To cite this article: Teruel R, Pé rez-Sá nchez C, Corral J, Herranz MT, Pé rez-Andreu V, Saiz E, García-Barberá N, Martínez-Martínez I, Roldá n V, Vicente V, Ló pez-Pedrera C, Martínez C. Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome. J Thromb Haemost 2011; 9: 1985-92.Summary. Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP-1 and MDA-MB-231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR-20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT-PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR-1792, for example miR-19b and miR-20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR-20a binds to TF mRNA. Finally, we measured levels of miR-19b and miR-20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down-regulation of miR-19b and miR-20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

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International audienc

MicroRNAs markedly affect the immune system, and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs’ expression was linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes, and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression, and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that are negative regulators in a crescent number of physiological and pathological processes. However, their role in haemostasis, a complex physiological process involving multitude of effectors, is just beginning to be characterized. We evaluated the changes of expression of miRNAs in livers of neonates (day one after birth) and adult mice by microarray and qRT-PCR trying to identify miRNAs that potentially may also be involved in the control of the dramatic change of hepatic haemostatic protein levels associated with this transition. Twenty one out of 41 miRNAs overexpressed in neonate mice have hepatic haemostatic mRNA as potential targets. Six of them identified by two in silico algorithms potentially bind the 3′UTR regions of F7, F9, F12, FXIIIB, PLG and SERPINC1 mRNA. Interestingly, miR-18a and miR-19b, overexpressed 5.4 and 8.2-fold respectively in neonates, have antithrombin, a key anti-coagulant with strong anti-angiogenic and anti-inflammatory roles, as a potential target. The levels of these two miRNAs inversely correlated with antithrombin mRNA levels during development (miR-19b: R = 0.81; p = 0.03; miR-18a: R = 0.91; p<0.001). These data suggest that miRNAs could be potential modulators of the haemostatic system involved in developmental haemostasis.

β1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased β1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a ~40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of β1-tubulin might not be critical to sustain platelet discoid shape.

Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silico studies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3'UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.

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