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Keywords:

  • estrogen;
  • human;
  • microRNA;
  • miR-494;
  • miRNA;
  • protein S

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.