ANA BELEN ARROYO RODRIGUEZ, SALAM SALLOUM ASFAR, CARLOS PEREZ SÁNCHEZ, RAÚL TERUEL MONTOYA, SILVIA NAVARRO, GINES LUENGO GIL, VANESSA ROLDAN SCHILLING, JOHN-BJARNE HANSEN, CHARY LÓPEZ PEDRERA, VICENTE VICENTE GARCÍA, ROCÍO GONZÁLEZ-CONEJERO HILLA, CONSTANTINO MARTÍNEZ GÓMEZ,

• [GI/IMIB/C001/2011] HEMATOLOGÍA Y ONCOLOGÍA MÉDICA CLÍNICO-EXPERIMENTAL

Tissue factor pathway Inhibitor (TFPI) expression in adults is regulated by testosterone. This regulation contributes to explain the protective effect of testosterone to cardiovascular events, and why their decline from age 50 increases endothelial dysfunction and the risk of thrombosis. The cellular mechanisms underlying this regulation are unknown. Our group and others have demonstrated the regulation of several haemostatic factors including tissue factor by microRNA (miRNA). However, miRNAs role regulating TFPI are unknown. The aim of this study was to evaluate TFPI regulation by testosterone through miRNA.

4 algorithms were used to select miRNA potential regulators of TFPI. EA.hy926 and HUVEC endothelial cells were transfected with precursors and inhibitors of miRNA (mimic and antimiR; 100nM). EA.hy926 cells were treated with 5?-dihydrotestosterone (DHT) (30nM, physiological doses for 24h and 72h). TFPI levels were quantified by qRT-PCR and western blot and miRNA by qRT-PCR. The validation of the results was performed ex vivo by analyzing miRNA levels in TFPI and 74 vein endothelial cells from human umbilical cord. TFPI anticoagulant activity in EA.hy926 and HUVEC supernatants was determined with Actichrome® TFPI activity assay kit (Sekisui).

4 potential miRNA regulators TFPI were selected: miR-19b, miR-24a, and miR-27a / b. Only miR-27a / b decreased significantly TFPI mRNA and protein in both endothelial cells. Moreover, anti-miR-27a/b increased significantly TFPI mRNA and protein again in both endothelial cells. Luciferase assays demonstrated a direct interaction between miR-27a / b and the 3'UTR of TFPI. Ex vivo study showed a significant inverse correlation between levels of TFPI mRNA and miR-27a. DHT 30nM increased levels of TFPI ~ 40% (mRNA and protein) and decreased miR-27a / b ~ 50% at 24 and 72h. Anticoagulant activity of TFPI decreased or increased significantly ~ 30-50% in both cells transfected with miR-27a or anti-miR27a, respectively.

The results show that TFPI expression is regulated directly by miR-27a/b in endothelial cells, with functional consequences. In addition, miR-27a / b are involved in the mechanism of up-regulation of TFPI-mediated by testosterone. Future studies are needed to evaluate the potential regulation of TFPI involvement of miRNA in cardiovascular diseases.