原因不明复发性自然流产患者外周血pDC、miR-6165水平变化及其调控机制

Changes in levels of pDCs and miR-6165 in peripheral blood of URSA patients and the underlying mechanism

目的观察原因不明复发性自然流产(URSA)患者外周血中浆细胞样树突状细胞(pDC)、微小RNA-6165(miR-6165)水平变化,并探讨二者间的调控机制。方法选取正常早孕妇女(正常妊娠组)和URSA患者(URSA组)各30例,空腹抽取静脉血,分离单个核细胞(PBMC),流式细胞术测定pDC细胞比例,q PCR法检测miR-6165表达水平;采用Target Scan生物信息软件,以Target Scan 7. 1程序预测miR-6165与信号传导及转录激活因子3(STAT3) 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miR-6165(空白对照NC)与野生型或突变型STAT3 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Lipofectamine2000转染miR-6165 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,分别采用q PCR、Western blotting法检测各组细胞STAT3 mRNA、STAT3总蛋白及磷酸化水平(p-STAT3)。结果与正常妊娠组比较,URSA组PBMC中pDC比例降低而miR-6165表达水平升高(P均<0. 05)。Target Scan 7. 1程序预测显示,miR-6165与STAT3 mRNA的3'UTR之间具有潜在的碱基互补结合位点;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STAT3 mRNA 3'UTR荧光素酶报告基因活性(P <0. 05),但对突变型STAT3 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STAT3 mRNA、STAT3与p-STAT3蛋白表达均降低(P均<0. 05),而抑表达组STAT3 mRNA、STAT3与p-STAT3蛋白表达均升高(P均<0. 05)。结论URSA患者外周血PBMC中pDC比例降低,而miR-6165表达水平升高; miR-6165与STAT3结合并抑制STAT3表达和功能,抑制pDC亚群分化,打破母胎免疫耐受状态进而导致URSA。

Objective To investigate the changes of plasmacytoid dendritic cells(pDCs)and microRNA-6165(miR-6165)in the the peripheral blood of patients with unexplained recurrent spontaneous abortion(URSA)and to explore the regulatory mechanism 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.Peripheral blood mononuclear cells(PBMCs)of all subjects were taken from each group.The proportion of pDC was detected by flow cytometry.In addition,the expression of miR-6165 was detected by qPCR.Target Scan 7.1 software was used to predict the binding site between miR-6165 and signal transducer and activator of transcription 3(STAT3);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,which were transfected with miR-6165 mimics(blank negative control)and wide-type or mutant STAT3 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-6165 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,STAT3 mRNA,STAT3 total protein and phosphorylation level(p-STAT3)were detected by qPCR and Western blotting,respectively.Results Compared with the normal pregnancy group,the proportion of pDCs decreased significantly,while the expression of miR-6165 increased significantly in the URSA group(both P<0.05).Target Scan 7.1 and luciferase reporter assay showed that miR-6165 bound with STAT3 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 STAT3 mRNA 3'UTR luciferase reporter gene as compared with the control group(P<0.05),but not the mutant STAT3 mRNA 3'UTR(P>0.05).Compared with the blank control group,the STAT3 mRNA and STAT3 and p-STAT3 protein levels of decreased in the overexpression group,but increased in the inhibition group(both P<0.05).Conclusions The proportion of pDC decreases while the miR-6165 expression increases in the peripheral blood of URSA patients.MiR-6165 inhibits the differentiation of pDC subset by binding and inhibiting STAT3 signaling pathway.Thereby,it breaks the materno-fetal immunotolerance and leads to URSA.