深静脉血栓形成患者外周血miR-374a-5p、IL-10水平变化及其调控关系

摘要

目的 观察深静脉血栓形成(DVT) 患者外周血微小RNA-374a-5p(miR-374a-5p) 、白细胞介素10(IL-10) 水平变化, 并分析二者在DVT发病中的关系.方法 收集DVT患者(DVT组) 和健康对照(健康对照组) 各30例, 抽取受试者空腹静脉血, 分离血清及单个核细胞(PBMC) , ELISA法检测血清IL-10蛋白, q PCR法检测PBMC中的miR-374a-5p.通过对差异基因的生物信息学分析, 用Target Scan 7.1程序预测miR-374a-5p与IL-10 mRNA 3'非翻译区(3'UTR) 是否存在结合位点;将293T细胞分为干预组和对照组, 干预组同时转染空白对照NC与野生型或突变型IL-10 3'UTR荧光素酶报告基因pmir GLO载体, 采用双荧光素酶报告基因系统检测荧光素酶报告基因活性.将He La细胞分为3组, 过表达组、抑表达组利用Lipofectamine 2000转染miR-374a-5p过表达及抑表达基因序列, 空白对照组转染阴性对照引物;转染24 h后收集细胞, q PCR检测各组细胞IL-10 mRNA水平, ELISA检测各组培养上清IL-10蛋白水平.结果 与健康对照组比较, DVT组血清IL-10蛋白水平降低而PBMC中miR-374a-5p表达升高(P均<0.05) .Target Scan 7.1程序预测显示, miR-374a-5p与IL-10 mRNA 3'UTR之间具有潜在碱基互补结合位点;双荧光素酶报告基因系统检测显示, 与对照组比较, 干预组能降低野生型IL-10 mRNA 3'UTR荧光素酶报告基因活性, 但对突变型IL-10 mRNA 3'UTR荧光素酶报告基因活性无明显影响(P> 0.05) .与空白对照组比较, 过表达组He La细胞IL-10 mRNA表达及上清液IL-10蛋白水平均降低(P均<0.05) , 抑表达组He La细胞IL-10 mRNA表达及上清液IL-10蛋白水平均升高(P均<0.05) .结论 DVT患者血清IL-10蛋白水平降低而PBMC中miR-374a-5p表达升高, miR-374a-5p通过碱基互补结合IL-10 mRNA 3'UTR抑制IL-10表达, 进而介导的炎症反应参与DVT形成, 靶向miR-374a-5p调控细胞因子表达可能成为DVT治疗的有效靶点与途径.%Objective To investigate the changes in levels of microRNA-374 a-5 p (miR-374 a-5 p) and IL-10 in the peripheral blood of patients with deep venous thrombosis (DVT) and to explore the relationship between them. Methods Thirty cases of DVT patients (DVT group) and 30 cases of healthy people (healthy control group) were selected. The serum and peripheral blood mononuclear cells (PBMCs) from the fasting venous blood of all subjects were isolated. The expression of IL-10 in serum was detected by ELISA and miR-374 a-5 p was detected by q PCR. Target Scan 7.1 software was used to predict the binding site between miR-374 a-5 p and IL-10; besides, 3'UTR luciferase reporter assay was used to confirm the binding between them. The 293 T cells were divided into the intervention group and control group, and cells in the intervention group were transfected with miR-374 a-5 p mimics and wide-type or mutant IL-10 mRNA 3'UTR fluorescent reporter gene pmir GLO vector, respectively. Luciferase reporter gene activity was detected by using dual luciferase reportergene system. In addition, 293 T cells were divided into 3 groups. The cells in the overexpression group and the inhibition group were transfected with miR-374 a-5 p mimics and inhibitor by using Lipofectamine 2000, respectively, and the cells in the blank control group were transfected with negative control primers. The cells were harvested 24 h after transfection.Then, the expression of IL-10 mRNA was detected by q PCR, and the protein level of IL-10 was detected by ELISA. Results Compared with the healthy control group, the serum IL-10 protein level decreased while the miR-374 a-5 p expression in the PBMCs increased significantly in the DVT group (both P < 0.05). Target Scan 7.1 and luciferase reporter assay showed that miR-374 a-5 p bound to IL-10 3'UTR via the complementary bases. The dual luciferase reporter gene system assay showed that the activity of the wild-type IL-10 mRNA 3'UTR luciferase reporter gene decreased in the intervention group as compared with that of the control group (P < 0.05) , but the mutant IL-10 mRNA 3'UTR did not change significantly (P> 0.05). Compared with the blank control group, IL-10 mRNA expression and IL-10 protein decreased in the overexpression group (both P < 0.05) , while increased in the inhibition group (both P < 0.05). Conclusions Serum IL-10 decreases while miR-374 a-5 p increases in the PBMCs of DVT patients. The miR-374 a-5 p inhibits the expression of IL-10 by binding to IL-10 3'UTR. Thereby, the mediated inflammation is involved in DVT. The miR-374 a-5 p may become a novel therapeutic target for DVT by regulating IL-10.