Diagnostic and Immunosuppressive Potential of Elevated Mir-424 Levels in Circulating Immune Cells of Ischemic Stroke Patients

Abstract

Our previous study demonstrated that microRNA-424 (miR-424) protected against experimental stroke through inhibition of microglial proliferation and activation by targeting cell cycle proteins. The purpose of this study was to further explore the clinical significance of miR-424 in peripheral immune cells of patients with acute ischemic stroke (AIS). Blood samples were collected from 40 patients within 6 hours of symptom onset and 27 control subjects. MiR-424 levels in lymphocytes, neutrophils and plasma were determined by quantitative realtime-PCR. The diagnostic sensitivity and specificity of miR-424 for stroke was evaluated by receiver operator characteristic (ROC) curve. The correlation between miR-424 levels and clinical data was analyzed using Pearson's correlation test. Plasma levels of inflammatory mediators (TNF-α, IL-10) and neurotrophic factor (IGF-1) were detected by ELISA. Notably, miR-424 expression levels in lymphocytes and neutrophils increased after stroke, suggestive of its diagnostic value in ischemic stroke. MiR-424 levels in neutrophils were negatively correlated with infarct volume. Lymphocytic miR-424 levels were negatively correlated with the number of lymphocytes and the expression of cyclin-dependent kinase CDK6. Moreover, plasma TNF-α and IGF-1 levels increased and decreased, respectively, in stroke patients, and miR-424 levels in lymphocytes and neutrophils were both inversely correlated with plasma TNF-α, IL-10, or IGF-1 levels. In summary, miR-424 levels in peripheral immune cells has diagnostic potential for ischemic stroke, and might affect the severity of acute stroke by depressing the peripheral inflammatory response through CDK6-dependent pathway in lymphocytes or CDK6-independent pathway neutrophils.

Keywords: CDK6; ischemia; lymphocytes; miR-424; neutrophils; stroke.

Figure 1.

Changes of circulating miR-424 levels and its diagnostic value in hyperacute ischemic stroke. Quantitative real-time PCR analysis of the expressions of miR-424 in (A) lymphocytes; (B) neutrophils; and (C) plasma from AIS patients within 6 hours of symptom onset (n=40) and control group (n=27). Data represent mean ± SEM. * p<0.05 compared to control; **p<0.01 compared to control. (D) The diagnostic sensitivity and specificity of miR-424 levels in lymphocytes for AIS patients (n=40) was evaluated by ROC curve. ROC= receiver operator characteristic.

Figure 2.

Correlations between miR-424 levels in circulating blood and cerebral infarct volume or NIHSS score. (A) Correlation between miR-424 levels in lymphocytes, neutrophils, and plasma and cerebral infarct volume within 6 hours of symptom onset in 24 AIS patients at admission; (B) Correlation between miR-424 levels in lymphocytes and neutrophils within 6 hours and NIHSS score in 40 AIS patients at admission; (C) Correlation between miR-424 levels in lymphocytes and neutrophils within 6 hours and NIHSS score in 40 AIS patients at 7 days after therapy. AIS, acute ischemic stroke.

Figure 3.

Correlations between miR-424 levels in circulating immune cells and in plasma. (A) Correlation between plasma miR-424 levels and its levels in lymphocytes from AIS patients. (B) Correlation between plasma miR-424 levels and its levels in neutrophils from AIS patients. AIS, acute ischemic stroke. N=40.

Figure 4.

Correlations between miR-424 levels in lymphocytes and neutrophils, and the number of lymphocytes and neutrophils and CDK6 expression. (A) Correlation between miR-424 levels in lymphocytes and the number of lymphocytes from 40 AIS patients; (B) Correlation between miR-424 and CDK6 levels in lymphocytes from 19 AIS patients; (C) Correlation between miR-424 levels in neutrophils and the number of neutrophils from 40 AIS patients; (D) Correlation between miR-424 and CDK6 levels in neutrophils from 19 AIS patients. AIS, acute ischemic stroke.

Figure 5.

Correlations between changes in plasma TNF-α and IL-10 levels with miR-424 levels in lymphocytes and neutrophils. (A) TNF-α levels in plasma from 33 stroke patients and 24 control volunteers; ***p<0.001 compared to control. (B) Correlation between miR-424 levels in lymphocyte and TNF-α levels in plasma from 33 AIS patients. (C) Correlation between miR-424 levels in neutrophils and TNF-α levels in plasma from 33 AIS patients; (D) IL-10 levels in plasma from 33 stroke patients and 24 control volunteers. (E) Correlation between miR-424 levels in lymphocytes and IL-10 levels in plasma from 33 AIS patients; (F) Correlation between miR-424 levels in neutrophils and IL-10 levels in plasma from 33 AIS patients. Data represent mean±SEM. TNF-α, tumor necrosis factor-alpha; IL-10, interleukin-10. AIS, acute ischemic stroke.

Figure 6.

Correlations between change in plasma IGF1 levels with miR-424 levels in lymphocytes and neutrophils. (A) IGF1 levels in plasma from 33 stroke patients and 24 control volunteers; **p<0.01 compared to control. (B) Correlation between miR-424 levels in lymphocytes and IGF1 levels in plasma from 33 AIS patients; (C) Correlation between miR-424 levels in neutrophils and IGF1 levels in plasma from 33 AIS patients. IGF1(insulin-like growth factor 1),