Diagnostic value of miR-186-5p for carotid artery stenosis and its predictive significance for future cerebral ischemic event

From January 2013 to May 2014, 67 cases diagnosed with asymptomatic CAS were enrolled, and another 60 healthy individuals were recruited as the control group. All study subjects gave written informed consents following the Declaration of Helsinki. The study protocols were approved by the Ethics Committee of Binzhou Medical University Hospital. All cases performed carotid artery color Doppler ultrasonography (CDUS), and the degree of CAS was determined according to the measurement results of computed tomography angiography (CTA). Patients who had conditions that can lead to stroke or CIE were excluded, including atrial fibrillation, myocardial infarction, cardiomyopathy, severe pulmonary disease, and arrhythmia. CAS refers to a > 50% stenosis of the extracranial internal carotid artery (ICA), and the stenosis severity was estimated according to the North American Symptomatic Carotid Endarterectomy Trial method [15]. An asymptomatic status was confirmed by a review of the patient’s history, a physical examination, and the numeric National Institutes of Health Stroke Scale [16]. The asymptomatic CAS patients had no history of stroke or transient ischemic attack, carotid revascularization, prior myocardial infarction, valvular heart disease, cardiomyopathy, severe pulmonary disease, arrhythmia. Among 67 cases with asymptomatic CAS, 36 cases had hypertension, 35 cases had diabetes and 38 cases had hypercholesterolemia. Hypertension was defined as the previous use of antihypertensive medications, a systolic pressure more than 140 mmHg or a diastolic pressure more than 90 mmHg for at least two separate measurements. Hypercholesterolemia was defined as total cholesterol at least 200 mg/dl. Diabetes was defined as a fasting blood glucose level of 126 mg/dL and above or current usage of oral antidiabetic drugs or insulin. Each control underwent carotid ultrasound to exclude critical CAS. Only individuals with normal Doppler ultrasound or evidence of carotid stenosis < 20% were included in the control group. All healthy controls had no history of diabetes, hypertension, hyperlipidemia, inflammatory diseases, cardiovascular diseases (CVDs), autoimmune disease and cancer.

Fasting blood samples were drawn from each individual, and serum samples were collected and stored at − 80 °C after centrifugation. The demographic and clinical information were recorded. Additionally,, all CAS patients were followed-up for 5 years to recorded the occurrence of CIEs, including strokes, transient ischemic attack (TIA) or sudden death. After the CIE occurs, the follow-up was ended.

Firstly, total RNA was extracted from the serum of each subject by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the serum samples were homogenized in Trizol regnant, then chloroform was added to the 1/5 volume of Trizol and mixed thoroughly. The mixture was centrifuged at 12,000 g (4 °C) for 15 min. The supernatant was collected and mixed with isopropanol at a ratio of 1:1 and incubation for 10 min and was then centrifuged at 14,000 g (4 °C) for 10 min. The supernatant was removed and 100 μl 75% ethanol was added to the tubes and mixed thoroughly. After centrifugation at 8000 g (4 °C) for 5 min, the supernatant was removed and the tubes were air-dried. Then total RNA was reverse transcribed into complementary DNA by using a PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). Then qRT-PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems, USA) by using a SYBR green I Master Mix Kit (Invitrogen, Carlsbad, CA, USA). And relative levels of serum miR-186-5p were normalized by U6 by using 2^−ΔΔCt method.

As shown in Table 1, the clinical characteristics of the study subjects were recorded. It was shown that 60 healthy controls and 67 asymptomatic CAS patients were collected, with the mean age of 62.62 ± 9.66 and 61.57 ± 10.14 years old, respectively. No significant difference was detected for age, gender, BMI (body mass index), total cholesterol, HDL (high density lipoprotein), LDL (low density lipoprotein), triglycerides, fasting glucose, and SBP (systolic blood pressure) between the CAS patients and control groups (P > 0.05). But DBP (diastolic blood pressure) level was significantly higher in CAS patients group compared with the control group (P < 0.05).

According to the results of qRT-PCR, the serum expression levels of miR-186-5p were compared between healthy individuals and asymptomatic CAS patients. It was found that the serum level of miR-186-5p was increased significantly in asymptomatic CAS patients (Fig. 1, P < 0.001).

According to the mean value (1.423) of serum levels of miR-186-5p, all asymptomatic CAS patients were divided into low expression group (n = 31) and high expression group (n = 36). And the demographic, clinical, and biochemical properties were compared between the two groups. It was found that patients in the high miR-186-5p expression group had high levels of total cholesterol and a high degree of carotid stenosis compared with patients in low miR-186-5p expression group, and a larger proportion of cases suffered from hypertension were found in the high miR-186-5p expression group (Table 2). But for age, gender, BMI, HDL, LDL, triglycerides and fasting glucose, there was no significant difference between the two groups. These results revealed that miR-186-5p might be associated with dyslipidemia, hypertension, and degree of carotid stenosis in CAS patients.

The association of different variables with the degree of carotid stenosis was evaluated by using logistic regression analysis. It was observed that miR-186-5p (OR = 4.250, 95%CI = 1.200–15.057, P = 0.025) and total cholesterol levels (OR = 3.570, 95%CI = 1.083–11.766, P = 0.036) were the most significant factors associated with the high degree of carotid stenosis in CAS patients (Table 3).

According to the serum levels of miR-186-5p in both asymptomatic CAS patients and healthy controls, the ROC curve was constructed to evaluate the diagnostic value of miR-186-5p for asymptomatic CAS. In the ROC curve analysis, the AUC (area under the curve) was 0.919, with the sensitivity of 89.6% and specificity of 81.7% at the cutoff value of 1.221 (Fig. 2). The data indicated that serum miR-186-5p could distinguish asymptomatic CAS patients from healthy controls.

According to the 5-year follow-up data, the Kaplan-Meier method was applied for the evaluation of the predictive value of miR-186-5p for the occurrence of CIE in asymptomatic CAS patients. All patients completed the fiver year follow-up, and among 67 asymptomatic CAS patients, 24 cases underwent CIE, including 19 TIAs and 5 strokes. Among 24 CIE patients, 18 cases were at high miR-186-5p expression and 6 were at low miR-186-5p expression. Kaplan-Meier method results revealed that high miR-186-5p level was associated with the occurrence of CIEs (Fig. 3). Multivariate Cox regression analysis was performed for all the variables measured in the study, including age, gender, BMI, dyslipidemia, diabetes, hypertension, degree of carotid stenosis and serum miR-186-5p expression. The results suggested that high miR-186-5p level (HR: 4.190, 95% CI: 1.166–15.061, P = 0.028) and high degree of carotid stenosis (HR: 3.143, 95% CI: 1.117–8.845, P = 0.030) were independent factors for the occurrence of CIEs in asymptomatic CAS patients (Table 4).