Do miRNAs Have a Role in Platelet Function Regulation?

Abstract

MicroRNAs (miRNAs) are a class of non-coding RNAs known to repress mRNA translation and subsequent protein production. miRNAs are predicted to modulate many targets and are involved in regulating various cellular processes. Identifying their role in cell function regulation may allow circulating miRNAs to be used as diagnostic or prognostic markers of various diseases. Increasing numbers of clinical studies have shown associations between circulating miRNA levels and platelet reactivity or the recurrence of cardiovascular events. However, these studies differed regarding population selection, sample types used, miRNA quantification procedures, and platelet function assays. Furthermore, they often lacked functional validation of the miRNA identified in such studies. The latter step is essential to identifying causal relationships and understanding if and how miRNAs regulate platelet function. This review describes recent advances in translational research dedicated to identifying miRNAs' roles in platelet function regulation.

Keywords

microRNA - platelet function - biomarker

Introduction

Platelets play a key role in maintaining hemostasis[1] and vascular integrity; they are also involved in regulating inflammation,[2] immunity,[3] and tumor metastasis.[4] Platelets' main role is stopping bleeding during vascular lesions by accumulating at the site of vessel injury, thanks to their adhesive properties. Contact with the subendothelial components exposed in damaged vessels promotes platelet activation, aggregation, and thrombus growth. Additionally, platelet activation induces phosphatidylserine exposure at the membrane surface, activating the coagulation cascade and formation of a stable clot.[5] Platelets' ability to promote hemostasis—platelet reactivity (PR)—is a variable phenotype in individuals taking or not taking aspirin,[6] [7] and may be associated with bleeding or thrombotic events. The determinants of PR have yet to be fully elucidated, but family-based studies point to a genetic origin.[8]

In recent decades, miRNAs' roles in the regulation of various biological processes and the progression of several diseases have gradually emerged. miRNAs are noncoding sections of RNA approximately 22 nucleotides long; they regulate gene expression at the post-transcriptional level by degrading mRNA or repressing its translation. Increasing numbers of clinical trials have shown correlations between PR and levels of circulating miRNA, revealing several platelet-derived miRNAs as putative biomarkers of platelet function or the recurrence of ischemic events.[9] [10] [11] [12] [13] [14] Nevertheless, a causal link between circulating miRNA and platelet function or thrombus formation remains elusive, and mechanisms involved in miRNA-mediated regulation of platelet function are poorly understood. This may be due to the many miRNA–mRNA duplexes potentially involved and the variety of biological pathways that could be either upregulated or downregulated by miRNA levels. Validating candidate miRNAs identified as being regulators of platelet function in association studies is a major challenge, and of utmost importance to select them as potential biomarkers and to identify the underlying mechanisms.[15]

miRNA Biogenesis

In humans, miRNA loci are mainly located in intronic regions, in both transcription coding or non-coding units. Approximately 10% of all miRNAs are encoded by exons.[16] The genes that encode for miRNAs are transcribed by either RNA polymerase II or III into primary miRNA transcripts (pri-miRNA), depending on the miRNA loci and their upstream promoters.[17] [18] Pri-miRNA consists of a stem and a terminal loop flanked by two single-stranded segments. The single-strand–double-strand junction of the pri-miRNA hairpin is cleaved by Drosha, an RNase III endonuclease coupled to the DiGeorge syndrome critical region 8 (DGCR8) at approximately 11 bp from the pri-miRNA stem, which allows the release of the precursor miRNA (pre-miRNA). Exportin-5 forms a complex with the guanine triphosphate (GTP)-bound form of the small nuclear guanine triphosphatase (GTPase) Ran (RanGTP). This complex recognizes the pre-miRNA structure,[19] protects pre-miRNA from degradation, and promotes the export of properly processed pre-miRNA into the cytoplasm.[19] [20] In the cytoplasm, the double-strand pre-miRNA is cleaved by the RNase III endonuclease Dicer, resulting in the generation of two partially complementary strands of an miRNA,[20] each with its own targets and regulating specific biological functions.[21]

Mature miRNA recognizes six to eight nucleotides located on the 3′-untranslated region (3′-UTR) of mRNA.[22] Moreover, one miRNA can target several mRNAs and regulate several biological functions. Conversely, one mRNA can have a binding sequence for multiple miRNAs; therefore, the same gene may be regulated by several miRNAs. The miRNA–mRNA duplex guides the binding of the Ago2 protein in an RNA-induced silencing complex,[23] allowing mRNA cleavage, mRNA decay, and/or translational repression ([Fig. 1]).

Fig. 1 miRNA biogenesis. miRNAs are mainly transcribed from the intronic region by polymerase II/III. The pri-miRNAs is processed into pre-miRNAs by the Drosha–DGCR8 complex and is then exported to the cytoplasm by exportin-5 for further maturation by the Dicer protein. Mature miRNA binds to a seed sequence by imperfect complementarity on the 3′-untranslated region (3′-UTR) of an mRNA. The miRNA–mRNA duplex is stabilized by Ago2 into an miRNA-induced silencing complex (RISC), thus triggering mRNA translational repression.

Platelets contain the necessary machinery for mRNA translation and protein synthesis.[24] In response to their activation, platelets synthesize proteins by signal-dependent translation, using newly spliced mRNA and mature mRNA.[25] Platelets contain more than 500 miRNAs inherited from their parent cell, the megakaryocyte.[26] Therefore, it seems reasonable to speculate that these miRNAs have a fine-tuning function in the modulation of PR.

Circulating miRNAs

After stimulating various cells, including cell fragments such as platelets, circulating miRNAs release their intracellular content into the extracellular medium. Current understanding points to microvesicles being the main transporters of circulating miRNAs[27] since miRNAs' profiles in the blood are close to those of microvesicles.[28] However, the composition of this pool of extracellular miRNAs also includes miRNAs bound to proteins. A recent study reported that miRNAs are more abundant in non-vesicular than in vesicular fractions,[29] and that these vesicle-free miRNAs were associated with the Ago2 protein.[30] The authors concluded that Ago2 as a miRNA carrier could account for all vesicle-free miRNAs; however, other miRNA complexes can also exist in plasma, such as with nucleoplasmin or high-density lipoproteins.[31] [32] The mechanism governing extravesicular biogenesis, transport, and cargo uptake in recipient cells is not entirely understood and is reviewed elsewhere.[33]

Since approximately 45% of microvesicles circulating in the blood are released from platelets,[28] the circulating miRNA profile may reflect the platelet miRNA content and provide information on platelet activation status in real time. This concept is illustrated by the impact of antiplatelet drugs on circulating miRNA,[34] meaning that the profile of circulating miRNA may provide important information on PR in diseases where platelets play major roles, such as in the progression of atherosclerosis,[35] cancer metastasis,[36] and inflammation.[3]

Associations between miRNAs and Platelet Reactivity in Cardiovascular Patients

Several studies have addressed correlations between circulating miRNAs and PR or cardiovascular events,[14] [34] [37] promoting the concept of using circulating, platelet-derived microRNAs (e.g., miR-223, miR-126, miR-197, miR-150, miR-21, miR-96, or miR-204) as potential biomarkers for the recurrence of cardiovascular events ([Fig. 2]).[15] [38] It is of note that miR-223–3p and miR-126–3p, among the most abundant platelet-derived miRNAs, have been particularly studied.

Fig. 2 Platelet-derived miRNA. Selected platelet-derived miRNAs and their experimentally validated mRNA targets (in brackets) associated with atherosclerosis, platelet reactivity, and cancer. For miR-197, no validated target related to these diseases was identified.

MiR-223 may mediate its effect on platelet function via the regulation of several genes. For example, miR-223 represses P2Y₁₂ mRNA translation, the ADP receptor targeted by antiplatelet drugs (thienopyridines and ticagrelor).[24] [39] Increased exosomal miR-223 was shown to be associated with the occurrence and severity of acute ischemic stroke and short-term outcomes.[40] In patients with acute coronary syndrome, levels of circulating miR-223 in plasma were statistically correlated with the PR determined using vasodilator-stimulated phosphoprotein phosphorylation flow cytometry after clopidogrel or dual-antiplatelet therapy. Moreover, a lower level of circulating miR-223–3p was the only independent predictor of poorer response to treatment, as determined using a PR index.[39] [41] These results indicated that miR-223 might be useful in predicting disease severity and assessing responsiveness to antiplatelet agents.

Zampetaki et al showed that diabetes was associated with a reduced level of miR-126 in plasma,[42] which may reflect a platelet dysfunction in diabetic populations. A study of patients with type 2 diabetes showed a direct correlation between platelet activation, as assessed by measuring soluble P-selectin, and the level of circulating miR-126. In addition, the administration of aspirin induced a decrease in the level of circulating miR-126 in this population.[43] Another study, of patients with diabetes mellitus, showed that miR-126 exhibited antithrombotic properties by regulating post-transcriptional tissue factor expression. Optimizing antidiabetic treatment using metformin or sulfonylurea was associated with the upregulation of miR-126 in plasma, which correlated with reduced thrombogenicity.[44] However, this association may not be true in nondiabetic patients.[45] In recent years, several hypotheses have emerged regarding the underlying mechanisms mediated by miR-126's impact on platelet function. These include the modulation of ADAM9, a mediator of platelet binding to collagen,[37] and of PLXNB2, a transmembrane protein known to support thrombus formation in mice.[46]

Other miRNA–mRNA duplexes have been described as putative regulators of PR, including miR-96–VAMP8, described as having a role in the heterogeneity of PR.[47]

Platelet-Derived miRNAs and Atherosclerosis

Accumulated evidence has indicated that many miRNAs, including platelet-derived miRNAs, can affect the initiation and progression of atherosclerosis and the development of its complications via endothelial cell (EC) function regulation, including the maintenance of vascular integrity (miR-126), EC proliferation (miR-126, miR-17, miR-18, miR-20a), migration (miR-218, miR-106b-25 cluster), and senescence (miR-34, miR-217, miR-146a; see [Fig. 2]).[48]

miRNAs are important regulators of lipoprotein homeostasis and lipid accumulation. Among them, miR-223 represses genes involved in cholesterol biosynthesis (HMGS1, SC4MOL) and HDL uptake (SRB1).[49] The miR-33 family targets the HDL–reverse cholesterol transport pathway.[50] [51]

Overexpression of miR-146a can delay both inflammatory response and oxidized low-density lipoprotein accumulation by inhibiting activation of the toll-like receptor 4–dependent signaling pathway.[52] MiR-30c has been shown to decrease the production of apoB-containing lipoproteins (very low density lipoprotein, low-density lipoprotein) by targeting both the microsomal triglyceride transfer protein (MTTP), a protein essential for the lipidation of nascent apoB, and lysophosphatidylglycerol acyltransferase 1 (LPGAT1), which reduces de novo lipogenesis.[53]

miRNAs can also play roles in the regulation of proinflammatory response. The inhibition of endogenous miR-125a-5p expression has been correlated with an increased inflammatory cytokines tumor growth factor-β level, tumor necrosis factor-α, interleukin 2 (IL-2), and interleukin 6 (IL-6).[54]

Additionally, other miRNAs, including miR-126–3p, miR-17–3p, and miR-31, regulate the development of vascular inflammation by controlling the expression of adhesion molecules, such as vascular cell adhesion molecule 1, intercellular adhesion molecule, and E-selectin, respectively.[54] [55] Other miRNAs facilitate calcification by targeting Ets1 (miR-125b) or, on the contrary, inhibit calcification (miR-29a and miR-29b) by suppressing the expression of ADAMTS-7.[56] [57] Furthermore, microRNAs play roles in plaque stability, with either stabilizing effects (miR-222, miR-24, miR-26a, miR-27b, let-7e) or destabilizing effects (miR-21, miR-181, miR-712, miR-29).[58] Finally, several microRNAs, including miR-155–5p, miR-483–5p, and miR-451a, have been reported as potential biomarkers for the early identification of plaque rupture.[59]

Among the microRNAs identified as playing roles in the development of atherosclerosis, several are found in platelets and in platelet-derived microvesicles, including miR-223, miR-126, miR-24, miR-155, and miR-21. Therefore, platelet-derived microRNAs may contribute to the development of atherosclerosis at every stage leading to the formation of atherosclerotic plaques.

Platelet-Derived miRNA and Cancer

Besides their potential role in PR and cardiovascular diseases, activated platelets and platelet-derived microvesicles are suggested to be implicated in the progression of cancers.[60] Cancer patients are also at an increased risk of platelet-driven venous thromboembolism.[61]

Several miRNAs enriched in platelet-derived microvesicles can target both tumor suppressor genes and oncogenes (in multiple cancer types), among them miR-27a, miR-24, miR-155, miR-195, let-7a/b, and miR-223.[60] [62] [63] [64] [65] [66] [67] [68] [69] The platelets and platelet-derived microvesicles of patients with non-small-cell lung cancer contain higher levels of miR-223 than those of healthy individuals. The incubation of A549 human lung cancer cells with platelet-derived microvesicles has been shown to result in the rapid delivery of miR-223 into those cells, thus inhibiting the EPB41L3 tumor suppressor and promoting A549 cell invasion.[67]

However, although platelet-derived microvesicles are generally considered to participate in cancer progression, recent data suggest that they may have a cancer-suppressive effect. Platelet-derived microvesicles infiltrate solid tumors in humans and mice and transfer their RNA content, including miRNAs promoting tumor cell apoptosis. MiR-24 was a significantly present species in this transfer. In another model, where human platelet-derived microvesicles were transfused to mice, mitochondrial mt-Nd2 and Snora75—two small, noncoding, nucleolar RNAs—were identified as direct targets of platelet-derived miR-24. These target RNAs were suppressed in platelet-derived, microvesicle-treated tumor cells, resulting in mitochondrial dysfunction and tumor growth inhibition.[60]

Analytical Pitfalls in Measuring Circulating miRNAs

miRNAs are highly stable in biological fluids[70] [71] and could therefore be interesting for developing a new type of biomarker. Over recent decades, numerous studies have investigated using circulating miRNAs as potential biomarkers,[37] [39] [72] [73] [74] raising several issues regarding sample types, extraction and quantification procedures, and clinically significant cut-off values.[75]

Sample selection is important. Indeed, serum samples may reflect the total miRNA content in platelets and miRNAs derived from the microvesicles of other blood cells that can be activated during the coagulation process. miRNA quantification using plasma samples is probably more likely to reflect basal platelet function status and to be better for evaluating the risk of ischemic event recurrence; thus, plasma samples are preferred.[75] [76] Freezing and thawing cycles can lyse residual cells and release all of their miRNA content, biasing miRNA quantification. Therefore, plasma samples should be prepared using a double centrifugation step, and measuring platelet-specific or leukocyte-specific proteins (e.g., ITGA2B or PTPRC, respectively) can validate the absence of any residual cell contamination.[77]

The miRNA extraction procedure is usually performed using a TRIzol-based method followed by (or not) a standardized column extraction process.[39] This only enables a small amount of miRNA to be extracted, however; so, a preamplification step is usually performed to optimize subsequent quantification. The ratio between sensitivity and the number of targets differs according to the quantification technique. Indeed, a complete miRNA profile can be evaluated using high throughput techniques such as RNA sequencing,[37] whereas custom-designed locked nucleic acid (Exiqon)[37] [78] and NanoString Technologies[73] [74] enable the quantification of approximately 100 miRNAs targeted in one sample. The most sensitive technique remains the TaqMan-based quantitative polymerase chain reaction on complementary DNA,[75] [79] which is considered the gold standard. Because only a few miRNAs can be evaluated at once, however, candidate miRNAs must have been identified in a previous study.

Finally, although the normalization procedure remains challenging, it is of utmost importance for comparing samples and studies. A wide range of strategies has been developed,[80] including using exogenous spike-in (e.g., UniSP6[14] and cel-miR-39[81]), endogenous, small, non-coding RNA (e.g., RNU6[82]) or endogenous miRNA(s). The spike-in is useful for evaluating the quality and reproducibility of the extraction and quantification process, whereas endogenous RNA or miRNAs are more adapted to normalize the level of quantified miRNA. However, endogenous normalizers from the same class of nucleotide sequence should be preferred. Endogenous miRNA normalizers differ according to the sample used—for example, miR-638, miR-93, and miR-484 are used for plasma,[83] [84] [85] whereas miR-23a, let7a, and miR-1260 are preferred for serum.[78] [86] [87] Using a combination of stable normalizers, determined using dedicated tools (e.g., GeNorm[88] and NormFinder[89]), enhances the efficiency of the normalization.[87]

Functional Validation of miRNAs

Although several miRNAs have been pinpointed as potential biomarkers of PR or the recurrence of ischemic events, the functional validation of candidate miRNAs as true regulators of platelet function remains a crucial step. It will be the cornerstone criterion formally linking a specific miRNA and platelet function regulation.[15] Platelets have a very short lifespan and only remain functional for a few hours after blood sampling. This precludes the modulation of their miRNA content via transfection; thus, alternative strategies should be used. Several in vitro models are available using megakaryocyte lines (MEG01, DAMI, or K562) or megakaryocytes from hematopoietic stem cells (CD34) differentiated into platelet-like structures (PLSs). The latter model has the advantage of producing PLSs that are functionally close to human platelets.[46] [90] However, this model's main limitation is the small number of cells recovered, which means that it is not compatible with all of the conventional methods dedicated to the study of platelet function. Modulation of the miRNA content in these in vitro models can be done using lipofection or nucleofection to transfect the megakaryocytes with synthetic miRNA mimics or miRNA inhibitors. An alternative method uses lentiviral transduction, where the lentiviral vector genome is integrated into the host genome, leading to stable miRNA expression.

Several in vivo models are available, including zebrafish and mouse models that have hemostatic systems close to that of humans. The zebrafish model is widely used. The transparency of zebrafish embryos and larvae between 24 hours and 5 days after fertilization facilitates the visualization of their vessels and real-time thrombocyte accumulation after laser injury.[15] [45] Modifications in thrombocyte miRNA content can be obtained by genetic modifications. Inserting a sequence coding for the miRNA of interest, under the control of CD41 promoter, results in the generation of a transgenic line that only overexpresses miRNA in thrombocytes without affecting the levels of other miRNAs.[45]

The mouse is a model of choice for studies related to hemostasis. It allows the performance of aggregation tests, monitoring the exposure of CD62P and the expression of activated GPIIb–IIIa on platelets using flow cytometry, and the assessment of tail bleeding time. Several gene-editing strategies can be used, including transgenesis, homologous recombination using the Cre/loxP,[91] [92] [93] and the TALEN[94] and CRISPR[95] systems that can successfully induce miRNA deletions. The transduction of a lentiviral vector specifically designed to stably overexpress a specific miRNA can be injected into mice.[96] Bidirectional miRNA reporter lentiviral vectors have shown promising results for post-transcriptional (miRNA-restricted) repression in hematopoietic stem cells in mice via the insertion of target sequences for hematopoietic stem cell–specific miRNA into the lentiviral cassette.[97]

In contrast, a transient modulation of a miRNA level can be obtained by systemic injection of locked nucleic acid, a chemical inhibitor.[37]

Generating transgenic lines has obvious advantages for the functional investigation of a miRNA's impact on PR and hemostasis. However, animal models must be used cautiously due to the possible non-conservation of miRNA–mRNA duplexes, which can differ from miRNA's function in humans.

Identification and Validation of miRNA Targets

The in silico identification of target miRNAs using databases such as Targetscan, MiRanda, or DIANA-microT represents the first step in deciphering the mechanisms linking miRNA and platelet function. However, different databases may identify variable numbers of putative targets. For example, the Targetscan database (Release 7.2) finds 423 putative targets for miR-223, whereas the MiRanda database finds 3,485. These databases are also known to provide high numbers of false-positive results[98]; therefore, any miRNA–mRNA duplexes predicted in silico must be validated. The reporter gene assay is one of the most widely used methods to identify an miRNA's binding site on the target gene's 3′-UTR and to validate the direct interaction between an miRNA and its target. In addition to P2Y12, calpain-2 (known to regulate the platelet signaling pathway)[99] has also been validated as a direct target of miR-223–3p.[100] Several other predicted targets of miR-223–3p have been validated using this method, including PDGFRβ,[101] [102] EPB41L3,[67] Septin-2, and Septin-6[103]; however, their direct role in platelet function remains poorly understood.

Conclusion

There is increasing evidence of associations between levels of specific miRNAs and PR and the recurrence of ischemic events.

The significant methodological differences between different clinical association studies, in terms of their biological samples, miRNA extraction methods, quantification, and clinical settings, make functional validation of the utmost importance when attempting to provide a causal link and a mechanistic insight into the role of candidate miRNAs in regulating PR.[15] These functional validation data are more limited but are critical to selecting the most promising miRNAs as biomarkers for diseases where platelets play a major role.

Taken together, in the near future, the profile of a patient's circulating miRNA could be evaluated for the biomarkers of various platelet-related diseases and contribute to the emergence of personalized medicine.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Private Foundation of the Geneva University Hospitals (Grant RC04–05).

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Publication History

Received: 30 November 2020

Accepted: 08 April 2021

Article published online:
30 June 2021