Micro‐Ribonucleic Acid 494 regulation of protein S expression

Summary Background Acquired protein S (PS) deficiency is highly associated with elevated circulating estrogen levels resulting from pregnancy, oral contraceptives, and estrogen replacement therapy; however, the mechanism of estrogen‐mediated acquired PS deficiency remains poorly understood. Increasing evidence indicates that estrogen receptor signaling can indirectly modulate the expression of target genes at the post‐transcriptional level by modulating the expression of microRNAs (miRNAs), and miRNAs have also been demonstrated to be involved in the regulation of hemostasis. Objectives To investigate the mechanism of estrogen‐mediated downregulation of PROS1 expression by the microRNA miR‐494. Methods Computational analyses of the PROS1 3′‐untranslated region (UTR) were performed to identify putative miRNA‐binding sites, and direct targeting of the PROS1 3′‐UTR by miR‐494 was determined with dual luciferase reporter assays in HuH‐7 cells. Reporter vectors containing the PROS1 3′‐UTR sequence with deleted miR‐494‐binding sites were also analyzed with luciferase reporter assays. The effects of estrogen on miR‐494 and PROS1 mRNA levels in HuH‐7 cells were determined by quantitative real‐time PCR, and estrogen‐mediated changes to secreted PS levels in culture supernatant of HuH‐7 cells were measured with an ELISA. Results The PROS1 3′‐UTR sequence contains three putative miR‐494‐binding sites. miR‐494 directly targets PROS1, and miR‐494 levels are upregulated following estrogen treatment in HuH‐7 liver cells in association with downregulated PROS1 mRNA and PS levels. Conclusions The results from this study provide the first evidence for miRNA downregulation of PROS1 by miR‐494, and suggest that miR‐494 is involved in the mechanism of estrogen‐mediated downregulation of PS expression.