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  

Changes in levels of miR-374a-5p and IL-10 in peripheral blood of DVT patients and the relationship between them

作　　者：刘婧 张云虹 赵霖 朱肖肖 张振[2] 魏然[2] 郭强[2] 尹训强 周宪宾[2] 王彬[4] 李霞[2] LIU Jing;ZHANG Yunhong;ZHAO Lin;ZHU Xiaoxiao;ZHANG Zhen;WEI Ran;GUO Qiang;YIN Xunqiang;ZHOU Xianbin;WANG Bin;LI Xia(University of Jinan,Shandong Academy of Medical Sciences,Jinan 250200,China)

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院,济南250200 [2]山东省医学科学院基础医学研究所 [3]临沂市兰山区妇幼保健院 [4]山东中医药大学附属医院

出　　处：《山东医药》2019年第5期13-16,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81673981;81873337);中国博士后特别资助项目(2017T100513);山东省自然科学基金项目(ZR2018PH042;ZR2013HQ038);山东省重点研发项目(2017GSF19118;2017G006018);山东省中医药科技发展计划(2015-112;2013-217)

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-374a-5p(miR-374a-5p)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-374a-5p was detected by qPCR.Target Scan 7.1 software was used to predict the binding site between miR-374a-5p and IL-10;besides,3′UTR luciferase reporter assay was used to confirm the binding between them.The 293T cells were divided into the intervention group and control group,and cells in the intervention group were transfected with miR-374a-5p mimics and wide-type or mutant IL-10 mRNA 3′UTR fluorescent reporter gene pmirGLO vector,respectively.Luciferase reporter gene activity was detected by using dual luciferase reporter gene system.In addition,293T cells were divided into 3 groups.The cells in the overexpression group and the inhibition group were transfected with miR-374a-5p 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 qPCR,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-374a-5p 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-374a-5p 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 m

关 键 词：深静脉血栓 白细胞介素10 微小核糖核酸374a-5p 表观遗传 炎症反应 

分 类 号：R392[医药卫生—免疫学]