Plasma microRNA-133a is a new marker for both acute myocardial infarction and underlying coronary artery stenosis
- Feng Wang†1,
- Guangwen Long†1,
- Chunxia Zhao1,
- Huaping Li1,
- Sandip Chaugai1,
- Yan Wang1,
- Chen Chen1, 2Email author and
- Dao Wen Wang1
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 17 July 2013
Accepted: 18 September 2013
Published: 23 September 2013
Previous study demonstrated that miR-133a was released into blood from injured myocardium in cardiovascular diseases. However, the dynamic change of circulating miR-133a level in the early phase of acute myocardial infarction (AMI) and the correlation between miR-133a and severity of coronary stenosis in coronary heart disease (CHD) patients are not clear.
Methods and results
Three different cohorts (including 13 AMI patients, 176 angina pectoris patients and 127 control subjects) were enrolled to investigate the expression levels of circulating miR-133a in patients with myocardial ischemia and also the relationship between plasma miR-133a and severity of coronary stenosis. Plasma miR-133a levels of participants were examined by real-time quantitative PCR. Simultaneously, plasma cardiac troponin I (cTnI) concentrations were measured by ELISA assays. The results showed that circulating miR-133a level was significantly increased in AMI patients in time-dependent manner, and achieved a 72.1 fold peak at 21.6 ± 4.5 hours after the onset of AMI symptoms and exhibited a similar trend to plasma cTnI level. We also found that plasma miR-133a levels were higher in CHD patients than control group. Importantly, the levels of circulating miR-133a positively correlated with the severities of the coronary artery stenosis. Receiver operating characteristic (ROC) analysis revealed that circulating miR-133a had considerable diagnostic accuracy for CHD with an AUC of 0.918 (95% confidence interval 0.877-0.960).
Circulating miR-133a may be a new biomarker for AMI and as a potential diagnostic tool. And increased miR-133a level may be used to predict both the presence and severity of coronary lesions in CHD patients.
Acute myocardial infarction (AMI) is the worst acute syndrome of coronary heart diseases (CHD) with high morbidity and mortality. An early and correct diagnosis is critical for providing appropriate therapy to improve the survival rate and prognosis . Blood biomarker cardiac troponin I (cTnI) is widely used in clinical practice as the gold standard for diagnosing acute myocardial infarction , but plasma cTnI concentrations may be falsely elevated in certain cardiac as well as non cardiac diseases such as severe heart failure, atrial fibrillation, chronic kidney disease, severe sepsis and septic shock [3–6]. Therefore, it is necessary to search novel biomarkers with high sensitivity and specificity for early diagnosis of AMI.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs with 21-25 nucleotides in length. By pairing with the 3’ UTR of target mRNAs, miRNAs can regulate protein-coding genes at the posttranscriptional level via degradation of mRNAs or repression of protein translation . At present, About 700 human miRNAs have been identified, and most of them were found to be tissue-/cell-specific . Mounting evidences suggest that miRNAs play crucial roles in various physiological and pathologic processes, and the dysfunctions of miRNAs are associated with various diseases and pathophysiologies [9–11]. Recently, studies showed that miRNAs are abundantly present in body fluid and can be used as biomarkers for some diseases [12–14]. MiR-133a is a muscle specific-miRNA and is expressed abundantly in myocardial cells [15–17]. It has been established that miR-133a plays important roles in myogenesis, cardiac development and hypertrophy [18–23]. Previous studies demonstrated that miR-133a had a low level presence in the plasma of healthy people , and it was expressed differentially in different cardiovascular diseases [15, 24]. Recently, it has been reported that the elevated miR-133a is released into peripheral circulation from the injured myocardium after Ca2+ stimulation . Although these studies demonstrated that the expression of circulating miR-133a increased in patients with CHD (including AMI and angina pectoris) and circulating miR-133a can be used as a marker for cardiomyocyte death, few clinical studies have reported on the dynamic change in circulating miR-133a level in the early phase of AMI, and also the correlation between miR-133a concentration and the severity of coronary stenosis in CHD patients is not clear.
In the present work, we aimed to confirm the role of plasma miR-133a as a biomarker for CHD, especially for AMI. Furthermore, we investigated the correlation between the levels of circulating miR-133a and the Gensini score (a numerical value for assessment the severity of coronary artery stenosis) in coronary heart disease patients.
Materials and methods
Characteristics of patients
Experiments were conducted in accordance with the Declaration of Helsinki. Three cohorts participated in this study.
The first cohort included 13 patients of AMI and 27 healthy volunteers. The inclusion criteria for AMI patients were based on the third Universal Definition of Myocardial Infarction . Briefly, AMI patients were clinically diagnosed by the following criteria: 1) acute ischemic chest pain within 24 hours; 2) electrocardiogram change of acute myocardial infarction (pathological Q wave, ST-segment elevation or depression); 3) plasma cTnI > 0.1 ng/mL. The initial blood sample (denoted by T0) was collected immediately after the AMI patient was admitted to Tongji hospital. Other 5 subsequent blood samples were obtained at 4, 12, 24, 48, 72 hours after the first collection, denoted by 4 h, 12 h, 24 h, 48 h and 72 h, respectively. The second cohort included 22 CHD patients with chest pain having single lesion of the left anterior descending coronary artery and 8 non-CHD patients with negative results of coronary angiography. The third cohort contained 246 patients with acute chest pain. Further, coronary angiography showed that 154 of them were CHD patients with complex lesions of coronary artery, and the remaining 92 patients were non-CHD patients with no coronary artery stenosis. A single blood sample from each participant in both second and third cohorts was obtained immediately after admission, and coronary angiography was used to confirm CHD and define the coronary artery lesions. Blood samples were collected via venous puncture. After isolation by centrifugation, the plasma were transferred to RNase-free tubes and stored at -80°C until further processing.
Participants were selected from inpatients or outpatients departments of Tongji hospital between October 2009 and June 2011 in Wuhan, China. The study was approved by the Medical Ethics Committee in Tongji Hospital and written informed consents were obtained from all the participants.
Total RNAs were isolated by TRIzol LS Reagent (Invitrogen) according to the manufacturer’s protocol as described previously . In brief, total RNA was purified from 500 μL of plasma and ultimately eluted into 25 μL of RNase-free water.
Detection and quantification of miRNAs by real-time PCR
Two microgram of total RNA was reverse-transcribed using Transcript First-strand cDNA synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. The Bulge-Loop™ miRNA qRT-PCR Detection Kit (Ribobio Co., Guangzhou, China) and SYBR Green PCR SuperMix Kit (TransGen Biotech, Beijing, China) were used in real-time PCR for examining the relative quantification of miR-133a according to the manufacturer’s protocol with the Rotor-Gene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany), and U6 was measured as endogenous control for normalizing the data of experimental qRT-PCR. Each specimen was measured in triplicate. The threshold cycle (Ct) was defined as the fractional cycle number at which fluorescence exceed the threshold. In our experiment the detection limit of Ct value was defined as 40. The Ct values from qRT-PCR assays over 40 were treated as 40 [15, 25, 28, 29].
Cardiac troponin I determination
The concentrations of cardiac Troponin I (cTnI) were measured by the Human Troponin I ELISA kit (Abnova, Taiwan) according to manufacturer’s protocol.
Real-time PCR assays were analyzed by 2-ΔΔct method, which is a widely used method to present relative gene expression by comparative Ct. All the data of patients’ clinical characteristics are described as mean ± SD, and the other data are described as mean ± SEM. The data of miR-133a and cTnI were analyzed by the Kolmogorov-Smirnov test to examine whether they followed the normal distribution. If the data fit the normal distribution, then student’s t test and the ANOVA are used. Otherwise, Mann–Whitney U test and two-tailed Kruskal-Wallis tests are performed. In this study, both the data of miR-133a and cTnI were found to follow the normal distribution by the Kolmogorov-Smirnov test and hence student’s t test and ANOVA were used. Categorical variables were compared by χ2 test. The correlation analyses were determined by linear regression analysis. The receiver operating characteristic (ROC) curve was used to assess the predictive power for diagnosing CHD. Multiple logistic regression analysis was carried out to investigate whether miRNA-133a was an independent predictor of CHD after adjustment for relevant co-variants (including age, sex, smoking and cardiovascular risk factors hypertension, diabetes, hyperlipidemia etc.) as previously described . All statistical analyses were accomplished by using SPSS 17.0 software, and the cutoff point of statistical significance was set at p < 0.05 (two-sided).
The real-time RT-PCR (qRT-PCR) assay for miRNA quantification
To ensure the method of qRT-PCR assay for miR-133a quantification is viable and suitable, the amplification curves for both miR-133a and U6 were provided (Additional file 1: Figure S1A and B). To verify primer specificities, melting curve analyses (Additional file 1: Figure S1C and D) and agarose gel electrophoresis images (Additional file 1: Figure S2A and B) were performed, the RNAs extracted from mouse heart and brain were treated as positive and negative control, respectively.
The pattern of plasma miR-133a levels in acute myocardial infarction
The correlation between plasma miR-133a levels and the severities of coronary lesion in CHD patients
The correlation between circulating miR-133a levels and the gensini score in a validation cohort with a large number of CHD patients
The plasma miR-133a is a sensitive predictor for coronary heart disease
Previous studies demonstrated that miRNAs are abundantly present in a remarkably stable form and they can be detected in peripheral circulation [12, 31]. Recently, more and more circulating miRNAs, including heart-, vascular- and muscle-specific miRNAs, have been reported as new biomarkers in multiple cardiovascular diseases [32, 33]. For example, circulating miR-423-5p is suggested as a biomarker for heart failure . And additionally, cardiac-related miRNAs (miR-208, miR-499 and miR-1) and stress-related miRNAs (miR-21 and miR-146a) may be potential biomarkers for acute coronary syndrome . Moreover, a recent study had reported that circulating miR-126, miR-223 and miR-197 were consistently and significantly related to incidence of myocardial infarction . These observations suggest that circulating miRNAs may be useful not only for prediction of cardiovascular events, but also serve as sensitive biomarkers for improving the diagnostic accuracy of cardiovascular diseases.
The present study demonstrated dynamic change in circulating miR-133a expression in the early phase of acute myocardial infarction. Furthermore, our data is the first to demonstrate a positive correlation between circulating miR-133a and the severity of coronary stenosis in CHD patients.
The results demonstrated that circulating miR-133a levels increased in time-dependent manner in the early phase of AMI and exhibited a similar trend as cTnI in AMI patients; both of them rapidly increased at first, achieved a peak at 21.6 ± 4.5 hours after the onset of AMI symptoms, and then gradually returned close to normal level on the following days. Importantly, the circulating miR-133a positively correlated with cTnI in AMI patients. These results strongly indicated that circulating miR-133a can be a biomarker for diagnosing acute myocardial infarction.
Furthermore, 22 CHD patients with single lesion of coronary artery were included in the second cohort to study the relationship between plasma miR-133a and the severity of coronary atherosclerosis. The results showed that circulating miR-133a increased in CHD patients compared with non-CHD patients, and the levels of elevated miR-133a positively correlated with the severities of coronary atherosclerosis. The results were further verified in a large validation cohort of 246 subjects (154 CHD patients and 92 non-CHD). Interestingly, we found a higher expression of circulating miR-133a in CHD patients with low cTnI expression compared with non-CHD patients and it correlated with Gensini score of these CHD patients. These results showed that circulating miR-133a is superior to cTnI in detecting the severity of coronary artery lesions.
Finally, the ROC curve of miR-133a and cTnI were plotted in CHD patients with an AUC of 0.918 and 0.741, respectively. The ROC curves of CHD subcategories revealed that circulating miR-133a is more informative for CHD diagnosis than cTnI in CHD patients. Importantly, the diagnostic accuracy for CHD became significantly raised when combining clinical model, miR-133a and cTnI with the AUC of 0.947. Interestingly, this addition effect of combination could be more valuable for cTnI to improve the diagnostic accuracy of CHD, while miR-133a appeared to be a strong and independent predictor for CHD. These results may provide theoretical foundation in improving the clinical diagnosis of CHD.
In summary, the present study measured the early changes in expressions of circulating miR-133a in AMI patients, and provided first insights into the relationship between plasma miR-133a and the severity of coronary atherosclerotic stenosis in CHD patients, our results suggested that circulating miR-133a was a sensitive predictor for diagnosing AMI and CHD.
All these results suggested that circulating miR-133a can be a novel and potent biomarker for CHD, especially for AMI. And its level in plasma can reflect the severity of coronary atherosclerosis in CHD patients.
We thank all the people who helped in collecting the blood samples.
This work was supported by grant from the National Natural Science Foundation of China (No. 81070236 and No. 31200594) and Key Project of Ministry of Health of the People's Republic of China.
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