Diagnostic and prognostic value of circulating miRNA‐499 and miRNA‐22 in acute myocardial infarction

Abstract Background Currently, acute myocardial infarction (AMI) represents a serious cardiovascular disease with high morbidity and mortality. Therefore, this study aimed to systematically evaluate the roles of miRNA‐499 and miRNA‐22 as potential biomarkers for AMI. Methods According to the inclusion and exclusion criteria, we measured circulating levels of miRNAs in 50 AMI patients and 50 non‐MI populations. The expression levels of plasma miRNA‐499 and miRNA‐22 were analyzed by real‐time fluorescent quantitative polymerase chain reaction (qRT‐PCR). A statistical analysis of clinical data of AMI patients was conducted by 90‐day follow‐up. Results Real‐time PCR analysis showed that the relative expression level of miRNA‐499 increased gradually among the three groups (P < .05). However, the expression of miRNA‐22 showed a downward trend (P < .05). According to logistic analysis, the relative levels of miRNA‐499 and miRNA‐22 were important predictors of AMI. When the miRNA‐499 and miRNA‐22 levels were 0.377 and 0.946 separately, the diagnostic value of miRNA‐499 and miRNA‐22 for AMI was 86.00% and 86.00% for sensitivity, and 98.00% and 94.00% for specificity, respectively. In addition, compared to the baseline GRACE scoring system, the combination of miRNA‐499, miRNA‐22, and GRACE scores had a stronger discriminating power for MACE occurrence, with a sensitivity of 100.00% and a specificity of 79.40%. Conclusions The results showed that plasma miRNA‐499 and miRNA‐22 were more sensitive and specific for the diagnosis of AMI, suggesting that they can be used as potential biomarkers for clinical diagnosis of AMI.

renal failure, and skeletal muscle tissue damage, also lead to elevated serum levels, which reduce the diagnostic specificity. 5 Consequently, it is especially important to explore and research new biomarkers in the diagnosis of AMI. 6 GRACE risk score is the current risk assessment tool recommended for acute coronary syndromes. It can effectively assess the risk of death or AMI in hospitalization or discharge for 6 months. 7,8 Recently, some studies have shown that combining new biomarkers with GRACE risk scores can improve cardiovascular risk prediction ability compared to biochemical indicators alone. 9 MicroRNAs (miRNAs) are a class of noncoding, small single-stranded RNAs. 10 Mature miRNAs can bind to the 3' untranslated region of the target gene mRNA 11 and ultimately inhibit gene expression by promoting mRNA degradation or inhibiting translation. 12 Many studies have shown that miRNAs are involved in a variety of gene regulation processes such as proliferation, development, differentiation, inflammation, and other physiological processes. 13,14 MiRNAs are considered to be biomarkers of certain diseases such as cardiovascular diseases because they are stable in blood and other body fluids. 15,16 Similarly, Wang et al 17 also believed that serum miRNAs may be promising as novel indicators for the diagnosis of AMI. In addition, many studies have shown that circulating miRNAs play an important role in the poor prognosis of patients with AMI. 18 Recently, miRNA-499 has been shown to have a crucial effect on the differentiation of cardiac stem cells into cardiomyocytes. And the literature indicated that the concentration of miRNA-499 was significantly elevated in patients with myocardial infarction. 19 Furthermore, Huang et al 20 found that miRNA-22 in the heart of high expression was an important regulatory factor of cardiac remodeling, highlighting miRNA-22 as a candidate biomarker for cardiovascular disease. However, other research reports on the diagnostic performance of serum miRNA-499 on AMI are still controversial, and the diagnostic value of miRNA-22 in AMI is rarely reported. Therefore, the main purpose of this study was to explore the diagnostic value of miRNA-499 and miRNA-22 in AMI. Furthermore, given the prognostic significance of GRACE score, we aim to identify the potential predictive value of miRNA-499 and miRNA-22 on the GRACE score to guide risk stratification and clinical treatment.

| Sample collection and storage
The venous blood of the patients after 6 hours of admission was collected using an EDTA-anticoagulative tube of about 5 mL. After centrifugation at 3000 g for 10 minutes, the supernatant was transferred to an RNase/DNase-free tube and stored at −80°C for analysis.

| Total RNA extraction
The total RNA was isolated from plasma using TRIzol LS Reagent

| Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
The reverse transcription reaction was carried out using the Bulge-LoopTM miRNA qRT-PCR primer set (RiboBio Co). Caenorhabditis elegans microRNA (cel-miR-39) was used as the control. The reverse transcription process was carried out at 42°C for 60 minutes and then at 70°C for 10 minutes. At last, the resulting cDNA was stored at −20°C until use.
The PCR was carried out according to the Bulge-LoopTM miRNA qRT-PCR Kit (RiboBio Co). The final reaction volume was 20 μL of reaction and was performed on a Roche Cobas z480 detection system (Roche Molecular Diagnostics). Amplification reaction program: After initial denaturation for 2 minutes at 95°C, and then 40 cycles at 95°C for 15 seconds, 60°C 30 seconds and 95°C 15 seconds. Finally, a melting curve is produced. The data were obtained directly from a real-time fluorescent quantitative PCR instrument using an amplification profile of cel-miR-39 as an internal standard. To calculate the relative expression levels of miRNAs, the 2 −ΔCt (ΔCt = Ct miRNAs −Ct cel-miR-39 ) method was used to assess miRNA expression. 22,23

| Follow-up and study end point
A 90-day follow-up was performed on patients with AMI and UAP by telephone or hospitalization. The study end point was the occurrence of cardiovascular adverse events, including MACE, allcause death, myocardial infarction, cardiogenic shock, and cardiac arrest/ventricular fibrillation.

| Calculation of GRACE risk score
GRACE scores were based on age, heart rate, systolic blood pressure, creatinine, Killip classification, prehospital cardiac arrest, ST-segment depression, and elevated myocardial enzymes in all patients. The scoring criteria were based on the GRACE risk score. 24

| Statistical analyses
Statistical analysis was performed using SPSS 25.0 software (SPSS).
All data are expressed as mean ± standard error (SE). All continuous variables were checked using the Kolmogorov-Smirnov normality test to show their distributions. One-way ANOVA was used to test for differences among the three groups, and independent-samples t test was used between the two groups. Binary logistic regression analysis was performed to show the associated variables in the data analysis to determine the independent predictors of AMI. Receiver operating characteristic curves further analyzed the diagnostic efficacy of the two indicators for AMI. Correlation between variables in AMI patients was analyzed by Spearman rank correlation. All statistical tests were two-tailed, and P < .05 was considered statistically significant.

| Clinical characteristics of the study population
In this study, 50 patients with AMI served as the experimental group (AMI). And 25 UAP patients (UAP) and 25 healthy people (HC) served as the control. As shown in Table 1, there were significant differences in white blood cell counts, red blood cell counts, and total cholesterol levels and triglycerides among the groups (P < .05). Table 2, 50 patients with myocardial infarction (AMI) and 50 patients with non-myocardial infarction (non-MI) (UAP + HC) were compared. White blood cell count was significantly higher in the AMI group compared with the non-MI group (P < .05).
As shown in Figure 1A, the serum miRNA-499 levels were significantly up-regulated in AMI patients compared to subjects in other groups including healthy controls and UAP patients. As shown in Among these patients, it was observed that the relative expression levels of miRNA-499 were significantly higher in the AMI group than in the non-MI group (P < .05) (Figure 2A). Serum miRNA-22 levels were reduced significantly in the AMI group compared to the non-MI group (P < .05) ( Figure 2B). Moreover, there was a statistically significant difference in miRNA-499 between non-MI and AMI (P < .05).

| Analysis of the sensitivity and specificity of miRNA-499 and miRNA-22
The receiver operating characteristic (ROC) curves were plotted with (1-specificity) as the abscissa and sensitivity as the ordinate. Furthermore, whether the miRNAs can be used as

| Logistic regression analysis
Logistic regression analysis showed that miRNA-499 and miRNA-22 were independent predictors of AMI (miRNA-499: P = .020; miRNA-22: P = .011) ( Table 3). Through the logical regression analysis, a regression model was constructed to diagnose AMI from non-MI population. The logistic regression model was as follows: Results showed that the cutoff value was 0.1511 with maximizing Youden index. According to the regression model, the sensitivity and specificity for the diagnosis of AMI were 98.00% and 96.00%, respectively ( Figure 4).
In order to further verify the ability of model testing AMI, 40 samples (20 AMI, 10 UAP + 10 HC) were tested by a blind method.
Finally, the true-positive rate was 90% and the true-negative rate was 100%.

| Correlation analysis
Spearman rank correlation analysis was used to analyze the relationship between miRNAs and various indicators in AMI and UAP patients. The results are shown in Table 4

| The predictive value of miRNAs, GRACE score, and combined indicator of miRNAs and GRACE score
Data analysis was performed between the MACE group and the non-MACE group. Compared with the non-MACE group, the MACE group had higher levels of serum miRNA-499 and GRACE score (P < .05) ( Figure 5A). Conversely, miRNA-22 levels in the MACE group were significantly lower than in the non-MACE group (P < .05) ( Figure 5B). As shown in Table 5 The ROC analysis results are shown in Table 5, and the AUC areas of miRNA-499, miRNA-22, and GRACE score were 0.822, 0.700, and  ( Figure 6).

Acute myocardial infarction (AMI) caused by myocardial ischemia
is the leading cause of morbidity and mortality in the world. 19 For patients with AMI, accurate diagnosis and timely treatment are of paramount importance. 25 At present, some common indicators, such as creatine kinase-MB (CK-MB) and highly sensitive cardiac troponin (hs-cnTnT), have become biomarkers for the diagnosis of AMI, 26 but their sensitivity and specificity are still not satisfactory. Some studies have shown that circulating miRNAs are specifically expressed in tissues and participate in the pathological process of myocardial infarction. 27,28 In addition, based on the remarkable stability of miR-NAs in plasma, the potential of miRNAs as markers of myocardial infarction is further illustrated. 29,30 Recently, it has been found that miRNA-499 is one of the miRNAs encoding myosin. 31 MiRNA-499 is located in the intron of b-myosin heavy chain 7B (Myh7b) gene in the human heart. 32 Moreover, miR-NA-499a is highly expressed in the heart. 33 Similarly, Wang et al 34 have shown that miRNA-499 is produced almost exclusively in the heart. Jia et al 35 found that miRNA-499 may be involved in myocardial injury and remodeling. In addition, miRNA-499 was shown to be involved in cardiomyocyte differentiation. 36

| CON CLUS IONS
In conclusion, miRNA-499 and miRNA-22 may be potential biomarkers for the diagnosis of AMI, and miRNA-22 may be a potential therapeutic agent for coronary atherosclerosis. And with the improvement of miRNAs detection technology, more accurate, fast, and inexpensive detection methods may appear. At the same time, taking into account the limitations of the detection time limit and the number of samples, further research is needed to confirm the results.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T S
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.