title: Micro-ribonucleic Acid 494 regulation of protein S expression.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Hematology, PathWest Laboratory Medicine, Royal Perth Hospital, Perth, WA, Australia.
authors.forename: J W
authors.initials: JW
authors.lastname: Tay
authors.forename: G
authors.initials: G
authors.lastname: Romeo
authors.forename: Q W
authors.initials: QW
authors.lastname: Hughes
authors.forename: R I
authors.initials: RI
authors.lastname: Baker
mesh_terms: D020413:3' Untranslated Regions; D001665:Binding Sites; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000737:chemistry; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004967:Estrogens / Q000737:chemistry; D005786:Gene Expression Regulation; D006487:Hemostasis; D006801:Humans; D008099:Liver / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D017293:Protein S / Q000235:genetics / Q000378:metabolism*; D018455:Protein S Deficiency / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D020413:3' Untranslated Regions; D017077:Culture Media, Conditioned; D004967:Estrogens; C547168:MIRN494 microRNA, human; D035683:MicroRNAs; D017293:Protein S
keywords: estrogen; human; miR-494; miRNA; microRNA; protein S