原因不明复发性自然流产患者外周血IL-6、let-7d-5p水平变化及其关系

Changes in levels of IL-6 and let-7d-5p in peripheral blood of URSA patients and the relationships between them

目的观察原因不明复发性自然流产(URSA)患者外周血白细胞介素6(IL-6)、微小核糖核酸(miRNA)let-7d-5p水平变化,探讨二者在URSA发病中的关系。方法选取正常早孕妇女(正常妊娠组)和URSA患者(URSA组)各30例,空腹抽取其外周静脉血,ELISA法检测血清IL-6蛋白,q PCR法检测单个核细胞(PBMC)中的let-7d-5p。采用Target Scan生物信息软件,以Target Scan 7. 1程序预测let-7d-5p与IL-6 mRNA的3'非翻译区(3'UTR)是否存在潜在结合位点;将293T细胞分为干预组和对照组,分别同时转染let-7d-5p(空白对照NC)与野生型或突变型IL-6 mRNA 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293T细胞分为3组,过表达组、抑表达组利用Lipofectamine 2000转染let-7d-5p mimics及inhibitor,空白对照组转染阴性对照引物;转染24 h后收集细胞,q PCR检测各组细胞中的IL-6 mRNA,ELISA检测各组培养上清液中的IL-6。结果与正常妊娠组比较,URSA组外周血IL-6蛋白水平升高,而PBMC中let-7d-5p表达降低(P均<0. 05)。Target Scan 7. 1程序预测显示,let-7d-5p与IL-6 mRNA的3'UTR之间具有潜在的碱基互补结合位点;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-6 mRNA 3'UTR荧光素酶报告基因活性(P <0. 05),但对突变型IL-6 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,过表达组细胞中IL-6 mRNA表达及细胞上清液中IL-6水平降低(P均<0. 05),抑表达组细胞中IL-6 mRNA表达及细胞上清液中IL-6水平升高(P均<0. 05)。结论 URSA患者血清IL-6蛋白水平升高,而PBMC中let-7d-5p表达降低; let-7d-5p与IL-6 mRNA 3'UTR结合并抑制IL-6表达,靶向let-7d-5p调控IL-6表达可能成为URSA治疗的有效途径。

Objective To investigate the changes of IL-6 and microRNA(miRNA)let-7d-5p in the peripheral blood of patients with unexplained recurrent spontaneous abortion(URSA)and to explore the relationships between them.Methods Thirty cases of normal pregnant women(normal pregnancy group)and 30 cases of URSA patients(URSA group)were included in this study.The serum and peripheral blood mononuclear cells(PBMCs)of all subjects were taken from each group.The protein level of IL-6 in plasma was detected by ELISA,and let-7d-5p expression was detected by qPCR.Target Scan 7.1 software was used to predict the binding site between let-7d-5p and IL-6;besides,3'untranslated region(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 were transfected with let-7d-5p mimics(blank negative control)and wide-type or mutant IL-6 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 leto-7d-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-6 mRNA was detected by qPCR,and the protein level of IL-6 was detected by ELISA.Results Compared with the normal pregnancy group,the serum IL-6 protein level increased significantly while the let-7d-5 expression in the PBMCs decreased significantly in the URSA group(both P<0.05).Target Scan 7.1 and luciferase reporter assay showed that let-7d-5p bound with IL-6 3'UTR via the complementary bases.The dual luciferase reporter gene system assay showed that the intervention group reduced the activity of the wild-type IL-6 mRNA 3'UTR luciferase reporter gene as compared with the control group(P<0.05),but not the mutant IL-6 mRNA 3′UTR(P>0.05).Compared with the blank control group,IL-6 mRNA expression and IL-6 protein level decreased in the overexpression group,but increased in the inhibition group(both P<0.05).Conclusions Serum IL-6 increases while let-7d-5p decreases in the PBMCs of URSA patients.Let-7d-5p inhibits the expression of IL-6 by binding IL-6 3'UTR.Thereby,it breaks the materno-fetal immunotolerance and leads to URSA.Let-7d-5p may become a novel therapeutic target for URSA by regulating IL-6.