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.

Objective To investigate the changes in levels of microRNA-374 a-5 p(miR-374 a-5 p) 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-374 a-5 p was detected by q PCR. Target Scan 7.1 software was used to predict the binding site between miR-374 a-5 p and IL-10; 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, and cells in the intervention group were transfected with miR-374 a-5 p mimics and wide-type or mutant IL-10 mRNA 3’UTR fluorescent reporter gene pmir GLO vector, respectively. Luciferase reporter gene activity was detected by using dual luciferase reportergene 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-374 a-5 p 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 q PCR, 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-374 a-5 p 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-374 a-5 p 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 mRNA 3’UTR did not change significantly(P> 0.05). Compared with the blank control group, IL-10 mRNA expression and IL-10 protein decreased in the overexpression group(both P < 0.05), while increased in the inhibition group(both P < 0.05). Conclusions Serum IL-10 decreases while miR-374 a-5 p increases in the PBMCs of DVT patients. The miR-374 a-5 p inhibits the expression of IL-10 by binding to IL-10 3’UTR. Thereby, the mediated inflammation is involved in DVT. The miR-374 a-5 p may become a novel therapeutic target for DVT by regulating IL-10. [ABSTRACT FROM AUTHOR]