Alteration of circulating platelet-related and diabetes-related microRNAs in individuals with type 2 diabetes mellitus: a stepwise hypoglycaemic clamp study

Abstract

Background: In patients with type 2 diabetes mellitus (T2DM) an association between severe hypoglycaemic episodes and the risk of cardiovascular (CV) morbidity and mortality has been previously established.

Methods: We aimed to investigate the influence of hypoglycaemia on several diabetes-related and platelet-related miRNAs selected based on bioinformatic analysis and literature search, including hsa-miR-16, hsa-miR-34a, hsa-miR-129-2, hsa-miR-15a, hsa-miR-15b, hsa-miR-106a, miR-223, miR-126. Selected miRNAs were validated by qRT-PCR in 14 patients with T2DM on metformin monotherapy, without established CV disease and antiplatelet therapy during a stepwise hypoglycaemic clamp experiment and a follow-up 7 days after the clamp event. In order to identify which pathways and phenotypes are associated with validated miRNAs we performed target prediction on genes expressed with high confidence in platelets.

Results: Circulating levels of miR-106a-5p, miR-15b, miR-15a, miR-16-5p, miR-223 and miR-126 were increased after euglycaemic clamp followed by hypoglycaemic clamp, each with its distinctive time trend. On the contrary, miR-129-2-3p, miR-92a-3p and miR-34a-3p remained unchanged. MiR-16-5p was negatively correlated with interleukin (IL)-6, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) (p = 0.002, p < 0.001, p = 0.016, respectively), whereas miR-126 was positively correlated with VCAM (p < 0.001). There were negative correlations between miR-16-5p, miR-126 and coagulation factors, including factor VIII and von Willebrand factor (vWF). Among all studied miRNAs, miR-126, miR-129-2-3p and miR-15b showed correlation with platelet function. Bioinformatic analysis of platelet-related targets of analyzed miRNAs showed strong enrichment of IL-2 signaling. We also observed significant enrichment of pathways and diseases related to cancer, CV diseases, hyperglycemia, and neurological diseases.

Conclusions: Hypoglycaemia can significantly influence the expression of platelet-enriched miRNAs, with a time trend paralleling the time course of platelet activation. This suggests miRNAs could be exploited as biomarkers for platelet activation in response to hypoglycaemia, as they are probably released by platelets upon activation by hypoglycaemic episodes. Should they hold their promise in clinical endpoint studies, platelet-derived miRNAs might become helpful markers of CV risk in subjects with diabetes. Trial registration The study was registered at clinical trials.gov; Impact of Hypoglycaemia in Patients With DIAbetes Mellitus Type 2 on PLATElet Activation (Diaplate), trial number: NCT03460899.

Keywords: Bioinformatic analysis; Biomarker; Diagnosis; Network; Platelet reactivity; Platelets; Prognosis; miRNA; miRNA-gene target interaction.

Fig. 1

Effect of hypoglycaemia on miRNAs expression levels. a miR-106a-5p; b miR-15b; c miR-16-5p; d miR-15a; e miR-223; f miR-126; g miR-129–2-3p; h miR-92a-3p; i miR-34a-5p. Two groups comparison p value was calculated by Wilcoxon paired test, multiple groups comparison was calculated based on One-way ANOVA. MiRNAs expression data was presented as log10 transformation. Only statistical significant p values between the groups are shown (i.e. p ≤ 0.05)

Fig. 2

Top ten Pathways (A) and Diseases (B) associated with targets expressed in platelets of each validated miRNAs. Ontological terms were selected based on adjusted p value, and ordered by the increasing summary rank order (top ranks count)