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. 2013 Dec 5;8(12):e80738.
doi: 10.1371/journal.pone.0080738. eCollection 2013.

Signature of circulating microRNAs as potential biomarkers in vulnerable coronary artery disease

Jingyi Ren  1 , Jing Zhang, Ning Xu, Guanping Han, Qiang Geng, Junxian Song, Sufang Li, Jianqing Zhao, Hong Chen
Affiliations

Signature of circulating microRNAs as potential biomarkers in vulnerable coronary artery disease

Jingyi Ren et al. PLoS One. .

Abstract

Aims: MicroRNAs (miRNAs) play important roles in the pathogenesis of cardiovascular diseases. Circulating miRNAs were recently identified as biomarkers for various physiological and pathological conditions. In this study, we aimed to identify the circulating miRNA fingerprint of vulnerable coronary artery disease (CAD) and explore its potential as a novel biomarker for this disease.

Methods and results: The Taqman low-density miRNA array and coexpression network analyses were used to identify distinct miRNA expression profiles in the plasma of patients with typical unstable angina (UA) and angiographically documented CAD (UA group, n = 13) compared to individuals with non-cardiac chest pain (control group, n = 13). Significantly elevated expression levels of miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126*, and miR-451 were observed in UA patients compared to controls. These findings were validated by real-time PCR in another 45 UA patients, 31 stable angina patients, and 37 controls. In addition, miR-106b, miR-25, miR-92a, miR-21, miR-590-5p, miR-126* and miR-451 were upregulated in microparticles (MPs) isolated from the plasma of UA patients (n = 5) compared to controls (n = 5). Using flow cytometry and immunolabeling, we further found that Annexin V(+) MPs were increased in the plasma samples of UA patients compared to controls, and the majority of the increased MPs in plasma were shown to be Annexin V(+) CD31(+) MPs. The findings suggest that Annexin V(+) CD31(+) MPs may contribute to the elevated expression of the selected miRNAs in the circulation of patients with vulnerable CAD.

Conclusion: The circulating miRNA signature, consisting of the miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126* and miR-451, may be used as a novel biomarker for vulnerable CAD.

Trial registration: Chinese Clinical Trial Register, ChiCTR-OCH-12002349.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Study flow diagram.
All patients were enrolled at Peking University People's Hospital between August 1, 2012 and April 18, 2013. UA, unstable angina; SA, stable angina. * 10 patients in UA group underwent intravascular ultrasound (IVUS) confirming plaque rupture.
Figure 2
Figure 2. Profile of circulating miRNAs in UA patients (n = 13) and controls (n = 13).
Heat map illustrates the levels of significantly changed miRNAs (fold change >8 and FDR <0.0001%) in UA patients compared with controls. Color intensity is scaled within each row, such that the highest expression value corresponds to bright red and the lowest to bright green.
Figure 3
Figure 3. Circulating miRNAs expression in the validation cohort.
Expression of selected miRNAs were analyzed in controls (n = 37), SA patients (n = 31), and UA patients (n = 45) by quantitative PCR. Red points in UA group indicate UA patients with IVUS-confirmed plaque rupture. Data represent the mean ± SEM. ** P<0.01, *** P<0.001 compared to control group; ## P<0.01, ### P<0.001 compared to SA group.
Figure 4
Figure 4. Receiver–operator characteristics (ROC) curves for selected miRNAs in validation cohort.
ROC curves regarding diagnostic power to distinguish UA patients from non-UA cases for 7 selected miRNAs in the validation cohort were shown.
Figure 5
Figure 5. Principle component analysis (PCA) of circulating miRNA profiling in the derivation cohort.
The miRNA expression profile was reduced to three main principal components. PCA showed that most UA cases (84.6%, 11/13) could be correctly classified from the control group (CON).
Figure 6
Figure 6. Principle component analysis (PCA) of circulating miRNA expression in the validation cohort.
PCA decomposition of the 7 selected miRNAs (miR-106b, miR-25, miR-92a, miR-21, miR-590-5p, miR-126*, and miR-451) could distinguish most UA cases (84.4%, 38/45) from the non-UA cases in the PCR validation cohort.
Figure 7
Figure 7. Coexpression network analysis of circulating miRNAs in UA patients and controls.
(A–B) Coexpression network constructed with all detected miRNAs. Nodes represent individual miRNAs, and edges represent coexpression relationships between miRNA pairs. PCR-validated miRNAs (miR-106b, miR-25, miR-92a, miR-21, miR-590-5p, miR-126*, and miR-451) and their first neighbors are labeled with red nodes. (C–D) Subnetwork constructed with PCR-validated miRNAs (red nodes) and their first neighbors.
Figure 8
Figure 8. Characterization of microparticles (MPs) from plasma of control and UA patients by flow cytometry.
(A) Trucount beads and calibrator beads (1 µm in diameter) were gated in the upper two windows respectively. MPs are particles with size between 0.1 and 1 µm and gated in window P2. (B) Representative flow cytometry plot displaying MPs from the plasma of controls and UA patients stained with AnnexinV-FITC and anti-CD31-phycoerythrin. Annexin V+ CD31+ MPs were gated in window P2.
Figure 9
Figure 9. Comparison of microparticles (MPs) counts isolated from the plasma of UA patients (n = 5) and controls (n = 5).
(A) Comparison of Annexin V+ MP counts in the plasma of UA patients and controls. (B) Comparison of Annexin V+ CD31+ MP counts in the plasma of UA patients and controls. (C) Comparison of Annexin V+ CD31− MP counts in the plasma of UA patients and controls. MP counts were determined by flow cytometry. Data represent the mean ± SEM. *** P<0.001; NS, non-significant.
Figure 10
Figure 10. Comparison of miRNA expression in microparticles (MPs) isolated from the plasma of UA patients (n = 5) and controls (n = 5).
Expression levels of selected miRNAs were analyzed by quantitative PCR. Data represent the mean ± SEM. * P<0.05; ** P<0.01.
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