Functional validation of microRNA-126-3p as a platelet reactivity regulator using human hematopoietic stem cells

Abstract

Platelets are an abundant source of microRNAs (miRNAs), which may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, are pointed out as potential biomarkers of platelet reactivity and recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain and functional validation of these candidate miRNAs on human-derived cells is lacking. To functionally validate miR-126-3p as regulator of platelet reactivity in platelet-like structures derived from human hematopoietic stem cells. The functional impact of miRNAs on platelet-like structures derived from human hematopoietic stem cells was evaluated using a flow-based assay. CD34+ derived megakaryocytes were transfected with selected miRNA or siRNA and were differentiated in platelet-like structures. Platelet adhesion phenotype was assessed by perfusion of differentiated cells in a microfluidic system under a constant shear rate. Overexpression of miR-126-3p increased platelet-like structures adhesion compared to control. Moreover, miR-126-3p transfection was associated with downregulation of ADAM9, a validated target of miR-126-3p, and of PLXNB2, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results than miR-126-3p transfection. Reporter gene assay confirm that miR-126-3p directly targets PLXNB2 3′UTR sequence. Taken together, using a flow-based assay, we functionally validate miR-126-3p as a regulator of platelet reactivity on platelet-like structures derived from human hematopoietic stem cells. Moreover, PLXNB2 was identified as a platelet reactivity regulator supporting the hypotheses that miR-126-3p modulates platelet reactivity at least in part by downregulation of PLXNB2.