Characterisation of oestrogen-responsive microRNAs regulating coagulation factors

Jiayin Tian

Oestradiol (E2) regulation of microRNAs (miRNAs) is well characterised in breast cancer, but poorly understood in regulating coagulation. We previously reported that miR-494 directly downregulates Protein S gene (PROS1) expression in E2-treated human liver carcinoma cells (HuH-7) (Tay et al. 2013). Subsequent miRNA array analyses (NanoString nCounter®) identified numerous E2-responsive miRNAs in HuH-7 cells, which may be involved in regulating thrombotic factors. Therefore, this study sought to validate the E2 responsiveness of the candidate miRNAs and investigate their direct effects on coagulation gene targets. Twelve E2-responsive miRNAs were selected for validation and their potential coagulation gene targets predicted using four computational tools. HuH-7 cells were cultured -/+ 10nM E2 for 12h followed by RT-qPCR quantitation of E2-mediated expression of the twelve selected miRNAs and predicted gene targets. The direct interaction between validated miRNAs and their common coagulation gene targets were measured via dual-luciferase reporter assays. In concordance with NanoString nCounter® results, expression of let-7f-5p, miR-26b-5p, miR-128-3p, miR-365a-3p, miR-455-3p and miR-548aa were significantly downregulated following E2 treatment (p<0.05). An associated increase in common predicted targets Tissue Factor (F3) and Factor VIII (F8) mRNA levels was also observed. Furthermore, miR-365a-3p was identified to have a direct binding site on F3-3’UTR. MiR- 365a-3p was down-regulated by E2 and it could directly bind to F3-3’UTR, suggesting downregulation of miR-365a-3p may lead to up-regulation of Tissue Factor to promote thrombosis in high circulating E2 levels during pregnancy or contraceptive pill use.

Characterisation of oestrogen-responsive microRNAs regulating coagulation factors

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