compatible with acute myocardial ischemia and includes unstable angina (UA), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI) 1. Acute coronary syndrome is one of the leading causes for morbidity and mortality worldwide; therefore, an accurate and early diagnosis of ACS can definitely help decrease the mortality rate 2. In the setting of typical symptoms and ST segment elevation on the electrocardiogram (ECG), the diagnosis and subsequent management is straightforward as in case of STEMI 1. In patient with NSTEMI or UA, the diagnosis cannot be based on clinical symptoms and ECG only 1. A diagnosis of NSTEMI can be made when the ischemia is sufficiently severe to cause myocardial damage results in the release of biomarkers of myocardial necrosis into the circulation (as cardiac-specific troponins T and I). In contrast, the patient is considered to have UA if no such biomarkers can be detected in the bloodstream hours after the initial onset of ischemic chest pain 3.

Up to date, the most commonly used biomarkers for myocardial infarction (MI) are cardiac troponins (cTns), cardiac troponin I and T (cTnI and cTnT). Unfortunately, these biomarkers are not consistently elevated within the first hours after onset of symptoms, demanding subsequent measurements and delaying early diagnosis 4. Rapid and correct diagnosis of ACS plays a crucial role in saving patients' life. So there is still a clinical need for novel biomarkers, which can reliably rule in or rule out ACS immediately on admission. This is of particular interest in patients with UA and NSTEMI in whom diagnostic uncertainty is high.

MicroRNAs (miRNAs) are endogenous, 19 to 25 nucleotide long, short noncoding RNAs that regulate genes post-transcriptionally 5. MiRNAs regulate target genes by repressing their translation or inducing their degradation 6. They exist in the serum and plasma in a consistent, reproducible and stable manner, opening the possibility of using them as diagnostic markers for various diseases including cardiovascular disorders 7, 8. In the cardiovascular system, miRNAs have been shown to play crucial roles in pathophysiological conditions, such as endothelial dysfunction, inflammation, apoptosis, and angiogenesis 9. Several studies highlighted the potential of circulating miRNAs as diagnostic and prognostic biomarkers in ACS 10-13. Yet, most of these studies used samples taken at the time of reperfusion to determine miRNAs, rather than samples taken upon initial admission to the Emergency Unit, when diagnostic uncertainty is most evident. Also, most of them studied the acute myocardial infarction (AMI); the straightforward cases 14-16.

The aim of the present study was to evaluate the potential role of miRNA-499; the cardiac enriched miRNA, and miRNA-210; the stress related miRNA as novel molecular biomarkers for early diagnosis of UA and NSTEMI suspected patients presented at the Emergency Unit.

Patients and Methods

This study was carried out at Intensive Care Units (ICU) of Internal Medicine and Cardiology Departments of Zagazig University Hospitals and Medical Biochemistry Department. A total of 110 patients presenting to the ICU within 24 h of onset of chest pain suggestive of ACS were enrolled in this cross-sectional study between May 2015 and March 2016. The protocol was approved by the ethics committee of Zagazig University and informed consent was obtained from all the patients included in the study. Patients were presented by chest pain of the following criteria: chest discomfort, pressure, tightness, or heaviness; pain that radiated to the neck, jaw, shoulders, back, or one or both arms; and persistent dyspnea. All patients underwent full history taking, clinical examination, complete 12-lead electrocardiography (ECG), chest radiology, and cardiac angiography. Immediately at enrollment, blood samples were taken for estimation of high-sensitive troponin T (Hs-cTnT) and other routine laboratory parameters. Part of serum was stored for analysis of serum miRNAs. The study included 37 UA, 48 NSTEMI, and 25 noncardiac chest pain (NCCP) patients (as proved later by investigations including cardiac angiography). Patients were diagnosed as typical UA if they presented with clinical symptoms of chest pain, the ECG did not show ST-segment elevations or new left bundle branch block (ST depression, transient ST-elevation, or new T-wave inversion was present in some patients), cardiac troponin negative and the coronary angiography done later documented coronary artery disease (CAD). Patients were diagnosed as NSTEMI if they presented with symptoms of chest pain, no ST segment elevation, elevated cardiac troponin with a clear rising or falling pattern and the coronary angiography done after that according to the guidelines revealed CAD. Criteria for NCCP individuals had been presented with chest pain, troponin assays were negative, no alternative cardiac diagnosis explaining the symptoms and we chose those who underwent coronary angiography that was done for them by any soft indications proven exclusion of any coronary stenosis 17. STEMI patients and patients with chronic kidney disease (CKD) were excluded from the study. In CKD, the elevated levels of cTnT due to impaired clearance may result in false positive Hs-cTnT assays 18.

Measurements of Routine Clinical Biomarkers

High-sensitive cardiac Troponin T (Hs-cTnT) was done on admission and 6 to 8 h later if the first set was negative. hs-cTnT was measured by the Elecsys troponin T high-sensitive assay fourth generation (Roche Diagnostics) with the 99th percentage cut-off point of 14 pg/mL. CK-MB was measured by the Elecsys CK-MB (Roche Diagnostics). Lipid profile (total cholesterol, HDL-cholesterol, and triglyceride) were measured by routine enzymatic methods (Spinreact, Girona, Spain), the LDL cholesterol level was calculated using the Friedewald formula, and blood glucose levels were measured using the glucose oxidase method (Spinreact, Girona, Spain); all were measured within 24 h from admission to assess patients' risk factors of ischemic heart disease.

MiRNA Extraction

Total RNA including miRNAs from serum was extracted using RNA extraction kit (Qiagen Inc., Valencia, CA) following the manufacturer's instructions and quantified spectrophotometrically at 260 nm.

cDNA Synthesis and Quantitative Real-Time PCR

100 ng of template RNA was reverse transcribed in 20 µL using the miScript II reverse transcription kit (Qiagen, Valencia, CA). For the PCR reaction, 20 ng of RT-product was used. The amplification was carried out using Real time qPCR StepOnePlus™ System (Applied Biosystems Inc., Foster, CA). SYBR Green Master Mix (Qiagen/SABiosciences Corporation, Frederick, MD) was used in the real-time PCR reaction according to the manufacturer's suggested protocol. Each reaction was performed in a final volume of 20 µL containing 5 µL of the cDNA, 100 pmol/µL of each primer (0.5 µL each) miRNA-499(Cat. no. MS00004375) and miRNA-210 (Cat. no. MS00003801), 10 µL 1× QuantiTect SYBR Green PCR Master mix (Qiagen) and 4 µL DdH2O. The amplification protocol was initial denaturation and polymerase activation at 95 °C for 15 min, then 40 cycles of denaturation 94 °C for 15 s; annealing at 55 °C for 30 s; extension at 70 °C for 30 s. The expression levels of miRNAs were normalized to RNU6 as U6 levels did not differ between ACS and non-ACS patients 19, 20. The expression level of miRNA was normalized by calculating the ΔCt value based on subtracting its Ct value from that of the internal control RNU6. The relative gene expression was calculated as ΔΔCt. The amplitude of change of the expression miRNA observed in patients in relation to control group was analyzed by the 2^−ΔΔCt method 21.

Statistical Analysis

All data were collected, tabulated and statistically analyzed using SPSS 20.0 for windows (SPSS Inc., Chicago, IL) and MedCalc 13 for windows (MedCalc Software bvba, Ostend, Belgium). Quantitative data were expressed as the mean ± SD & median (range). Continuous data were checked for normality by using Shapiro-Walk test. Independent Student's t-test was used to compare two groups of normally distributed data. Mann-Whitney U test was used to compare two groups of non-normally distributed data. Receiver operating characteristic (ROC) curve analysis was used to identify optimal cut-off values of miRNA-499, miRNA-210, and their combinations with maximum sensitivity and specificity for diagnosis of unstable angina, non STEMI and ACS patients with symptoms <3 h. Area under curve (AUC) was also calculated, criteria to qualify for AUC were as follows: 0.90–1 = excellent, 0.80–0.90 = good, 0.70–0.80 = fair; 0.60–0.70 = poor; and 0.50–0.6 = fail. The optimal cutoff point was established at point of maximum accuracy. Multivariate logistic regression models were built to study add value of miRNA to clinical model and cardiac hs-treponin as independent predictors for ACS, adjust was done for clinical co-variates and cardiac hs-treponin, we use predicted probability of different models to find AUC then compare between miRNAs and cardiac hs-treponin diagnostic performance. All tests were two sided. P < 0.05 was considered statistically significant.

Results

Patient Characteristics

In 110 suspected ACS patients at enrollment, 85 were subsequently classified as having UA (37 patients) and NSTEMI (48 patients). The others represented as NCCP (25 patients). Our results showed that the mean values of age, male/female ratio, BMI, serum creatinine, and onset of chest pain before admission in the suspected ACS patients were nonsignificantly different between the studied groups. Moreover, there was no significant difference in the percentage of smokers or diabetic patients among the studied groups. However, the frequency of hypertensive and hyperlipidemic patients were significantly higher in both NSTEMI and UA groups when compared with NCCP patients. There was significant higher increase of CK-MB values in NSTEMI patients than in UA and NCCP (P < 0.001).

The mean value of Hs-cTnT was 11.5 ± 3.2 pg/mL (range: 8.5–13.2) in UA patients, 83.6 ± 43.2 pg/mL (range: 40–211.5) in NSTEMI patients and 6.1 ± 2.5 pg/mL (range: 4.5–9.2) in controls. There was significant elevation of Hs-cTnT in NSTEMI group when compared either with UA group (P < 0.001) or NCCP patients (P < 0.001) (Table 1).

Table 1. Characteristics of the studied groups

	Noncardiac chest pain (NCCP) (N = 25)	Unstable angina (UA) (N = 37)	NSTEMI (N = 48)	P
Age (yr)	49.2 ± 10.2	53.5 ± 9.8	54.3 ± 8.3	0.09
Male/female	17/8	26/11	32/16	0.94
BMI (kg/m²)	26.7 ± 6.1	28.2 ± 5.7	27.8 ± 5.3	0.58
Onset of chest pain (h)	6.35 ± 4.2	6.24 ± 4.3	6.13 ± 3.9	0.97
Hypertension (N, %)	5 (20%)	18 (48.6%)	22 (45.8%)	0.04
Diabetes mellitus (N, %)	7 (28%)	19 (51.3%)	19 (39.6%)	0.18
Smokers (current or ex-smokers)	8 (32%)	12 (32.4%)	18 (37.5%)	0.88
Hyperlipidemia (N, %)	3 (12%)	14 (37.8%)	20 (41.7%)	0.03
Serum creatinine (mg/dL)	0.87 ± 0.5	0.92 ± 0.4	0.8 ± 0.3	0.42
CK-MB (ng/mL)	2.16 ± 0.6	3.92 ± 2.7	19.53 ± 7.5	<0.001
Hs-cTnT (pg/mL)	6.1 ± 2.5	11.5 ± 3.2	83.6 ± 43.2	<0.001

NSTEMI, non-ST-segment elevation myocardial infarction; BMI: body mass index; CK-MB: creatine kinase MB; Hs-cTnT: high-sensitive cardiac troponin T.

Circulating Levels of miRNA-499 and miRNA-210 in Studied Groups

The quantitative expression of miRNA-499 and miRNA-210 in serum were significantly higher in UA and NSTEMI patients compared with NCCP controls (each, P < 0.001). In UA, serum expression levels of miRNA-210 were significantly higher than in NSTEMI (P = 0.002) while there was no significant changes between both groups concerning miRNA-499 (Table 2).

Table 2. Expression levels of circulating miRNA-499 and miRNA-210 in serum of studied patients

	Noncardiac chest pain (NCCP) (N = 25)	Unstable angina (UA) (N = 37)	NSTEMI (N = 48)	ACS patients symptoms <3 h (N = 27)
miRNA-499
Mean ± SD	1.33 ± 0.30	18.87 ± 7.798*	20.77 ± 8.51*	15.12 ± 11.14*
Median (range)	1.27 (1–2.23)	21.70 (1.50–28.90)	23.25 (1.10–29.90)	19.80 (1–28.90)
miRNA-210
Mean ± SD	1.12 ± 0.21	3.91 ± 2.50*	2.57 ± 1.08* **	2.39 ± 1.25*
Median (range)	1.20 (0.40–1.35)	4.90 (0.90–8.40)	2.45 (0.80 −5.20)	2.30 (1–5.50)

*P < 0.001 when compared UA or NSTEMI with NCCP.
**P < 0.05 when compared UA with NSTEMI.
NSTEMI, non-ST-segment elevation myocardial infarction; ACS: acute coronary syndrome.

Expression Levels of miRNAs in ACS Suspected Patients Presenting within 3 h of Symptoms Onset

From 85 suspected ACS patients, 27 patients (16 men and 11 women, mean age 53.7 ± 6.8 years), the onset of chest pain was <3 h before the enrollment. In this subgroup, circulating expression levels of miRNA-499 and miRNA-210 were also higher than NCCP group (each; P < 0.001) (Table 2).

Receiver Operating Characteristic (ROC) Curve Analysis

At enrollment, ROC curve analysis of Hs-cTnT revealed that the area under curve (AUC) was low in UA (0.54, 95% CI: 0.44–0.62) as all of UA patients were troponin negative and also was poor in NSTEMI group (0.67, 95% CI: 0.58–0.77) as some of NSTEMI showed initially negative troponin.

ROC curve analysis was used to identify optimal cut-off values of miRNA-499, miRNA-210, and their combinations with maximum sensitivity and specificity for diagnosis of unstable angina, non-STEMI, and ACS patients with symptoms <3 h. ROC curve analysis revealed that miRNA-499 and miRNA-210 could differentiate either UA or NSTEMI patients from NCCP controls.

MiRNA-499 ROC analysis showed an AUC of 0.98 (95% CI: 0.92–1, P <0.001) in UA (Fig. 1A) and 0.97 (95% CI: 0.91–0.99, P < 0.001) in NSTEMI (Fig. 2A). Also, miRNA-210 ROC analysis showed an AUC of 0.84 (95% CI: 0.72–0.92, P < 0.001) in UA (Fig. 1B) and 0.90 (95% CI: 0.81–0.96, P < 0.001) in NSTEMI (Fig. 2B). These findings suggested that miRNA-210 or miRNA-499 could be predictor diagnostic biomarkers for ACS.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Receiver operating characteristic (ROC) curve of miRNA-499, miRNA-210, and their combinations as a diagnostic marker for unstable angina (UA): (A) miRNA-499; (B) miRNA-210; (C) miRNA-499 + miRNA-210.

Combining miRNA-499 and miRNA-210 significantly improved the diagnostic value by increasing the AUC to 0.99 (95% CI: 0.93–1, P < 0.001) in UA patients (Fig. 1C) and to 0.98 (95% CI: 0.92–1, P < 0.001) in NSTEMI patients (Fig. 2C) (Tables 3 and 4).

Table 3. miRNA-499, miRNA-210, and their combinations as diagnostic biomarkers for unstable angina (UA); ROC curve analysis

Cut-off values

SN % (95% CI)

SP % (95% CI)

PPV % (95% CI)

NPV % (95% CI)

Accuracy (95% CI)

AUC (95% CI)

miRNA-499

>2.23

91.90%

(78.1–98.3)

100%

(86.3–100)

100%

(89.7–100)

100%

(71.8–97.7)

95.20%

(81.4–99)

0.98

(0.92–1.00)

<0.001

miRNA-210

>1.35

70.30%

(53–84.1)

100%

(86.3–100)

100%

(86.3–100)

69.40%

(51.9–83.7)

82.30%

(66.4–90.5)

0. 84

(0.72–0.92)

<0.001

miRNA-499 + miRNA-210

>3.26

94.60%

(81.8–99.3)

100%

(86.3–100)

100%

(90–100)

92.60%

(75.7–99.1)

96.8

(83.6–99.6)

0.99

(0.93–1.00)

<0.001

ROC curve: receiver operating characteristic curve; SN: sensitivity, SP: specificity, PPV: positive predictive value, NPV: negative predictive value, AUC: area under curve, 95% CI: 95% confidence interval.

Table 4. miRNA-499, miRNA-210, and their combinations as diagnostic biomarkers for non-ST-segment elevation myocardial infarction (NSTEMI); ROC curve analysis

Cut-off values	SN % (95% CI)	SP % (95% CI)	PPV % (95% CI)	NPV % (95% CI)	Accuracy (95% CI)	AUC (95% CI)	P
miRNA-499 >2.23	93.4% (82.8–98.7)	100% (86.3–100)	100% (92.1–100)	89.3% (71.8–97.7)	95.7% (83.7–99.1)	0.97 (0.91–0.99)	<0.001
miRNA-210 >1.35	83.3% (69.8–92.5)	100% (86.3–100)	100% (92.1–100)	75.8% (57.7–88.9)	89% (75.4–95.1)	0.90 (0.81–0.96)	<0.001
miRNA-499 + miRNA-210 >3.26	97.9% (88.9–99.9)	100% (86.3–100)	100% (92.1–100)	96.2% (80.4–99.9)	98.6% (88–99.9)	0.98 (0.92–1.00)	<0.001

ROC curve: receiver operating characteristic curve; SN: sensitivity, SP: specificity, PPV: positive predictive value, NPV: negative predictive value, AUC: area under curve, 95% CI: 95% confidence interval.

The optimal cut-off value of miRNA-499 for diagnosis of UA and NSTEMI was >2.23 with a sensitivity of 91.9% and 93.4% for UA and NSTEMI, respectively, and a specificity of 100% for each. Notably; the optimal cut-off value of miRNA-210 for diagnosis of UA and NSTEMI was >1.35 with a sensitivity of 70.3% and 83.3% for UA and NSTEMI, respectively, and a specificity of 100% for each (Tables 3 and 4).

In ACS patients with symptoms <3 h, the AUC for miRNA-499 was 0.89 (95% CI: 0.77–0.96) (Fig. 3A) and for miRNA-210 was 0.86 (CI: 0.74–0.94) (Fig. 3B). Interestingly, the combined expression of the two miRNAs showed an increase of AUC to 0.96 (95% CI: 0.86–0.99) (Fig. 3C) (Table 5). In ACS patients with symptoms <3 h, the AUC for Hs-cTnT was 0.59 (95% CI: 0.55–0.61), which is low compared with miRNAs.

Table 5. miRNA-499, miRNA-210, and their combinations as diagnostic biomarkers for acute coronary syndrome patients with symptoms <3 h; ROC curve analysis

Cut-off values

SN % (95% CI)

SP % (95% CI)

PPV % (95% CI)

NPV % (95% CI)

Accuracy (95% CI)

AUC (95% CI)

miRNA-499

>2.23

74.10%

(53.7–88.9)

100%

(86.3–100%)

100%

(83.2–100)

78.10%

(59.7–90.9)

86.60%

(69.4–94.2)

0.89

(0.77–0.96)

<0.001

miRNA-210

>1.35

74.10%

(53.7–88.9)

100%

(86.3–100%)

100%

(83.2–100)

78.10%

(59.7–90.9)

86.60%

(69.4–94.2)

0.86

(0.74–0.94)

<0.001

miRNA-499 + miRNA-210

>3.26

85.20%

(66.3–95.8)

100%

(86.3–100)

100%

(85.2–100)

86.20%

(67.9–96.2)

92.30%

(75.9–97.8)

0.96

(0.86–0.99)

<0.001

ROC curve: receiver operating characteristic curve; SN: sensitivity, SP: specificity, PPV: positive predictive value, NPV: negative predictive value, AUROC: area under curve, 95% CI: 95% confidence interval.

Circulating miRNA-499 and miRNA-210 are Independent Predictors for ACS

The clinical model with age, sex, and cardiovascular risk factors resulted in an AUC of 0.83 (95% CI 0.74–0.89) for ACS patients which increased to 0.91 after addition Hs-cTnT. Addition of miR-499 or miR-210 to the clinical model with Hs-cTnT significantly increased the AUC to 0.97 and 0.96 respectively (each; P < 0.001). Importantly, the combination of the two miRNAs resulted in an even higher AUC of 0.99 (95% CI 0.94–0.99) (Table 6). Interestingly, after correction for the clinical model and Hs-cTnT, miRNA-499, and miRNA-210 were still strong predictors for ACS (OR: 6.16, 95% CI: 1.33–8.56 for combined miRNA-499 and miRNA-210) (Table 6).

Table 6. AUCs and odds ratios of miRNAs in suspected ACS patients in a clinical model (N = 110)

Marker	AUC	95% CI	ORa	95% CI
Clinical model (CM)	0.83	0.74–0.89	NA	NA
CM + Hs-cTnT	0.91	0.85–0.96	NA	NA
CM + Hs-cTnT with
miRNA-499	0.97*	0.92–0.99	3.19	1.33–7.67
miRNA-210	0.96*	0.91–0.99	3.17	0.31–26.49
miRNA-499+ miRNA-210	0.99*	0.94–0.99	6.16	1.33–8.56

CM: clinical model (age, sex, hypertension, hyperlipidemia, smoking and diabetes mellitus); AUC: area under curve; Hs-cTnT: high-sensitive cardiac troponin T; ACS: acute coronary syndrome 95% CI: 95 confidence interval; OR: odds ratio; NA: not applicable.
^a Adjusted for clinical model and Hs-cTnT.
*P < 0.001 versus Hs-cTnT.

Discussion

Circulating miRNAs are short noncoding RNAs resistant to endogenous ribonuclease activity and can be present in human serum in a stable form 22. Several studies indicated that circulating miRNAs provide novel and promising biomarkers for ACS diagnosis 10-13. Even though, most of these studies used healthy controls as references and displayed a large variation in the time of blood sampling after the chest pain. In the present study, we analyzed two miRNAs, miRNA-499 and miRNA-210 in suspected ACS patients at the time of their enrollment at the emergency department.

We selected miRNA-499 because of its known high expression in cardiac muscle 23, while miRNA-210 is implicated in endothelial cell response to hypoxia 24. A deliberately chosen selection of miRNAs, e.g., cardiac muscle and vascular wall may increase the diagnostic power.

In the present study we found that the levels of miRNA-499 and miRNA-210 were markedly elevated in UA and NSTEMI patients than NCCP patients. There were no significant differences between UA and NSTEMI patients for miRNA-499 expression values. Yet, miRNA-210 expression levels could be discriminate between UA and NSTEMI patients as there was an increase of miRNA-210 levels in UA than NSTEMI patients.

MiRNA-499 is one of cardiac expressed miRNAs 23. MiRNA-499 is located in an intron of the Myh7b gene and almost exclusively expressed in the heart 25, although may be limited in skeletal muscles 25. MiRNA-499 regulates the myosin gene and mitochondrial dynamics 26, 27. Also, miRNA-499 was demonstrated to be participated in cardiomyocyte differentiation 26. These data could explain the elevation of miRNA-499 expression levels in NSTEMI patients as it might be released from damaged cardiac cells to the circulation. The release mechanism of miRNAs is unclear, but they may be freed to the bloodstream as a consequence of passive release of the cell contents as apoptotic bodies, exosomes and microparticles 28. Microparticles containing miRNAs may be formed during myocardial ischemia as well as during necrosis 28. Therefore, we hypothesized that the elevated levels of miRNA-499 in the state of UA could result from its passive release from ischemic myocardial tissue.

Recent studies 29, 30 have shown that miRNA-499 may be a clinically practicable biomarker for AMI but it was below the limit of detection for ACS 23. However, many studies were in consistent with ours. Recently, Chen et al. found that plasma miRNA-499 expression levels in UA patients were higher than those in the control group 31. Circulating miRNA-499-5p, a family member of miR-499, was increased nearly 80-fold in geriatric patients with acute NSTEMI compared with healthy controls 32. The ROC curve analysis showed that diagnostic accuracy of miRNA-499-5p was higher than that of troponin T (AUC of miRNA-499-5p vs. cTnT vs. Hs-cTnT: 0.86 vs. 0.68 vs. 0.70) 32.

In the present study, we found that NSTEMI patients had higher miRNA-499 expression levels than UA patients, although there was no statistically difference between them. Widera et al. 33 proposed that there was no difference in the levels of miRNA-499 between UA and AMI patients. In the contrary, Oerlemans et al. found relatively higher levels in UA patients than NSTEMI 20. Larger diagnostic studies will be indispensable to explain these controversies.

MiRNA-210 is the only miRNA consistently upregulated in all published studies, in both normal and transformed hypoxic cells 24 Camps et al. identified only three miRNAs (miRNA-210, ambi-miRNA-7105, and mmu-miRNA-322–3p), which showed at least twofold induction in response to hypoxia 34. An upregulation of miRNA-210 in myocardial infarction had been reported 35. Likewise, a crucial cardiac role has also been implicated for miRNA-210 in the endothelial cell response to hypoxia 36. These data could explain the elevation of miRNA-210 levels in UA and NSTEMI patients in our study. Surprisingly, there was an increase of expression levels in UA patient than those of NSTEMI suggesting that miRNA-210 could be contributory in discriminating UA from NSTEMI patients. Eight miRNAs (miRNA-210 was one of them) showed significantly differential levels in UA patients compared with noncoronary chest pain or healthy controls in the profiling and replication phases and were selected for the validation phase in three-phase approach study 37. In the validation phase, Zeller et al. found the AUC for miRNA-210 was 0.43in UA patients at the time of admission 37.

When we analyzed miRNAs levels in ACS patients with the onset of chest pain less than 3 h before presentation, we found also an elevation of both miRNA-499 and miRNA-210 expression levels. We suggested that miRNA-499 and miRNA-210 might be helpful for accelerating the diagnosis of ACS patients in Emergency Department, so the better management could be allowed. Muscle-derived miRNAs were shown to be elevated as early as 1 h after induction of coronary artery ligation in rats 38 and miRNA-499-5p was already significantly 1.7-fold increased after 15 min of permanent coronary artery ligation in mice 39. In humans, miRNA-1 and miRNA-133a/b were elevated as early as 156 min after onset of symptoms and declined thereafter, whereas miRNA-499 further increased achieving maximal levels approximately 9 h after symptom onset 39. Moreover, Oerlemans et al. 20 showed that in patients suspected of ACS presenting to the ED, circulating miRNAs levels (miR-1, miR-208a, miR-499, miR-21, and miR-146a) are higher in suspected ACS patients with initially negative troponin levels and in those presenting within 3 h of symptom onset.

In our study, the individual ROC analysis revealed that miRNA-499 had an AUC of 0.98 and miRNA-210 had an AUC of 0.84, both were higher than hs-cTnT which had an AUC of 0.54 for UA patients. Moreover, miRNA-499 had an AUC of 0.97 and miRNA-210 had an AUC of 0.90, while hs-cTnT had an AUC of 0.67 for NSTEMI patients. For early diagnosis of ACS, a single miRNA as a biomarker might not be as powerful as the combination of several miRNAs in a biomarker panel. The combined detection of miRNA-499 and miRNA-210 can predict the ACS diagnosis (AUC of 0.99 for UA and of 0.98 for NSTEMI patients) more accurately than either marker alone. The important finding in our study was that the AUC of miRNA-499 for the diagnosis of ACS patients with symptoms onset <3 h was 0.89 while the AUC of miRNA-210 was 0.86. Interestingly, combining miRNA-499 and miRNA-210 significantly improved the diagnostic value by increasing the AUC to 0.96, P < 0.001.

Based on ROC curves, we screened the cut-off values to define one that could best discriminate ACS patients from non-ACS patients, and we found that miRNA-499 >2.23 and miRNA-210 >1.35 may serve as a determinant for the diagnosis of UA and NSTEMI patients, these important cut-off values may help provide evidence for future research in this area.

We confirmed that miRNAs are detectable in the circulation at an earlier time point than Hs-cTnT in ACS. cTn is part of the myofibril apparatus, being large protein complexes, whereas the majority of miRNAs are also protein bound, but relatively smaller and are released in a controlled way, which can explain the time difference in release in ACS 40. Compared with cTnT and other conventional markers, miRNAs have specific advantages, for example, it is not affected by renal function 41 and has high stability in vitro 42. In addition, cTnT is currently detected by immunological methods and susceptible to interference of some specimen factors such as giant troponin 43 and troponin antibodies 44. Thus, miRNAs are still an expected marker of diagnosis of ACS.

There are some limitations of this study. First, the sample size of current study is relatively small and larger trials to retest the efficacy of these biomarkers should be done. Second, the generalizability of our findings is limited by the demographic differences and treatment characteristics of ACS patients.

In conclusion, serum miRNA-499 and miRNA-210 are upregulated in UA and NSTEMI and might be potentially promising biomarkers for accelerating the diagnosis of ACS patients in emergency department. The combined detection of miRNA-499 and miRNA-210 can predict the ACS diagnosis more accurately than either marker alone.

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Citing Literature

Volume68, Issue8

August 2016

Pages 673-682

Serum miRNA-499 and miRNA-210: A potential role in early diagnosis of acute coronary syndrome

Abstract

Introduction

Patients and Methods