Functional Validation of microRNA-126-3p as a Platelet Reactivity Regulator Using Human Haematopoietic Stem Cells

Abstract

Background Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking. Objective This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells. Materials and Methods CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay. Results CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p. Conclusion We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation.

Circulating MicroRNA Profiling in Non-ST Elevated Coronary Artery Syndrome Highlights Genomic Associations with Serial Platelet Reactivity Measurements

“…In contrast, miR-126-3p, the 3' strand of pre-miR-126 was positively correlated with PR in the Singapore cohorts. Both the 5' and 3' strand of miR-126 have been shown to be functional in platelet and vascular biology, with miR-126-3p showing significant effects in platelet activation and coagulation 31,32 , while miR-126-5p showing activity in regulating endothelial inflammation and promoting vascular health [33][34][35] . It is likely that both miR-126-3p and miR-126-5p play a role in post-ACS inflammatory regulation at the platelet and endovascular levels respectively, and additional functional work will be needed to delineate the modes and locations of miR-126 modulation within this dynamic time period following an ischemic event.…”

Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis

“…Recently, the potential role of miRNAs in platelet and MK function has also got in focus [25,33]. For instance, platelet miR-27b can regulate platelet synthesis of trombospondin-1 [19], or miR-15a modulates GPVI-mediated αIIbβ3 activation and α-granule release in MKs [34].…”

MicroRNA-126 is a regulator of platelet-supported thrombin generation

“…Indeed, the secretion of PLS after agonist stimulation, as well as their function in relation to fibrinogen receptors, is comparable to the results observed with human platelets [22,23]. PLS were derived from megakaryocytes produced from human hematopoietic stem cells as described elsewhere [24]. Briefly, human CD34+ cells were isolated from the buffy coats of healthy adult human donors provided by the Geneva University Hospitals' blood bank using a CD34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany).…”