Plasma Exosomal S1PR5 and CARNS1 as Potential Non-invasive Screening Biomarkers of Coronary Heart Disease

Abstract

Background: Early diagnosis and treatment significantly improve the prognosis of coronary heart disease (CHD), but no convenient screening tools are available. This study aims to find potential non-invasive screening biomarkers of coronary heart disease.

Method: We performed microarray analysis to investigate the mRNA expression levels in Small extracellular vesicles (sEVs) and screen significantly differentially expressed mRNAs in CHD patients vs. non-CHD patients. We then performed quantitative real-time polymerase chain reaction (qRT-PCR) to validate the microarray results, and we calculated the correlations between expression levels and clinicopathological data. Microarray analysis identified 72 downregulated mRNAs and 31 upregulated mRNAs in CHD patients relative to non-CHD patients.

Results: From the study, we found that upregulated sphingosine-1-phosphate receptor 5 (S1PR5) and downregulated carnosine synthase 1 (CARNS1) had the most significant differences between the patient group and the control group. S1PR5 expression was correlated with diabetes, heart rate, triglycerides, total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, and fasting blood glucose (P < 0.05). CARNS1 level was correlated with uric acid (UA) (P < 0.05). Overexpressed S1PR5 and downregulated CARNS1 were independent risk factors for CHD. The area under the receiver operating characteristic curve (AUC) of S1PR5 was 0.838 for diagnosing CHD; the AUC of CARNS1 was 0.883 for non-CHD; and the AUC of S1PR5 plus CARNS1 was 0.921 for CHD.

Conclusions: Microarray analysis showed that upregulated S1PR5 and downregulated CARNS1 in sEVs have the potential to become non-invasive biomarkers for CHD screening.

Keywords: coronary heart disease; genomics; lipid metabolism; mRNA; sEVs.

Figure 1

Identification of plasma exosomes in CHD patients. (A) The representative images of plasma exosomes derived from CHD group and the control group patients analyzed by transmission electron microscopy. Scale bar, 100 nm. (B) The positive markers of exosomes, CD63 and CD81 were detected in of plasma exosomes by Western blot. (C) Nanosight particle tracking analysis (NTA) of plasma exosomes derived from CHD group and the control group patients.

Figure 2

Identification of differentially expressed mRNAs in plasma exosomes derived from CHD patients. (A) Heat map showing hierarchical clustering analysis of mRNAs detected in CHD group and the control group patients. (B) Volcano map showing the distribution of differential mRNAs according to their p-values and fold-changes. Candidates with p < 0.05 and |log 2(fold-change) |≥1 are considered differential. The molecules with the most significant differences are marked on the volcano map. (C) Differential expression of 10 mRNAs was validated in plasma exosomes derived from CHD group and the control group patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t-test. *P < 0.05; **P < 0.01 and ***P < 0.005; ns, not significant.

Figure 3

KEGG Pathway and mRNA interaction network analysis based on the validated mRNA candidates. (A) KEGG pathway enrichment analysis of these mRNAs with the 20 highest enrichment scores. (B) mRNA interaction network based on the validated mRNA candidates. Every circle represents a mRNA. The darkness of the color denotes the strength of interaction. The key molecule we studied, S1PR5, has been highlighted in uppercase green fonts on the picture.

Figure 4

Diagnostic value of plasma exosomal S1PR5 and CARNS1 in CHD patients. qRT-PCR analysis of expression of S1PR5 (A) and CARNS1 (B) in a large sample of CHD patients (n = 120) and healthy controls (n = 121). ROC curve analysis of S1PR5 (C), CARNS1 (D) and combination of S1PR5 and CARNS1 (E) for discrimination of CHD patients (n = 120) from healthy controls (n = 121). ***P < 0.005.