Estradiol-Responsive miR-365a-3p Interacts with Tissue Factor 3′UTR to Modulate Tissue Factor-Initiated Thrombin GenerationFunding This study was supported by Murdoch University Small Grants and Perth Blood Institute.
Background High estradiol (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 characterize 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 downregulation in two independent miR quantification platforms, and tissue factor protein was upregulated 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 induce a hypercoagulable state via a miR network specific for coagulation factors.
J.T.: Drafted and edited the manuscript, performed the experiments, and analyzed the data. M.J.A.: Conceptualized, evaluated the data, edited and revised the manuscript. J.W.T.T.: Conceptualized, drafted the method sections of the manuscript, performed the experiments, and analyzed the data. I.J.: Performed statistical analyses on NanoString nCounter data and evaluated the data. S.P.: Performed the experiments and evaluated the data. Q.W.H.: Conceptualized, evaluated the data, and edited the manuscript. G.G.: Performed the experiments and evaluated the data. R.I.B.: Conceptualized, evaluated the data, and edited the manuscript. J.Y-H.T.: Conceptualized the experiments, evaluated the data, edited and revised the manuscript.
* Current affiliation: PathWest Laboratory Medicine, WA, Australia.
Eingereicht: 14. Juli 2020
Angenommen: 02. Februar 2021
04. Februar 2021 (online)
© 2021. Thieme. All rights reserved.
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
- 1 Parker MG. Transcriptional activation by oestrogen receptors. Biochem Soc Symp 1998; 63: 45-50
- 2 Bleker SM, Coppens M, Middeldorp S. Sex, thrombosis and inherited thrombophilia. Blood Rev 2014; 28 (03) 123-133
- 3 Gialeraki A, Valsami S, Pittaras T, Panayiotakopoulos G, Politou M. Oral contraceptives and HRT risk of thrombosis. Clin Appl Thromb Hemost 2018; 24 (02) 217-225
- 4 Beyer-Westendorf J, Bauersachs R, Hach-Wunderle V, Zotz RB, Rott H. Sex hormones and venous thromboembolism - from contraception to hormone replacement therapy. Vasa 2018; 47 (06) 441-450
- 5 Australian Institute of Health Welfare. Maternal deaths in Australia 2016. Canberra: AIHW; 2018. . Accessed February 19, 2021 at: https://www.aihw.gov.au/getmedia/558ae883-a888-406a-b48f-71f562db3918/aihw-per-99-printable-PDF-of-web-report.pdf.aspx
- 6 Abe K, Kuklina EV, Hooper WC, Callaghan WM. Venous thromboembolism as a cause of severe maternal morbidity and mortality in the United States. Semin Perinatol 2019; 43 (04) 200-204
- 7 Klinge CM. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 2001; 29 (14) 2905-2919
- 8 Di Bitondo R, Hall AJ, Peake IR, Iacoviello L, Winship PR. Oestrogenic repression of human coagulation factor VII expression mediated through an oestrogen response element sequence motif in the promoter region. Hum Mol Genet 2002; 11 (07) 723-731
- 9 Adams B, Western AK, Winship PR. Identification and functional characterization of a polymorphic oestrogen response element in the human coagulation factor IX gene promoter. Br J Haematol 2008; 140 (02) 241-249
- 10 Farsetti A, Narducci M, Moretti F. et al. Inhibition of ERalpha-mediated trans-activation of human coagulation factor XII gene by heteromeric transcription factor NF-Y. Endocrinology 2001; 142 (08) 3380-3388
- 11 Ali HO, Stavik B, Myklebust CF. et al. Oestrogens downregulate tissue factor pathway inhibitor through oestrogen response elements in the 5′-flanking region. PLoS One 2016; 11 (03) e0152114
- 12 Castellano L, Giamas G, Jacob J. et al. The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response. Proc Natl Acad Sci U S A 2009; 106 (37) 15732-15737
- 13 Jia J, Zhou H, Zeng X, Feng S. Estrogen stimulates osteoprotegerin expression via the suppression of miR-145 expression in MG-63 cells. Mol Med Rep 2017; 15 (04) 1539-1546
- 14 Bartel DP. Metazoan microRNAs. Cell 2018; 173 (01) 20-51
- 15 Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75 (05) 855-862
- 16 Ma YS, Lv ZW, Yu F. et al. MicroRNA-302a/d inhibits the self-renewal capability and cell cycle entry of liver cancer stem cells by targeting the E2F7/AKT axis. J Exp Clin Cancer Res 2018; 37 (01) 252
- 17 Li T, Li M, Xu C. et al. miR–146a regulates the function of Th17 cell differentiation to modulate cervical cancer cell growth and apoptosis through NF–κB signaling by targeting TRAF6. Oncol Rep 2019; 41 (05) 2897-2908
- 18 Tay J, Tiao J, Hughes Q, Jorritsma J, Gilmore G, Baker R. Circulating microRNA as thrombosis sentinels: caveats and considerations. Semin Thromb Hemost 2018; 44 (03) 206-215
- 19 Li NX, Sun JW, Yu LM. Evaluation of the circulating MicroRNA-495 and Stat3 as prognostic and predictive biomarkers for lower extremity deep venous thrombosis. J Cell Biochem 2018; 119 (07) 5262-5273
- 20 Kanuri SH, Ipe J, Kassab K. et al. Next generation microRNA sequencing to identify coronary artery disease patients at risk of recurrent myocardial infarction. Atherosclerosis 2018; 278: 232-239
- 21 Klinge CM. miRNAs regulated by estrogens, tamoxifen, and endocrine disruptors and their downstream gene targets. Mol Cell Endocrinol 2015; 418 (Pt 3): 273-297
- 22 Howard EW, Yang X. MicroRNA regulation in estrogen receptor-positive breast cancer and endocrine therapy. Biol Proced Online 2018; 20 (01) 17
- 23 Tay JW, Romeo G, Hughes QW, Baker RI. Micro-ribonucleic Acid 494 regulation of protein S expression. J Thromb Haemost 2013; 11 (08) 1547-1555
- 24 Ali HO, Arroyo AB, González-Conejero R. et al. The role of microRNA-27a/b and microRNA-494 in estrogen-mediated downregulation of tissue factor pathway inhibitor α. J Thromb Haemost 2016; 14 (06) 1226-1237
- 25 Wedderburn RWM. Quasi-likelihood functions, generalized linear models, and the Gauss-Newton method. Biometrika 1974; 61 (03) 439-447
- 26 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3 (06) 1101-1108
- 27 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120 (01) 15-20
- 28 Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res 2008; 36 (Database issue): D149-D153
- 29 Huang HY, Chien CH, Jen KH, Huang HD. RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. Nucleic Acids Res 2006; 34 (Web Server issue): W429-34
- 30 Wong N, Wang X. miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res 2015; 43 (Database issue): D146-D152
- 31 Rosenkranz A, Hiden M, Leschnik B. et al. Calibrated automated thrombin generation in normal uncomplicated pregnancy. Thromb Haemost 2008; 99 (02) 331-337
- 32 McLean KC, Bernstein IM, Brummel-Ziedins KE. Tissue factor-dependent thrombin generation across pregnancy. Am J Obstet Gynecol 2012; 207 (02) 135.e1-135.e6
- 33 Kovac MK, Lalic-Cosic SZ, Dmitrovic JM, Djordjevic VJ, Radojkovic DP. Thrombin generation, D-dimer and protein S in uncomplicated pregnancy. Clin Chem Lab Med 2015; 53 (12) 1975-1979
- 34 Westhoff CL, Pike MC, Cremers S, Eisenberger A, Thomassen S, Rosing J. Endogenous thrombin potential changes during the first cycle of oral contraceptive use. Contraception 2017; 95 (05) 456-463
- 35 Adams BD, Furneaux H, White BA. The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines. Mol Endocrinol 2007; 21 (05) 1132-1147
- 36 Iorio MV, Ferracin M, Liu CG. et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005; 65 (16) 7065-7070
- 37 McFall T, McKnight B, Rosati R. et al. Progesterone receptor A promotes invasiveness and metastasis of luminal breast cancer by suppressing regulation of critical microRNAs by estrogen. J Biol Chem 2018; 293 (04) 1163-1177
- 38 Kodahl AR, Lyng MB, Binder H. et al. Novel circulating microRNA signature as a potential non-invasive multi-marker test in ER-positive early-stage breast cancer: a case control study. Mol Oncol 2014; 8 (05) 874-883
- 39 Klinge CM. Estrogen regulation of microRNA expression. Curr Genomics 2009; 10 (03) 169-183
- 40 Bhat-Nakshatri P, Wang G, Collins NR. et al. Estradiol-regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Res 2009; 37 (14) 4850-4861
- 41 Masri S, Liu Z, Phung S, Wang E, Yuan Y-C, Chen S. The role of microRNA-128a in regulating TGFbeta signaling in letrozole-resistant breast cancer cells. Breast Cancer Res Treat 2010; 124 (01) 89-99
- 42 Tay J, Tiao J, Hughes Q, Gilmore G, Baker R. Therapeutic potential of miR-494 in thrombosis and other diseases: a review. Aust J Chem 2016; 69 (10) 1078-1093
- 43 Teruel R, Pérez-Sánchez C, Corral J. et al. Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome. J Thromb Haemost 2011; 9 (10) 1985-1992
- 44 Sahu A, Jha PK, Prabhakar A. et al. MicroRNA-145 impedes thrombus formation via targeting tissue factor in venous thrombosis. EBioMedicine 2017; 26: 175-186
- 45 Seitz H. Issues in current microRNA target identification methods. RNA Biol 2017; 14 (07) 831-834
- 46 Osman A, Fälker K. Characterization of human platelet microRNA by quantitative PCR coupled with an annotation network for predicted target genes. Platelets 2011; 22 (06) 433-441
- 47 Chen YC, Lin FY, Lin YW. et al. Platelet microRNA 365-3p expression correlates with high on-treatment platelet reactivity in coronary artery disease patients. Cardiovasc Drugs Ther 2019; 33 (02) 129-137
- 48 Guo D, Liu J, Wang W. et al. Alteration in abundance and compartmentalization of inflammation-related miRNAs in plasma after intracerebral hemorrhage. Stroke 2013; 44 (06) 1739-1742
- 49 Adesanya MA, Maraveyas A, Madden L. Differing mechanisms of thrombin generation in live haematological and solid cancer cells determined by calibrated automated thrombography. Blood Coagul Fibrinolysis 2017; 28 (08) 602-611
- 50 Marchetti M, Diani E, ten Cate H, Falanga A. Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography. Haematologica 2012; 97 (08) 1173-1180