Role of microRNA-26a in the diagnosis of lower extremity deep vein thrombosis in patients with bone trauma

Abstract

The present study aimed to investigate the role and mechanism of action of microRNA (miR)-26a in deep vein thrombosis (DVT). Peripheral blood was collected from 45 patients with DVT and 40 healthy controls. Levels of miR-26a, chemokine C-C motif ligand (CCL)2 mRNA and CCL7 mRNA were detected using reverse transcription-quantitative polymerase chain reaction and the value of miR-26a in the clinical diagnosis of DVT was assessed using receiver operating characteristic curve analysis. The correlation of miR-26a with CCL2 and CCL7 levels was analyzed using Spearman's rank correlation. In addition, miR-26a and protein kinase C δ (PRKCD) were overexpressed in human umbilical vein endothelial cells (HUVECs) and PRKCD expression was knocked down by small interfering (si)RNA. Western blotting was conducted to detect the expression of PRKCD and p65. Furthermore, a dual-luciferase reporter gene assay was performed. The results of the current study demonstrated that the expression of miR-26a was significantly downregulated in the peripheral blood of patients with DVT compared with healthy controls (P<0.05) and negatively correlated with CCL2 and CCL7 levels (P<0.05). Furthermore, it was demonstrated that miR-26a markedly inhibited the expression of PRKCD, significantly decreased levels of CCL2 and CCL7 mRNA (P<0.05) and inhibited activation of the NF-κB signaling pathway. Overexpression of PRKCD in HUVECs inhibited the effects of miR-26a and markedly upregulated the phosphorylation of p65. The present study indicated that miR-26a directly targets PRKCD mRNA and that miR-26a may be a useful biomarker in the clinical diagnosis of DVT. Thus, the present findings suggest that miR-26a regulates the NF-κB signaling pathway by binding to PRKCD mRNA, inhibits the expression of CCL2 and CCL7 and reduces the risk of DVT.

Keywords: clinical diagnosis; deep vein thrombosis; microRNA.

Figure 1.

Levels of miR-26 in the peripheral blood of patients with DVT. The expression of miR-26 was detected by reverse transcription-quantitative polymerase chain reaction. (A) Levels of miR-26 in the peripheral blood of patients with DVT compared with normal controls. (B) Levels of miR-26 in the peripheral blood of patients with DVT complicated by PTE, patients with DVT alone and normal controls. *P<0.05 vs. normal group; ^#P<0.05 vs. DVT group. (C) Receiver operating characteristic curve indicated the value of miR-26a in the clinical diagnosis of DVT. miR-26, microRNA-26; DVT, deep vein thrombosis; PTE, pulmonary thromboembolism.

Figure 2.

Levels of CCL2 and CCL7 mRNA and their correlation with miR-26a. (A) Levels of CCL2 and CCL7 mRNA in the peripheral blood of patients with DVT and normal individuals were measured using reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. corresponding normal group. (B) Correlation of CCL-2 mRNA with miR-26. (C) Correlation of CCL-7 mRNA with miR-26. DVT, deep vein thrombosis; miR-26, microRNA-26; CCL, chemokine C-C motif ligand 2.

Figure 3.

miR-26a regulates the expression of PRKCD, CCL2, CCL7 and p65. (A) Effect of miR-26a on the expression of PRKCD and p-p65 was assessed using western blotting. GAPDH was used as an internal control. (B) The effect of miR-26a on the levels of CCL2 and CCL7 mRNA was detected by RT-qPCR. (C) Fluorescence was detected following transfection with PRKCD expression vector. Magnification, ×400. (D) Effect of PRKCD overexpression on the phosphorylation of p65 was detected by western blotting. (E) Effect of PPKCD knockdown on the expression of p65 and p-p65 was assessed by western blotting. GAPDH was used as an internal control. (F) Effect of PRKCD knockdown on the levels of CCL2 and CCL7 mRNA was detected by RT-par. *P<0.05 vs. NC. NC, negative control; p-p65, phosphorylated p65; miR-26, microRNA-26; CCL, chemokine C-C motif ligand 2; PRKCD, protein kinase C δ; GFP, green fluorescent protein; siR, small interfering RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 4.

Identification of the miR-26a target gene using a dual-luciferase reporter gene assay. (A) Wild-type and mutant PRKCD sequences. (B) Fluorescence intensity changed when miR-26a mimic cells were co-transfected with pMIR-REPORT-wild-type or pMIR-REPORT-mutant type. *P<0.05 vs. NC. PRKCD, protein kinase C δ; NC, negative control.