Micro-Ribonucleic Acid 494 regulation of protein S expression

Tay, J.W., Romeo, G., Hughes, Q.W. and Baker, R.I. (2013) Micro-Ribonucleic Acid 494 regulation of protein S expression. Journal of Thrombosis and Haemostasis, 11 (8). pp. 1547-1555.

Abstract

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.

Item Type:	Journal Article
Murdoch Affiliation(s):	Centre for Haemophilia and Thrombosis Research
Publisher:	Wiley-Blackwell
Copyright:	© 2013 International Society on Thrombosis and Haemostasis
URI:	http://researchrepository.murdoch.edu.au/id/eprint/17661

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