Methods to Investigate miRNA Function: Focus on Platelet Reactivity

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs modulating protein production. They are key players in regulation of cell function and are considered as biomarkers in several diseases. The identification of the proteins they regulate, and their impact on cell physiology, may delineate their role as diagnostic or prognostic markers and identify new therapeutic strategies. During the last 3 decades, development of a large panel of techniques has given rise to multiple models dedicated to the study of miRNAs. Since plasma samples are easily accessible, circulating miRNAs can be studied in clinical trials. To quantify miRNAs in numerous plasma samples, the choice of extraction and purification techniques, as well as normalization procedures, are important for comparisons of miRNA levels in populations and over time. Recent advances in bioinformatics provide tools to identify putative miRNAs targets that can then be validated with dedicated assays. In vitro and in vivo approaches aim to functionally validate candidate miRNAs from correlations and to understand their impact on cellular processes. This review describes the advantages and pitfalls of the available techniques for translational research to study miRNAs with a focus on their role in regulating platelet reactivity.

MiR-221/222 Ameliorates Deoxynivalenol-Induced Apoptosis and Proliferation Inhibition in Intestinal Epithelial Cells by Targeting PTEN

“…MiRNAs are small non-coding RNA molecules that could hybridize with complementary sequences in the mRNA 3′ untranslated regions of its target gene. Then the miRNAs silence gene expression by inducing mRNA degradation or translation inhibition ( Garcia et al, 2020 ). Exosomal miR-125a/b inhibits intestinal epithelial cell proliferation and promotes cell apoptosis by suppressing myeloid cell leukemia-1 ( Cheng et al, 2020 ).…”

An Ex Vivo and In Silico Study Providing Insights into the Interplay of Circulating miRNAs Level, Platelet Reactivity and Thrombin Generation: Looking beyond Traditional Pharmacogenetics

“…The selection criteria for the miRNAs quantified in this study was based on their expression profile in platelets; miR-126-3p, miR-150-5p and miR-223-3p were amongst the most expressed miRNAs [21] (http://www.plateletomics.com/, version 1.0, accessed 2 April 2021), and they were consistently associated with platelet reactivity in clinical studies [13,17]. We also investigated miR-204-5p, which was first described by our group as associated with platelet reactivity [22]; this association was confirmed subsequently in an independent cohort [23].…”

“…Correlations between circulating miRNA levels and platelet aggregation and thrombin generation markers in stable cardiovascular patients. * The association between miR-126-3p and miR-150-5p with thrombin generation markers are derived from Zapilko et al[17]. AA: arachidonic acid; ADP 5: adenosine diphosphate 5 µM; ADP 20: adenosine diphosphate 20 µM; F1+2: prothrombin fragment 1 + 2; NS: not significant; TAT: thrombin-antithrombin complex (Spearman correlation test).…”

MicroRNA as Potential Biomarkers of Platelet Function on Antiplatelet Therapy: A Review

“…However, due to the high cost and preanalytical bias of those methods, qPCR remains the one recommended. To lessen the risk of the influence of technical variability on the miRNA levels measurements in qPCR, a synthetic oligonucleotide should be added as an exogenous normalizing target (Garcia et al, 2020). MiRNAs might potentially replace currently used platelet function tests and provide us with more specific and reliable data on platelet activity.…”