Diagnostic Performance of Circulating miRNAs and Extracellular Vesicles in Acute Ischemic Stroke

Abstract

Background: Increased inflammation activates blood coagulation system, higher platelet activation plays a key role in the pathophysiology of ischemic stroke (IS). During platelet activation and aggregation process, platelets may cause increased release of several proinflammatory, and prothrombotic mediators, including microRNAs (miRNAs) and extracellular vesicles (EVs). In the current study we aimed to assess circulating miRNAs profile related to platelet function and inflammation and circulating EVs from platelets, leukocytes, and endothelial cells to analyse their diagnostic and predictive utility in patients with acute IS. Methods: The study population consisted of 28 patients with the diagnosis of the acute IS. The control group consisted of 35 age- and gender-matched patients on acetylsalicylic acid (ASA) therapy without history of stroke and/or TIA with established stable coronary artery disease (CAD) and concomitant cardiovascular risk factors. Venous blood samples were collected from the control group and patients with IS on ASA therapy (a) 24 h after onset of acute IS, (b) 7-days following index hospitalization. Flow cytometry was used to determine the concentration of circulating EVs subtypes (from platelets, leukocytes, and endothelial cells) in platelet-depleted plasma and qRT-PCR was used to determine several circulating plasma miRNAs (miR-19a-3p, miR-186-5p and let-7f). Results: Patients with high platelet reactivity (HPR, based on arachidonic acid-induced platelet aggregometry) had significantly elevated platelet-EVs (CD62+) and leukocyte-EVs (CD45+) concentration compared to patients with normal platelet reactivity at the day of 1 acute-stroke (p = 0.012, p = 0.002, respectively). Diagnostic values of baseline miRNAs and EVs were evaluated with receiver operating characteristic (ROC) curve analysis. The area under the ROC curve for miR-19a-3p was 0.755 (95% CI, 0.63–0.88) p = 0.004, for let-7f, it was 0.874 (95% CI, 0.76–0.99) p = 0.0001; platelet-EVs was 0.776 (95% CI, 0.65–0.90) p = 0.001, whereas for leukocyte-EVs, it was 0.715 (95% CI, 0.57–0.87) p = 0.008. ROC curve showed that pooling the miR-19a-3p expressions, platelet-EVs, and leukocyte-EVs concentration yielded a higher AUC than the value of each individual biomarker as AUC was 0.893 (95% CI, 0.79–0.99). Patients with moderate stroke had significantly elevated miR-19a-3p expression levels compared to patients with minor stroke at the first day of IS. (AUC: 0.867, (95% CI, 0.74–0.10) p = 0.001). Conclusion: Combining different biomarkers of processes underlying IS pathophysiology might be beneficial for early diagnosis of ischemic events. Thus, we believe that in the future circulating biomarkers might be used in the prehospital phase of IS. In particular, circulating plasma EVs and non-coding RNAs including miRNAs are interesting candidates as bearers of circulating biomarkers due to their high stability in the blood and making them highly relevant biomarkers for IS diagnostics.

The expression levels of circulating miR‐140‐3p, miR‐130a‐3p, and miR‐320b as diagnostic biomarkers in acute ischemic stroke

“…In this study, the area under the curve (AUC) values for plasma miR‐140‐3p, miR‐130a‐3p, and miR‐320b were 0.790, 0.831, and 0.907, respectively, indicating their potential as diagnostic biomarkers for AIS. Researchers have also attempted to combine circulating biomarkers into panels to improve their diagnostic properties 8,23 . Combining multiple miRNAs yielded better diagnostic performance than using individual miRNAs alone, suggesting the potential benefit of utilizing multiple miRNAs as biomarkers to offer more comprehensive and precise information about the disease state.…”

“…Researchers have also attempted to combine circulating biomarkers into panels to improve their diagnostic properties. 8,23 Combining multiple miRNAs yielded better diagnostic performance than using individual miRNAs alone, suggesting the potential benefit of utilizing multiple miRNAs as biomarkers to offer more comprehensive and precise information about the disease state. The negative correlation between miR-140-3p/miR-320b and miR-130a-3p may suggest that these two miR-NAs could be involved in opposing pathways or regulatory networks in AIS.…”

“…MicroRNAs (miRNAs) are small, noncoding, endogenous RNA molecule ranging in length from 18 to 25 nucleotides. Several clinical studies have reported that miRNAs are highly stable and differentially expressed in specific conditions, making them specifically associated with AIS 7,8 . MiRNAs can be easily isolated from different liquid biopsies with low invasiveness, such as whole blood, plasma, serum, circulating exosomes, peripheral blood cells and cerebrospinal fluid 8 .…”

“…7,8 MiRNAs can be easily isolated from different liquid biopsies with low invasiveness, such as whole blood, plasma, serum, circulating exosomes, peripheral blood cells and cerebrospinal fluid. 8 Therefore, many studies have taken advantage of these tools and suggested a panel of plasma miRNAs for AIS diagnosis due to their stability in plasma and ease of access. 9 This approach has drastically reduced treatment delays.…”

Thrombosis-related circulating miR-16-5p is associated with disease severity in patients hospitalised for COVID-19

“…MiRNAs have been studied as diagnostic and prognostic biomarkers for many diseases including myocardial infarction, liver failure, sepsis, or ischaemic stroke [ 39 , 40 ]. Whilst protein-based biomarkers for COVID-19 have been highly studied [ 41 , 42 ], however, limited number of studies aimed to analyse preselected circulating miRNAs in COVID-19 patients thus far [ 43–45 ].…”