Li Zi, Ni Jiangdong
Department of Orthopedics, Xiangya Second Hospital of Central South University, Changsha, Hunan 410011, P.R. China.
Exp Ther Med. 2017 Nov;14(5):5069-5074. doi: 10.3892/etm.2017.5183. Epub 2017 Sep 22.
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.
本研究旨在探讨微小RNA(miR)-26a在深静脉血栓形成(DVT)中的作用及作用机制。收集45例DVT患者和40例健康对照者的外周血。采用逆转录-定量聚合酶链反应检测miR-26a、趋化因子C-C基序配体(CCL)2 mRNA和CCL7 mRNA的水平,并采用受试者工作特征曲线分析评估miR-26a在DVT临床诊断中的价值。采用Spearman等级相关分析miR-26a与CCL2和CCL7水平的相关性。此外,在人脐静脉内皮细胞(HUVECs)中过表达miR-26a和蛋白激酶Cδ(PRKCD),并通过小干扰(si)RNA敲低PRKCD表达。进行蛋白质印迹法检测PRKCD和p65的表达。此外,进行双荧光素酶报告基因测定。本研究结果表明,与健康对照相比,DVT患者外周血中miR-26a的表达显著下调(P<0.05),且与CCL2和CCL7水平呈负相关(P<0.05)。此外,结果表明miR-26a显著抑制PRKCD的表达,显著降低CCL2和CCL7 mRNA水平(P<0.05),并抑制NF-κB信号通路的激活。在HUVECs中过表达PRKCD可抑制miR-26a的作用,并显著上调p65的磷酸化水平。本研究表明,miR-26a直接靶向PRKCD mRNA,且miR-26a可能是DVT临床诊断中的一种有用生物标志物。因此,本研究结果提示,miR-26a通过与PRKCD mRNA结合来调节NF-κB信号通路,抑制CCL2和CCL7的表达,并降低DVT的风险。