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

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

doi:10.3969/j.issn.1002-266X.2019.02.006

Abstract：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 q PCR.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 293 T cells were divided into the intervention group and control group, and cells were transfected with let-7d-5p mimics ( blank negative control) and widetype or mutant IL-6 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 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 q PCR, 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 mRNA3'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.