Increased Circulating Exosomal miRNA-223 Is Associated with Acute Ischemic Stroke

Abstract

Recent studies have demonstrated that exosomal microRNAs (miRNAs) are novel biomarkers and therapeutic targets for various diseases including vascular disease. However, specific exosomal miRNAs expression in stroke patients has not been reported yet. Here, we explored whether circulating exosomal miRNAs can serve as potential biomarkers for the diagnosis of acute ischemic stroke and discussed the potential for clinical application. Blood samples were collected from acute ischemic stroke patients within the first 72 h (n = 50). Circulating exosomes were exacted by Exoquick exosome isolation kit and characterized by transmission electron microscopy. Western blot was performed to assess the expression of exosomal protein makers. Exosomal miRNA-223 (miR-223) was detected by RT-PCR assay. The relationship between the expression levels of miR-223 and National Institutes of Health Stroke Scale (NIHSS) scores, brain infarct volume, and neurological outcomes were analyzed. Circulating exosomes were isolated and the size of vesicles ranged between 30 and 100 nm. The identification of exosomes was further confirmed by the detection of specific exosomal protein markers CD9, CD63, and Tsg101. Exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group (p < 0.001). Exosomal miR-223 level was positively correlated with NIHSS scores (r = 0.31, p = 0.03). Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (p < 0.05). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. Future studies with large sample are needed to assess the clinical application of exosomal miR-223 as a novel biomarker for ischemic stroke diagnosis.

Keywords: acute ischemic stroke; biomarker; blood; diagnosis; exosomes; humans; microRNA; prognostic.

Figure 1

(A) The identification of serum exosomes by transmission electron microscope. (B) The identification of exosomes specific proteins by western blot.

Figure 2

(A) The expression of miR-223 in serum exosomes of acute ischemic stroke patients was upregulated significantly. The difference of exosomal miR-223 level between stroke and control group were compared with Student’s t-test. Box plots showed the expression of exosomal miR-223 in patients and control. The y-axis indicated miR-223 expression levels by log10 change. ***p < 0.001, stroke patients vs. control. Data are median (25% percentile, 75% percentile), n = 50 in stroke group, n = 33 in control group. (B) Serum exosomal miR-223 expression of stroke patients between different onset time. Exosomal miR-223 levels of different onset times were compared with one-way ANOVA analysis. Box plots showed the expression of exosomal miR-223 in stroke patients with different onset times. The y-axis indicated miR-223 expression levels by log10 change. Data are median (25% percentile, 75% percentile), n = 7 in 24 h subgroup, n = 25 in 48 h subgroup, and n = 18 in 72 h subgroup.

Figure 3

The receiver operating characteristic curve of serum exosomal miR-223 diagnosing ischemic stroke.

Figure 4

Correlation analysis between miR-223 and clinical information. (A) Serum exosomal miR-223 expression of stroke patients between different Trial of Org 10172 in Acute Stroke Treatment (TOAST) subtypes. Exosomal miR-223 level of different TOAST subtypes were compared with one-way ANOVA analysis. The y-axis indicated miR-223 expression levels by log10 change. Box plots showed the expression of exosomal miR-223 in different TOAST subtypes. Data are median (25% percentile, 75% percentile), n = 26 in LA, n = 7 in CE, n = 17 in SA. LA, large artery atherosclerotic stroke; CE, cardioembolism; SA, small artery stroke. (B) The correlation between exosomal miR-223 level and NIHSS score of stroke patients. The correlations between exosomal miR-223 level and NIHSS score was estimated by Pearson’s correlation test (r = 0.31, p = 0.03). The y-axis indicated miR-223 expression levels. NIHSS, National Institutes of Health Stroke Scale. (C) The correlation between exosomal miR-223 level and infarct volume. The correlations between exosomal miR-223 level and infarct volume was estimated by Pearson correlation test (r = 0.20, p = 0.19). The y-axis indicated miR-223 expression levels. (D) The correlation between exosomal miR-223 level and prognosis of stroke patients. The difference of exosomal miR-223 level between good prognosis patients [modified Rankin Scale (mRS) ≤2] and poor prognosis patients (mRS > 2) were compared with Student’s t-test. Box plots showed the expression of exosomal miR-223 in patients and control. The y-axis indicated miR-223 expression levels by log10 change. *p < 0.05, good prognosis patients vs. poor prognosis patients. Data are median (25% percentile, 75% percentile), n = 30 in good prognosis group, n = 20 in poor prognosis group.