Oestradiol-responsive miR-365a-3p interacts with tissue factor 3'-UTR to modulate tissue factor-initiated thrombin generation.

Background High oestradiol (E2) levels are linked to an increased risk of venous thromboembolism, however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494-3p that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. Objectives To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterise novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. Methods Ceveron® Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM on miR expression in HuH-7 cells was compared using NanoString nCounter® and validated with independent assays. The effect of tissue factor interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays and TGA. Results Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2-down-regulation in two independent miR quantification platforms, and tissue factor mRNA (F3) was up-regulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3'UTR was confirmed and overexpression of miR-365a-3p led to a decrease of 1) tissue factor mRNA transcripts, 2) protein levels, 3) activity and 4) tissue factor-initiated thrombin generation. Conclusion miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induces a hypercoagulable state via a miR-network specific for coagulation factors.