Hyperglycaemia suppresses microRNA expression in platelets to increase P2RY12 and SELP levels in type 2 diabetes mellitus

Abstract

Megakaryocyte (MK)-derived miRNAs have been detected in platelets. Here, we analysed the expression of platelet and circulating miR-223, miR-26b, miR-126 and miR-140 that might be altered with their target mRNAs in type 2 diabetes mellitus (DM2). MiRNAs were isolated from leukocyte-depleted platelets and plasma samples obtained from 28 obese DM2, 19 non-DM obese and 23 healthy individuals. The effect of hyperglycaemia on miRNAs was also evaluated in MKs using MEG-01 and K562 cells under hyperglycaemic conditions after 8 hours up to four weeks. Quantitation of mature miRNA, pre-miRNAs and target mRNA levels (P2RY12 and SELP) were measured by RT-qPCR. To prove the association of miR-26b and miR-140 with SELP (P-selectin) mRNA level, overexpression or inhibition of these miRNAs in MEG-01 MKs was performed using mimics or anti-miRNAs, respectively. The contribution of calpain substrate Dicer to modulation of miRNAs was studied by calpain inhibition. Platelet activation was evaluated via surface P-selectin by flow cytometry. Mature and pre-forms of investigated miRNAs were significantly reduced in DM2, and platelet P2RY12 and SELP mRNA levels were elevated by two-fold at increased platelet activation compared to controls. Significantly blunted miRNA expressions were observed by hyperglycaemia in MEG-01 and K562-MK cells versus baseline values, while the manipulation of miR-26b and miR-140 expression affected SELP mRNA level. Calpeptin pretreatment restored miRNA levels in hyperglycaemic MKs. Overall, miR-223, miR-26b, miR-126 and miR-140 are expressed at a lower level in platelets and MKs in DM2 causing upregulation of P2RY12 and SELP mRNAs that may contribute to adverse platelet function.

Platelet-derived miR-223 promotes a phenotypic switch in arterial injury repair

“…DM mice have reduced miR-223 and increased intimal hyperplasia. It has been reported that the levels of miRNAs in platelets are altered under pathological conditions, such as in DM (24,25). Assessment of both human and mouse DM platelets demonstrated consistent reductions in miR-223 expression ( Figure 5A).…”

“…We identified a number of miRNAs that were differ entially expressed in VSMCs when cocultured with APs, including key miRNAs with established roles in regulating VSMC pheno type, such as miR-223, as well as the VSMC master miRNA cluster miR-143/145. These changes were independently validated by quantitative PCR (qPCR) at various time points (2,4,8,24, and 48 hours) following coculture with APs. We found significant upreg ulation of miRNA expression in the AP compared with the control group, with expression peaking at 4 hours for miR-223 or 24 hours for miR-143/145 ( Figure 2B).…”

Circulating MicroRNA Levels Indicate Platelet and Leukocyte Activation in Endotoxemia Despite Platelet P2Y12 Inhibition

“…MiR-223 is known to target P2Y 12 mRNA and lower levels may therefore convey increased resistance to P2Y 12 inhibition. Reduced expression of miR-223 in platelets has also been shown in diabetes; which is a strong risk factor for coronary artery disease and can be associated with high on-treatment platelet reactivity [34,35].…”

Platelet RNA as a circulating biomarker trove for cancer diagnostics

“…The dynamic property of platelet RNA to rapidly respond to external stimuli provides the opportunity to potentially identify surrogate RNA biomarker signatures to detect diseases such as CVD, cancer, and other diseases [9,37,46,69,[91][92][93][94][95]98]. The platelet RNA repertoire contains distinct spliced mRNA signatures for different tumor types located in different organs, allowing for tumor type-specific classification of patients with cancer using tumor-educated platelet RNA profiles [37].…”