A Systems Biology Approach to Characterize Biomarkers for Blood Stasis Syndrome of Unstable Angina Patients by Integrating MicroRNA and Messenger RNA Expression Profiling

Abstract

Blood stasis syndrome (BSS) has been considered to be the major type of syndromes in unstable angina (UA) patients. The aim of this study was to find the systems biology-based microRNA (miRNA) and mRNA expression biomarkers for BSS of UA. We identified 1081 mRNAs and 25 miRNAs differentially expressed between BSS of UA patients and healthy controls by microarrays. We used DAVID, miRTrail, and the protein-protein interactions method to explore the related pathways and networks of differentially expressed miRNAs and mRNAs. By combining the results of pathways and networks, we found that the upregulation of miR-146b-5p may induce the downregulation of CALR to attenuate inflammation and the upregulation of miR-199a-5p may induce the downregulation of TP53 to inhibit apoptosis in BSS of UA patients. The expression patterns of miR-146b-5p, miR-199a-5p, CALR, and TP53 were confirmed by qRT-PCR in an independent validation cohort including BBS of UA patients, non-BBS of UA patients, and healthy controls. miR-146b-5p, miR-199a-5p, CALR, and TP53 could be significant biomarkers of BSS of UA patients. The systems biology-based miRNA and mRNA expression biomarkers for the BSS of UA may be helpful for the further stratification of UA patients when deciding on interventions or clinical trials.

Figure 1

Heat maps of mRNAs and miRNAs differentially expressed between UA patients with BSS and healthy controls. (a) Heat map and cluster analysis of the 1,206 differentially expressed probes between UA patients with BSS and healthy control. Red and green represented, respectively, differentially upregulated and downregulated mRNAs in UA patients with BSS. (b) Heat map and cluster analysis of the 25 differentially expressed miRNAs between UA patients with BSS and healthy control. Red and green represented, respectively, differentially upregulated and downregulatedmiRNAs in UA patients with BSS. Gray was for missing values. UA-BSS: unstable angina patients with Blood stasis syndrome; C: control group.

Figure 2

NOD-like receptor signaling pathway. Upregulated mRNAs were depicted in red by Search & Color Pathway in KEGG.

Figure 3

Apoptosis pathway. Upregulated mRNAs were depicted in red by Search & Color Pathway in KEGG.

Figure 4

Cytokine-cytokine receptor interaction pathway. Upregulated mRNAs were depicted in red by Search & Color Pathway in KEGG.

Figure 5

The regulation of upregulated miRNAs on the downregulated mRNAs in antigen processing and presentation pathway and p53 signaling pathway. Upregulated miRNAs were depicted in red and downregulated mRNAs were depicted in green on the maps.

Figure 6

The miRNA/mRNA interactive network. Round represented mRNAs and rectangular represented miRNAs. Red color indicated upregulation and green color downregulation, respectively. Sizes of the mRNAs were according to their degrees.

Figure 7

The PPIs network of downregulated mRNAs of the miRNA/mRNA interactive network. Rectangular represented downregulated mRNAs and round represented added interactive mRNAs. The interactions between two mRNAs extracted from pathways were shown as solid lines while those predicted interactions were shown as dashed lines. Extracted interactions involved in activation, expression regulation, or catalysis were shown with an arrowhead on the end of the line, while interactions involved in inhibition were shown with a “T” bar.

Figure 8

Hubs of the PPIs network.

Figure 9

Validation of differentially expressed miRNAs and mRNA among UA patients with BSS, UA patients with non-BSS, and healthy control by RT-PCR analysis.