Clinical impact of circulating miR-133, miR-1291 and miR-663b in plasma of patients with acute myocardial infarction

Acute coronary syndrome (ACS) is a very common health problem in the all the world. Acute myocardial infarction (AMI) is one of the leading causes for death in both developed and developing countries and it is the single largest cause of death in the United States, responsible for 1 out of every 6 deaths [1]. Up to 10 million Americans report to an Emergency Department (ED) for chest pain yearly and this number is expected to climb with continued aging of the United States (U.S.) population [2]. The substantial number of emergent hospitalizations for ACS, including ST-elevation myocardial infarction (STEMI), Non ST-elevation myocardial infarction (NSTEMI), and unstable angina (UA), represents a significant financial strain to both patients and the healthcare system [3]. This burden could be ameliorated, and more efficient treatment administered, if a more rapid, sensitive, and specific biomarker could differentiate between the spectrum of ACS (i.e. STEMI, NSTEMI, and UA) as well as differentiate from other less concerning causes of chest pain, such as gastroesophageal reflux (GERD). Rapid and correct diagnosis of AMI plays an important role in therapy and prognosis for this disease [4]. Over the past two decades, huge progress has been made in the diagnosis, treatment, and prognosis of AMI. Particularly, the progress in biomarkers for AMI attracted a great deal of attention. Currently, cardiac troponins are the most common biomarkers used for diagnosis of AMI in clinical practice [5]. However, there is still a clinical need for novel biomarker, which is able to reliably rule in or rule out AMI immediately on admission.

MicroRNAs (miRNAs) are small (19-22-nt) single stranded non-coding RNA molecules that are derived from hairpin-structured precursors [6, 7]. These microRNAs function by directly binding to heir potential target site in the 3′ untranslated region (3′ UTRs) of specific target mRNA, leading to the repression of mRNA translation or the degradation of target mRNAs. Small non-coding RNAs were also recently implicated in control of DNA damage response. Currently, there are 24521 mature miRNA sequences of both human and other species listed in the miRNA registry (Sanger miRBase; http://​www.​mirbase.​org/​). Over recent years, many miRNAs have been investigated in various human disease, such as kinds of cancer and cardiovascular diseases [7, 8]. The deregulation of expression of microRNAs has been shown to contribute to the multistep processes of carcinogenesis in human by modification either of oncogenic or suppressor gene function [9]. Nowadays, microRNA expression patterns are known to characterize the developmental origins of tumors more effectively than mRNA expression signatures and thus can be a useful tool for the diagnosis and prognosis of human disease [9]. It has long been a goal of cardiovascular research to search for non-invasive tools for the diagnosis and management of AMI that can greatly reduce its worldwide health burden [10]. Recently, it has been reported that microRNAs are circulating in serum/plasma and AMI related microRNAs such as miR-1, miR-133a, miR-208b, miR-223, miR-320a, miR-451 and miR-499 have been detected in the plasma or serum of AMI patients [11, 12]. These circulating microRNAs can be considered as a new class of effective biomarkers where their abundance profile might reflect physiological and/or pathological conditions. Accordingly, several subsequent studies have proven that miRNAs can serve as potential biomarkers for various cardiovascular diseases, such as AMI.

The purpose of the present study was to characterize the levels of miRNAs including miR-133, miR-1291 and miR-663b in the circulation of AMI patients. Accordingly, an extensive detection of miRNAs was screened for in the AMI patients and the results were compared with an age-, gender-, and ethnically matched control group. We aimed to develop find the diagnostic value of there miRNAs for the AMI.

There is a growing requirement for the detection of a new tool for the AMI. MicroRNAs are small molecules approximately 22 nucleotides in length and have been well conserved evolutionally. Although they are noncoding genes, they are involved in the regulation of a wide range of biologic processes through translational modulation. In cardiovascular pathology, they have been used to classify heart failure and hypertension [13, 14]. As well, it was also suggested that miRNA might be a potential biomarker for the diagnosis and prognosis of AMI [12]. In this study, we initially identified the potential biomarker for the diagnosis of AMI. By using qRT-PCR, we confirmed that miR-133, miR-1291 and miR-663b levels were significantly overexpressed in AMI compared with Non-AMI. Although the ROC analyses, we found that there three miRNAs might be potential diagnostic markers for the AMI. Besides, we found that the three miRNAs were significantly decreased post-operatively and which provided a suggestion of the prognosis value for there miRNAs.

MiRNAs have recently been detected in serum or plasma, which are referred to circulating miRNAs [15]. Despite intense research, the origin of circulating miRNAs remains largely unknown. A number of studies have reported that miRNAs are actively secreted in microvesicles or exosomes from different cell types, which area likely source of circulating miRNAs. So far, around 121 miRNAs were identified in exosomes from mast cells, and sphingomyelinase 2 (nSMase2), a rate-limiting enzyme for ceramide biosynthesis, controls the secretion of exosomes to the extracellular matrix [16]. In addition to microvesicles or exosomes, microparticles and lipoprotein complexes (such as high-density lipoprotein complexes) are other possible sources of circulating miRNAs Human miRNAs isolated from plasma are highly stable in boiling water, in solution with very high or low pH [17]. Plasma miRNA levels remain stable when it is subjected to prolonged room temperature incubation or freeze-thawed multiple times. It has been reported that endogenous plasma miRNAs exist in a form that is resistant to plasma Rnase activit. Compared to the endogenous plasma miRNAs, rapid degradation was observed within minutes when synthetic miRNAs (corresponding to caenorhabditis elegans miRNAs cel-miR-39, cel-miR-54, and cel-miR-238) were added into human plasma.

Rapid and correct diagnosis is crucial to treatment and prognosis of AMI. Up to date, cardiac troponins and creatine kinase-MB are the most commonly used biomarkers for AMI, but their clinical value is limited in many cases. Secreted by cardiac cells and accumulated in blood, miRNAs are expected to reflect cardiac injury in response to cardiovascular risk factors and various pathological conditions. Thus, the circulating cardiac-specific or -enriched miRNAs (such as miR-208, miR-499, and miR-133) may provide unique biomarkers for diagnostic and therapeutic interventions of AMI [18]. MiRNA profiling studies in AMI and other diseases have been vastly reported. For instance, a recent tissue miRNA profiling study by Kuwabara Y et al. alrevealed that serum levels of miR-1 and miR-133a were increased significantly in patients not only with acute myocardial infarction but also with unstable angina pectoris and Takotsubo cardiomyopathy without elevation of serum creatine phosphokinase or cardiac troponin [19]. An investigation by By Wang GK, through verifying their tissue expression patterns with real-time PCR analysis, muscle-enriched miRNAs (miR-1, miR-133a, and miR-499) and cardiac-specific miR-208a were selected as candidates for this study. With miRNA microarray and real-time PCR analyses, miR-1, miR-133a, and miR-499 were present with very low abundance, and miR-208a was absent in the plasma from healthy people. In the AMI rats, the plasma levels of these miRNAs were significantly increased [20]. In a microarray study by peripheral total blood samples, identified 121 miRNAs, which are significantly dysregulated in AMI patients in comparison to healthy controls. Among these, miR-1291 and miR-663b show the highest sensitivity and specificity for the discrimination of cases from controls. Using a novel self-learning pattern recognition algorithm, we identified a unique signature of 20 miRNAs that predicts AMI with even higher power (specificity 96%, sensitivity 90%, and accuracy 93%). In addition, we show that miR-30c and miR-145 levels correlate with infarct sizes estimated by Troponin T release [21].

Besides, in this study, we found that the expression of miR-133, miR-1291 and miR-663b were significantly reduced after the surgery. These findings were first reported in this study. The reduction of these miRNAs might indicate a sign of reconditioning of ischemic cardiac muscle. The dynamic expression of these molecular biomarkers may be used as a prognostic marker. Advanced well-designed studies with larger cohort and longer follow-up are required.

In this study, we identified plasma miR-133, miR-1291 and and miR-663b were significantly increased in the AMI cases. Through ROC analyses, it was found that miR-133, miR-1291 and and miR-663b were potential predictors of AMI. Circulating levels of the same miRNAs also were correlated with the presence of AMI. Advanced well-designed studies are required.