Thrombin‐reduced miR‐27b attenuates platelet angiogenic activities in vitro via enhancing platelet synthesis of anti‐angiogenic thrombospondin‐1

Abstract

Background Platelets can synthesize proteins upon activation. Platelets contain a number of microRNAs (miRNA) and a fully functional miRNA effector machinery. It is, however, unclear if platelet miRNAs can regulate protein synthesis of platelets, and whether the regulation may produce a physiological impact. Objectives To investigate if and how platelet miRNAs regulate de novo syntheses of angiogenic regulators and subsequently modulate platelet angiogenic activities. Methods and Results Microarray-based miRNA profiling showed that thrombin stimulation in vitro down- or up-regulated a number of platelet miRNAs, both in the total platelet miRNAs and in Ago2-associated miRNAs. Among those altered miRNAs, miR-27b was down-regulated in both the total and Ago2-immunoprecipitated miRNA profiles of platelets, which was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Using western blotting assays, we showed that thrombin induced platelet de novo synthesis of thrombospondin-1, and that the level of thrombospondin-1 synthesis could reach a level of 3-5-fold higher than that before thrombin stimulation. With either the platelet precursor megakaryocyte cell line MEG-01 cells or mature platelets, we demonstrated that transfection of miR-27b mimic, but not the negative control of miRNA mimic, markedly reduced thrombospondin-1 protein levels. The latter subsequently enhanced platelet-dependent endothelial tube formation on matrigel. Conclusions Thrombin stimulation in vitro reduces platelet miR-27b levels that may markedly enhance thrombin-evoked platelet de novo synthesis of thrombospondin-1. Elevation of platelet miR-27b by transfection inhibits thrombospondin-1 synthesis, and subsequently enhances platelet pro-angiogenic activities. Hence, platelet activation-dependent reduction of miR-27b levels may represent a novel negative regulatory mechanism of platelet angiogenic activities.

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]. In sepsis, there was only one former publication, which reported impaired miRNA levels in exosomes and pooled human blood cells but not in platelets [35].…”

“…Expression of altered miRNAs correlates with platelet reactivity [17], while Dicer1-mediated miRNAs are able to regulate target mRNAs and de novo synthesis of proteins being important for the hemostatic function of platelets [18]. Accordingly, the expression of RNAs and related proteins are not static in platelets and can be modulated upon platelet activation [19]. Among in vitro septic conditions, mRNA levels of IL1B (interleukin-1β, IL-1β) and F3 (tissue factor) are increased, and resulting proteins are translated and accumulated in human platelets in response to TLR4 agonist lipopolysaccharide (LPS) [20,21].…”

MiR‐222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene

“…Several previous studies have identified a number of miRNAs that regulate the THBS1 gene in different types of cells, e.g., miR‐487b, the miR‐17‐92 cluster, miR‐194, miR‐18 and miR‐19, miR‐27b, and miR‐let‐7d (Asama et al, ; Dogar, Semplicio, Guennewig, & Hall, ; Farberov & Meidan, ; Feng et al, ; Miao et al, ; Sundaram et al, ; van Almen et al, ). In a recent study, a quantitative proteomic analysis of PK‐15 cells that were transfected with miR‐222 mimics and infected with transmissible gastroenteritis virus (TGEV) indicated that THBS1 was regulated by miR‐222 (Zhao et al, ).…”

The “Janus Face” of Platelets in Cancer

“…MVs also deliver angiogenic signals [27]: Transfer of miRNA let-7a or miR-27b in endothelial cells down-regulates the expression of the anti-angiogenic modulator thrombospondin-1, thus enhancing platelet-dependent endothelial tube formation [168,169].…”