Global Surveillance of Circulating MicroRNA for Diagnostic and Prognostic Assessment of Acute Myocardial Infarction Based on Plasma Small RNA Sequencing

Academic Background

Acute Myocardial Infarction (AMI) is one of the most severe cardiovascular diseases leading to human mortality and morbidity worldwide. The pathological mechanism of AMI primarily involves the rupture of atherosclerotic plaques or endothelial erosion in the coronary arteries, resulting in myocardial ischemia and necrosis. AMI is traditionally categorized into ST-segment Elevated Myocardial Infarction (STEMI) and Non-ST-segment Elevated Myocardial Infarction (NSTEMI). Early and accurate diagnosis of AMI is crucial for timely reperfusion therapy to reduce adverse events such as cardiac death and heart failure.

Traditional AMI diagnosis relies on biomarkers such as Creatine Kinase-MB (CK-MB), serum Myoglobin (Myo), and cardiac Troponin (cTnI, cTnT). However, these biomarkers suffer from insufficient specificity or the long duration required from symptom onset to reaching diagnostic levels in the blood. Therefore, exploring novel biomarkers with better time-effectiveness, diagnostic sensitivity, and specificity has been a research focus.

In recent years, circulating microRNAs (miRNAs) have garnered significant attention as non-invasive biomarkers for cardiovascular disease diagnosis and prognosis. miRNAs are small RNA molecules approximately 22 nucleotides in length, known for their exceptional stability in plasma. This stability is attributed to their association with microparticles (e.g., exosomes, microvesicles, and apoptotic bodies), RNA-binding proteins (e.g., Argonaute2), or lipoprotein complexes (e.g., high-density lipoprotein), which protect them from degradation by plasma RNases. Additionally, miRNA levels in the plasma of AMI patients rise faster than cTnI, making them potential early diagnostic markers.

Although previous studies have shown that certain miRNAs (e.g., miR-208a, miR-499, miR-1, and miR-133) are significantly elevated in the plasma of AMI patients, these findings are inconsistent and controversial across independent studies. Therefore, there is an urgent need for global surveillance of circulating miRNAs at the transcriptome level in healthy individuals, AMI patients, and those undergoing reperfusion therapy to reveal miRNA differences among these populations and their dynamic changes during myocardial infarction and reperfusion.

Study Source

This study was conducted by Xiaomin Wang, Yaojun Lu, Ruiping Zhao, Bing Zhu, and Jian Liu, among others, from Baotou Central Hospital and the First Affiliated Hospital of Wenzhou Medical University. The research was published in 2024 in the journal Biomarker Research under the title “Global surveillance of circulating microRNA for diagnostic and prognostic assessment of acute myocardial infarction based on the plasma small RNA sequencing.”

Research Process and Results

Research Process

1. Study Subjects and Sample Collection
   The study enrolled patients who visited the Department of Cardiology at Baotou Central Hospital between November 2018 and April 2019 with symptoms of “chest discomfort.” The subjects were divided into three groups: healthy controls (27 individuals), AMI patients (64 individuals, including 37 STEMI and 27 NSTEMI patients), and AMI patients who underwent reperfusion therapy (20 individuals). All AMI patients were admitted within 48 hours of symptom onset, and those who had previously received intravenous thrombolytic or anticoagulant therapy were excluded. Healthy controls were individuals without severe or uncontrolled medical conditions, malignancies, or infectious diseases.

2. RNA Extraction and Sequencing
   Total RNA was extracted from each plasma sample using the miRNeasy Micro Kit (Qiagen), and RNA quality and concentration were assessed using the Agilent 2100 Bioanalyzer. Small RNA libraries were constructed using the TruSeq Small RNA Sample Prep Kit (Illumina), and single-end sequencing was performed on the Illumina HiSeq2500 platform with a sequencing depth of 20M reads per sample.

3. miRNA Quantification and Differential Analysis
   Sequencing data were aligned to the human genome (hg19) using the miRDeep2 software, and miRNA levels were normalized and analyzed for differential expression using the DESeq2 software. Differentially expressed miRNAs were filtered based on an adjusted p-value of less than 0.05 and a fold change of at least two (|log2FoldChange|>1).

4. Diagnostic miRNA Screening and Functional Analysis
   The diagnostic performance of all detected miRNAs was evaluated using ROC curve analysis, and the Least Absolute Shrinkage and Selection Operator (LASSO) was used for dimensionality reduction to identify the optimal diagnostic miRNA panel. Additionally, the correlation between miRNAs and cTnI levels was analyzed, and the functional roles of these miRNAs were explored through KEGG pathway enrichment analysis.

5. Prognostic miRNA Screening
   A five-year follow-up was conducted for AMI and reperfusion patients, and the prognostic value of circulating miRNAs was assessed using the Cox regression model, ROC curves, and Kaplan-Meier curves.

Key Results

1. miRNA Transcriptome Differences
   A total of 1659 known mature miRNAs were detected, with 288 miRNAs showing significant differences between AMI patients and healthy controls, including 58 upregulated and 230 downregulated miRNAs. Additionally, the miRNA expression patterns in reperfusion-treated patients differed significantly from those in AMI patients.

2. Diagnostic miRNA Screening
   ROC curve analysis identified 40 miRNAs with high diagnostic performance (AUC>0.85), including 32 upregulated and 8 downregulated miRNAs in AMI patients. Among these, miR-296-5p exhibited the best diagnostic performance, with an AUC of 0.983. LASSO dimensionality reduction ultimately identified a diagnostic model consisting of miR-296-5p and miR-660-3p, which achieved an AUC of 0.998 in the discovery cohort and 0.90 in the validation cohort.

3. miRNA Correlation with cTnI
   Twenty-nine miRNAs were significantly correlated with plasma cTnI levels, with 20 overlapping with high diagnostic performance miRNAs. These miRNAs were significantly enriched in pathways such as HIF-1 signaling, AMPK signaling, and fluid shear stress and atherosclerosis.

4. Prognostic miRNA Screening
   Cox regression analysis identified 18 miRNAs associated with survival, among which miR-548ap-5p and miR-4716-3p showed significant prognostic value in Kaplan-Meier curves.

Conclusions and Significance

This study is the first to globally monitor circulating miRNAs at the transcriptome level for the development of diagnostic and prognostic biomarkers for AMI. A subset of miRNAs with potential diagnostic and prognostic value was identified, which not only effectively distinguishes AMI patients from healthy individuals but also reflects the pathological mechanisms of myocardial infarction and cellular transcriptional and energetic responses to hypoxia. These findings provide new biomarkers for the early diagnosis and prognostic assessment of AMI, offering significant scientific and clinical value.

Research Highlights

1. Global Surveillance: This study is the first to globally monitor circulating miRNAs at the transcriptome level in AMI patients, reperfusion-treated patients, and healthy individuals, revealing the potential value of miRNAs in AMI diagnosis and prognosis.
2. High Diagnostic Performance miRNAs: Forty miRNAs with high diagnostic performance were identified, with the diagnostic model consisting of miR-296-5p and miR-660-3p demonstrating excellent diagnostic performance in both discovery and validation cohorts.
3. Prognostic miRNAs: Through long-term follow-up, miR-548ap-5p and miR-4716-3p were identified as potential prognostic markers for AMI.
4. Pathway Enrichment Analysis: These miRNAs were significantly enriched in pathways such as HIF-1 signaling, AMPK signaling, and fluid shear stress and atherosclerosis, further elucidating their roles in the pathological mechanisms of AMI.

Other Valuable Information

The study also explored the impact of age and rhythm status on miRNA diagnostic performance, finding that these factors did not significantly affect the predictive performance of the diagnostic model. Additionally, the study provided NCBI Sequence Read Archive (SRA) accession numbers for all high-quality sequencing data, facilitating validation and extension of subsequent research.

Summary

This study, through global surveillance of circulating miRNAs, identified a subset of miRNAs with potential diagnostic and prognostic value, providing new biomarkers for the early diagnosis and prognostic assessment of AMI. These findings hold significant scientific importance and offer new tools and insights for clinical practice.