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.
BackgroundAccumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma.ResultsWe compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups.ConclusionThe miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-017-2636-3) contains supplementary material, which is available to authorized users.
The small noncoding RNAs, microRNAs (or miRNAs), have been implicated in a myriad of diseases and accumulating evidence indicate their potential high value as diagnostic biomarkers. Although their roles in hemostasis and coagulation pathways are less defined, many studies have demonstrated their participation in regulating key factors of hemostasis. However, the mounting challenges associated with the accurate measurement of circulating miRNAs and the involvement of platelet activation in contributing to the circulating miRNA expression profile introduce further complexity to the study of thrombosis-associated miRNAs. This review outlines the current knowledge of miRNAs that have been postulated to regulate key hemostatic factors, and miRNA diagnostic panels in thrombotic disease, with a focus on experimental fundamentals, such as selecting condition-specific reference controls, considerations that are crucial for accurate evaluation of miRNAs in the context of disease biomarkers.
Functional nucleic acids, such as microRNAs (miRNAs), have been implicated in the pathophysiology of many diseases. The miRNA expression profiles of various cancers including haematological malignancies are well defined, but the role of miRNAs in haemostasis and the regulation of coagulation is poorly understood. We identified that miR-494 is oestrogen responsive and directly targets the anticoagulant protein, Protein S, as a mechanism for acquiring Protein S deficiency under high oestrogenic conditions such as during pregnancy and oral contraceptive use. Furthermore, previous studies have also characterised miR-494 to be involved in many biological processes. This paper reviews the current knowledge in the role of miRNAs in regulating haemostatic proteins and the known biological functions of miR-494, highlighting miR-494 as an emerging therapeutic target, with an overview of the strategy we have employed in identifying functional nucleic acids such as miRNAs that target haemostatic factors and the therapeutic potential of miR-494-directed therapy for the treatment of thrombotic disorders.
Background: High oestradiol (E2) levels are linked to an increased risk of venous thromboembolism, however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494-3p that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. Objectives: To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterise novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. Methods: Ceveron® Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM on miR expression in HuH-7 cells was compared using NanoString nCounter® and validated with independent assays. The effect of tissue factor interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays and TGA. Results: Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2-down-regulation in two independent miR quantification platforms, and tissue factor mRNA (F3) was up-regulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3’UTR was confirmed and overexpression of miR-365a-3p led to a decrease of 1) tissue factor mRNA transcripts, 2) protein levels, 3) activity and 4) tissue factor-initiated thrombin generation. Conclusion: miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induces a hypercoagulable state via a miR-network specific for coagulation factors.
In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ TM , and Applied Biosystems TM PrepFiler TM DNA extraction protocols. The DNA IQ TM and PrepFiler TM methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlexâ 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ TM method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler TM chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.