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

ZHENG Xiangzhao; YIN Xunqiang; WANG Dongmei; ZHANG Yunhong; ZHANG Zhen; WEI Ran; ZHU Xiaoxiao; GUO Qiang; ZHOU Xianbin; CHU Chu; ZHAO Lin; LI Xia

doi:10.3969/j.issn.1002-266X.2019.02.005

Abstract：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 q PCR. 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 293 T 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 pmir GLO vector, respectively. Luciferase reporter gene activity was detected by using dual luciferase reporter gene 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-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 q PCR 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.