Clinical Values and Underlying Mechanism Analysis of Serum miR-455-5p in Carotid Artery Stenosis

Abstract

Purpose: Carotid artery stenosis (CAS) is a leading cause of cerebral infarction, its early diagnosis and intervention are necessary. In light of the important role of microRNAs (miRNAs) in cerebrovascular disease, this study aimed to investigate the expression pattern and clinical significance of serum miR-455-5p in the onset and development of CAS, as well as its underlying mechanism.

Patients and methods: Seventy patients with asymptomatic CAS were recruited, and the development of cerebral ischemia events (CIEs) was recorded during the five-years follow-up. qRT-PCR was performed for the serum miR-455-5p detection. ROC curve was applied for the diagnostic ability evaluation. By constructing multivariable logistic or cox regression model, odds ratio (OR) or hazard ratio (HR) were calculated to assess the impact of each risk factor on independent variables. Human aortic endothelial cells (HAECs) were treated with ox-LDL to induce endothelial cell damage. The role of miR-455-5p in the cell viability, apoptosis, oxidative stress and inflammatory response was detected.

Results: Serum miR-455-5p showed low expression in cases with CAS, and had an independent influence on the degree of CAS. The diagnostic ability of serum miR-455-5p to diagnose CAS was determined via ROC curve, with the AUC of 0.927. During follow-up, patients with low miR-455-5p expression showed high incidence of CIEs. In multivariable cox regression model, degree of CAS and miR-455-5p were significant risk factors for the development of CIEs in the CAS patients. In vitro, miR-455-5p was at a low expression in HAECs cell models and can prevent cells from ox-LDL induced cell apoptosis, oxidative stress and inflammatory response. SOCS3 was a target gene of miR-455-5p and upregulated in ox-LDL treated cells.

Conclusion: Down-regulated expression of serum miR-455-5p is hopeful to be used as a biomarker for the early diagnosis of CAS. MiR-455-5p is an independent risk factor for the degree of CAS, and has a certain predictive value for the development of CIEs. That might be associated with the protective role of miR-455-5p against ox-LDL-induced endothelial cell injury via targeting SOCS3.

Keywords: carotid artery stenosis; cerebral ischemia events; endothelial cell injury; microRNA-455-5p.

Figure 1

Levels of serum miR-455-5p between different groups. (A) Comparison of serum miR-455-5p levels between CAS group and healthy controls (HC). (B) A decreased expression of serum miR-455-5p was detected in cases with hypertension compared with the non-hypertension group, but the difference did not reach a significant level. ***Means P < 0.001 when compared with the HC group.

Figure 2

Clinical values of serum miR-455-5p in CAS and the development of CIEs. (A) Analysis of the clinical diagnostic ability of miR-455-5p in CAS. Serum miR-455-5p showed the diagnostic potential to identify CAS from healthy individuals with the area under the curve (AUC) of 0.927, the diagnostic sensitivity and specificity were 82.9% and 90.8%, respectively. (B) The Kaplan-Meier (K-M) curve that drawn based on the follow-up results. The incidence of CIEs was higher in people carrying low miR-455-5p levels, the Log rank test indicated a significant difference between the low and high miR-455-5p expression group (P = 0.002).

Figure 3

MiR-455-5p overexpression can protect against ox-LDL-induced cell apoptosis. (A) Levels of miR-455-5p decreased gradually with increased ox-LDL concentration. (B) After miR-455-5p mimic transfection, the levels of miR-455-5p were increased. (C) In ox-LDL treated HAECs, miR-455-5p mimic transfection also increased the levels of miR-455-5p. (D) Ox-LDL led to the inhibition of HAECs viability, which was reversed by miR-455-5p overexpression. (E) miR-455-5p overexpression inhibited ox-LDL-induced cell apoptosis. *Means P< 0.05, ***Means P < 0.001 when compared with the HC group; ^&&&Means P < 0.001 when compared with the ox-LDL group.

Figure 4

MiR-455-5p overexpression can reverse the influence of ox-LDL on cell oxidative stress and inflammatory response. (A–C) ox-LDL treatment led to the decreasing of SOD and increasing in ROS and MDA, which was reversed by miR-455-5p overexpression. (D–F) Levels of inflammatory factors were also elevated after ox-LDL treatment, and miR-455-5p played the opposite role by inhibiting the release of sICAM-1, IL-1β, and IL-6. ***Means P < 0.001 when compared with the HC group; ^&&&Means P < 0.001 when compared with the ox-LDL group.

Figure 5

SOCS3 is a target gene of miR-455-5p. (A) The binding sites between miR-455-5p and SOCS3. (B) Luciferase activity of cells in different groups. (C) Levels of SOCS3 in different cell groups. ***Means P < 0.001 when compared with the HC group; ^&&&Means P < 0.001 when compared with the ox-LDL group.