Human Circulating MicroRNA-1 and MicroRNA-126 as Potential Novel Indicators for Acute Myocardial Infarction

Circulating miRNAs have been shown as promising biomarkers for various pathologic conditions. The aim of this study was to clarify that circulating miR-1 and miR-126 in human plasma might be useful as biomarkers in acute myocardial infarction (AMI). In our study, after pre-test, two candidate miRNAs were detected by using real-time RT-PCR. Cardiac troponin I (cTnI) concentrations were measured by ELISA assay in plasma from patients with AMI (n=17) and healthy subjects (n=25), simultaneously. Increased miR-1 and decreased miR-126 in plasma from patients with AMI after the onset of symptoms compared with healthy subjects were found. A remarkable finding in this study is that miR-1, miR-126 and cTnI expression levels exhibited the same trend. Our results suggest that the plasma concentrations of miR-1 and miR-126 may be useful indicators for AMI.


Introduction
MicroRNAs (miRNAs) are a relatively novel class of endogenous, non-protein coding small RNA molecules that can regulate hundreds of genes through binding to the 3' UTR of target mRNAs [1][2][3]. MiRNAs have been proven to play important roles in self-renewal, cellular development, differentiation, proliferation and apoptosis [4,5]. Mature miRNAs bind to specific regions of mRNA targets by a mechanism that is not completely understood, resulting in translational repression or mRNA degradation [6][7][8][9].
The human genome encodes more than 1,000 miRNAs with tissue or cell type specific expressions [10][11][12][13]. Studies have shown that miRNAs have been observed in a wide range of human diseases, includ-ing autoimmune, inflammatory, neurodegenerative and cardiovascular diseases [14,15]. There are mounting evidences showing that cardiac-specific miRNAs, miR-1, miR-133a/b and miR-208, are involved in heart development and cardiovascular diseases, including acute myocardial infarction (AMI) in experimental animals [16]. Recent studies have shown that some miRNAs are present in the systemic circulation, both in humans and animals, and are associated with exosomes and microparticles [17][18][19][20]. The levels of some circulating miRNAs have been reported to be differentially expressed during pregnancy and in the presence of a variety of cancers or cardiovascular diseases.

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High mortality and morbidity is common in AMI. Some biomarkers have been applied in diagnosis of AMI; for example, cardiac troponin I (cTnI), MB and so on. New approaches that can improve current diagnosis for AMI are still lacking. However, the expression and possible roles of miRNAs in AMI are less well studied. Cardiac injury, which occurs after AMI increases the circulating levels of several myocardial-derived miRs (eg, miR-1, miR-133, miR-499, miR-208), whereas patients with coronary artery disease or diabetes showed reduced levels of endothelial-enriched miRs, such as miR-126 [21]. In the present study, we assessed the hypothesis that miR-1 and miR-126 might be useful for identifying and evaluating AMI. Our aim was to look for miRNA-based modulation of gene expression in AMI.

Blood Samples
This study was supported by the Ethics Committee of Tongji Hospital, and was performed in accordance with the principles of the Declaration of Helsinki. After gaining their written informed consents, we gathered blood samples from 17 patients and 25 healthy adults at Tongji Hospital between October 2009 and May 2010. AMI was diagnosed based on combination of several criteria: 1) ischemic symptoms; 2) creatine kinase-MB (CK-MB); 3) pathological Q wave; 4) increased cTnI; and 5) ST-segment elevation or depression [22]. In addition, 25 healthy adult volunteers (normal electrocardiogram and no history of cardiovascular diseases) were enrolled in this study. The blood samples of patients with AMI were acquired at 4h (± 30min), 8h (± 30min), 12h (± 30min), 24h (± 30min), 48h (± 30min), 72h (± 30min) and 1w (± 60min) after the onset of symptoms. Plasma was isolated by centrifugation and conserved at -80°C until purification.

RNA Purification
Total RNA was extracted from plasma with TRIzol LS Reagent as described previously [23].

Plasma cardiac troponin I determine
Plasma cTnI concentrations were measured by ELISA assay according to the manufacturer's protocol (Abnova, Taiwan).

MiRNA qRT-PCR
Two μg of total RNA were reverse-transcribed using Transcript First-strand cDNA Synthesis Su-perMix (TransGen Biotech, Beijing, China) in accordance to the manufacturer's protocol. In short, the 50 uL reactions were incubated for 60 min at 42°C, 10 min at 70°C, and then stored at 4°C.
qRT-PCR were analyzed using the Bulge-Loop™ miRNA qRT-PCR Detection Kit (Ribobio Co., Guangzhou, China) and TransStart TM Green qPCR SuperMix (TransGen Biotech, Beijing, China) in accordance with the manufacturer's protocol with the Rotor-Gene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany). Briefly, the reactions were incubated at 95°C for 30s, followed by 40 cycles of 95°C for 30s, 60°C for 20s, 70°C for 1s. The relative expression level for each miRNA was computed using the comparative CT method. It is important to note that to control for possible diversity in the amount of starting RNA, miRNA expression was normalized to small nucleolar RNA U6.

Statistical analysis
A widely used method to present relative expression of miRNA is the 2 -ΔΔct method. Relative expression of miRNA presents the data of the miRNA of interest (CtmiRNA of interest) relative to internal control gene (Ctinternal control miRNA), termed Δct. Results are calculated as Δct± standard deviation (SD). Different groups of AMI patients and healthy adults were compared with Δct±SD of the control group (healthy adults) and tested for statistical significance. When the differences in Δct±SD of the tested groups reached or were below p = 0.05, the difference in miRNA expression was considered statistically significant. The data were presented as fold change graphically. Fold change of the tested group is calculated relative to the control group (healthy adults) using the 2 -ΔΔct equation.
All values of miRNAs are expressed as mean±SD. Comparisons of parameters among ≥ 3 groups were analyzed by repeated-measures ANOVA. Independent-sample T test was used for 2-group comparisons. For categorical variables, Chi-Square test was used. Time-course trends of miRNAs and cTnI were analyzed by repeated-measures ANOVA. Differences were considered statistically significant at a value of P<0.05.
A score (miRNA-score) was defined as the level of miR-1 and miR-126 in the AMI group as compared with the control group. The miR-1 and miR-126-score of each sample was calculated as the sum of the inverted-normalized signals of the miR-1 and miR-126-scores and adjusted by subtracting a constant (the minimal score), so that the range of scores starts at 0 [24].
For all statistical analyses, the statistical software SPSS 13.0 for Windows was used.

Statistical analysis of patients' characteristics
Among 17 patients with AMI, 13 were males and 4 females; their age ranged between 40 and 70 years (mean 53±12.5). All patients had a transmural MI. There were no statistical differences between the control group and the AMI patients for any of the considered variables such as LDL cholesterol, total cholesterol, total triglycerides, HDL cholesterol, white blood cell count, systolic blood pressure, diastolic blood pressure, creatinine and history of diabetes and smoking status were recorded respectively. Details were shown in Table 1.

Specificity and sensitivity of miR-1 and miR-126
Since the detected levels of miR-1 and miR-126 in plasma may be affected by both technical and biological variation, we combined the levels of miR-1 and miR-126 into a single score to provide an improved signal to noise ratio. The miR-1-score and miR-126-score stand for the plasma levels of the miR-1 and miR-126 with P<0.0001 for the comparison between AMI and control group, was calculated as described in the methods section. The miR-1 and miR-126-scores allowed a significant separation between the AMI and control groups (P = 1e-7). The median score of miR-1 is 2.16, 2.55, 2.85, 2.58, 2.52 and 2.10 in the AMI group. It is 1.04, 1.39, 1.25, 1.1, 0.96 and 1.08, respectively, in the control group at 4h, 8h, 12h, 24h, 48h and 72h (Figure 2). The ability of the miR-1-score to differentiate the AMI group from the control group is demonstrated by the ROC curve with an AUC of 0.92, 0.90, 0.94, 0.92, 0.96 and 0.90 (Figure 3 and Table 2). By using a threshold score of 1.57, 2.03, 2.26, 1.47, 1.83 and 1.74 above which patients are predicted to belong to the AMI group, we achieved a sensitivity of 93%, 93%, 94%, 93%, 93% and 90% and a specificity of 90%, 90%, 93%, 90%, 90% and 90%, respectively, for the identification of AMI patients. The median score of miR-126 is 2.51, 2.55, 2.5 2.6, 2.4 and 2.2 in the AMI group. It is 1.31, 1.31, 1.30, 1.35, 1.21 and 1.20 in the control group at 4h, 8h, 12h, 24h, 48h and 72h (Figure 4). The ROC curve of miR-126 showed moderate ability to distinguish between the AMI group and the healthy control group at 4h, 8h, 12h, 24h, 48h and 72h with an AUC of 0.86, 0.87, 0.88, 0.87, 0.84 and 0.83 ( Figure 5 and Table 2), respectively. Using a threshold score of 1.6, 1.59, 1.88, 1.7, 1.2 and 1.25 in the AMI group, we gained a specificity of 78%, 84%, 88%, 77%, 80% and 70% and a sensitivity of 81%, 81%, 81%, 81%, 81% and 81%, respectively, for the diagnosis of AMI in patients.

Discussion
MiRNA regulates gene expression by binding and modulating the translation of specific miRNAs [25][26][27][28]. In recent years, tremendous efforts have been made to use a miRNA microarray screening approach to address the miRNA expression profiling in infarted hearts from animal models [28][29][30]. Recent evidences suggested that miRNAs are excellent promising disease biomarkers [31][32][33]. MiRNAs have been consistently detected in serum, plasma and other bodily fluids and specific miRNA patterns have been associated with pregnancy and with certain diseases [34][35][36]. MiRNAs have been shown to play important roles in development, stress responses, angiogenesis and oncogenesis. Accumulating evidence also points to important roles of miRNAs in the cardiovascular system [8,37]. However, the role of miRNA in AMI is just emerging.
In this study, we reported the circulating expressions of miR-1 and miR-126 in AMI patients, in comparison to healthy adults. The most significant finding was that plasma levels of miR-126 were down-regulated in acute myocardial infarction at 4h (± 30min), 8h (± 30min), 12h (± 30min), 24h (± 30min), 48h (± 30min), 72h (± 30min) and 1w (± 60min) after the onset of AMI symptoms. Importantly, miR-1 was up-regulated at 4h (± 30min), 8h (± 30min), 12h (± 30min), 24h (± 30min), 48h (± 30min), 72h (± 30min) and 1w (± 60min) after the onset of AMI symptoms. A remarkable finding in this study is that miR-1, miR-126 and cTnI exhibited the same trends, however, with no significant interaction effects. The plasma concentration of miR-1 and miR-126 showed a good correlation with the plasma concentration of cTnI, a classic biomarker of myocardial injury. The validity of circulating cTnI as a marker of myocardial injury has been reported [36]. In our study, the plasma concentrations of cTnI and miR-1, miR-126 were highly correlated and exhibited similar time courses. Using the levels of the miR-1 and miR-126, we were able to define a score with a high sensitivity and specificity for the detection of AMI patients at 4h, 8h, 12h, 24h and 72h relative to control group. Furthermore, to look into the possible mechanisms of miR-1/miR-126 in AMI, computational predictions as described at MICROCOSM, miRanda, TargetScan and Pic Tar were employed. These analyses yielded 901/937, 7204/815, 790/25 and 535/21 potential candidates. Some of them were reported to be associated with coronary diseases, such as GJA1, KCNJ2 [38], tanshinone IIA [39], LRP6 [40]. Taken together, our results clearly support that miR-1 and miR-126 may be useful potential biomarker of AMI.
To avoid possible confounders derived from patient selection, age, gender, total triglyceride, creatinine, HDL cholesterol, LDL cholesterol, white blood cell count, total cholesterol, systolic blood pressure, diastolic blood pressure, history of diabetes and smoking status, all these were recorded in our study. Statistical analyses showed that they have no influence on plasma miRNA levels. This suggested that miR-1 and miR-126 were potential biomarkers for certain diseases.
The present study confirmed that miR-1 and miR-126 can be used as biomarkers of AMI in humans. The next question is whether assessment of plasma miR-1 and miR-126 concentration has any clinical significance. MiRNAs might be an especially rich source of diagnostic, prognostic and predictive information as biomarkers in AMI patients [41].
It should be noted that the present study of circulating miR-1 and miR-126 as potential biomarkers for AMI was conducted in a relatively small sample size and future evaluation in larger clinical studies will be needed to confirm the utility as a biomarker. Nonetheless, our study provides a solid basis to identify and develop miR-1 and miR-126 as a novel class of blood-based biomarkers for AMI in the future.
In summary, we found that circulating miR-1 and miR-126 of patients with AMI were significantly changed in a time dependent manner. This suggests that miR-1 and miR-126 have potential utility as novel potential biomarkers for clinic diagnosis of AMI.