Background: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS. Methods: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1–3 and days 4–7); one for the subacute phase IS (SIS, days 8–14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron -microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction. Results: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1–3) were lower than that in controls (p < 0.05). In AIS (days 1–3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570–0.859, p = 0.007), 0.734 for RIS (95% CI 0.596–0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665–0.988, p = 0.001), 0.438 for AIS (days 1–3; 95% CI 0.240–0.635, p = 0.516). Conclusions: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy.

1.
Volny O, Kasickova L, Coufalova D, Cimflova P, Novak J: microRNAs in cerebrovascular disease. Adv Exp Med Biol 2015; 888: 155–195.
2.
Sahota P, Savitz SI: Investigational therapies for ischemic stroke: neuroprotection and neurorecovery. Neurotherapeutics 2011; 8: 434–451.
3.
Prabhakaran S, Ruff I, Bernstein RA: Acute stroke intervention: a systematic review. JAMA 2015; 313: 1451–1462.
4.
Stefanovic Budimkic M, Pekmezovic T, Beslac-Bumbasirevic L, Ercegovac M, Berisavac I, Stanarcevic P, Padjen V, Jovanovic DR: Long-term prognosis in ischemic stroke patients treated with intravenous thrombolytic therapy. J Stroke Cerebrovasc Dis 2017; 26: 196–203.
5.
Uno J, Kameda K, Otsuji R, Ren N, Nagaoka S, Maeda K, Ikai Y, Gi H: Mechanical thrombectomy for acute basilar artery occlusion in early therapeutic time window. Cerebrovasc Dis 2017; 44: 217–224.
6.
Pang XM, Liu JL, Li JP, Huang LG, Zhang L, Xiang HY, Feng LB, Chen CY, Li SH, Su SY: Fastigial nucleus stimulation regulates neuroprotection via induction of a novel microRNA, rno-miR-676-1, in middle cerebral artery occlusion rats. J Neurochem 2015; 133: 926–934.
7.
Jickling GC, Sharp FR: Biomarker panels in ischemic stroke. Stroke 2015; 46: 915–920.
8.
Vijayan M, Reddy PH: Peripheral biomarkers of stroke: focus on circulatory microRNAs. Biochim Biophys Acta 2016; 1862: 1984–1993.
9.
Sepramaniam S, Tan JR, Tan KS, DeSilva DA, Tavintharan S, Woon FP, Wang CW, Yong FL, Karolina DS, Kaur P, Liu FJ, Lim KY, Armugam A, Jeyaseelan K: Circulating microRNAs as biomarkers of acute stroke. Int J Mol Sci 2014; 15: 1418–1432.
10.
Bhalala OG, Srikanth M, Kessler JA: The emerging roles of microRNAs in CNS injuries. Nat Rev Neurol 2013; 9: 328–339.
11.
Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K: The microRNA spectrum in 12 body fluids. Clin Chem 2010; 56: 1733–1741.
12.
Tan KS, Armugam A, Sepramaniam S, Lim KY, Setyowati KD, Wang CW, Jeyaseelan K: Expression profile of MicroRNAs in young stroke patients. PLoS One 2009; 4:e7689.
13.
Piehl F, Jagodic M: MicroRNAs as promising novel biomarkers and potential drug targets for inflammatory neurological diseases. J Neurol Sci 2015; 356: 3–4.
14.
Mirzaei H, Momeni F, Saadatpour L, Sahebkar A, Goodarzi M, Masoudifar A, Kouhpayeh S, Salehi H, Mirzaei HR, Jaafari MR: MicroRNA: relevance to stroke diagnosis, prognosis, and therapy. J Cell Physiol 2018; 233: 856–865.
15.
Qu S, Guan J, Liu Y: Identification of microRNAs as novel biomarkers for glioma detection: a meta-analysis based on 11 articles. J Neurol Sci 2015; 348: 181–187.
16.
Tkach M, Thery C: Communication by -extracellular vesicles: where we are and where we need to go. Cell 2016; 164: 1226–1232.
17.
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange -between cells. Nat Cell Biol 2007; 9: 654–659.
18.
Khalyfa A, Gozal D: Exosomal miRNAs as potential biomarkers of cardiovascular risk in children. J Transl Med 2014; 12: 162.
19.
Long G, Wang F, Li H, Yin Z, Sandip C, Lou Y, Wang Y, Chen C, Wang DW: Circulating miR-30a, miR-126 and let-7b as biomarker for ischemic stroke in humans. BMC Neurol 2013; 13: 178.
20.
Wang W, Sun G, Zhang L, Shi L, Zeng Y: Circulating microRNAs as novel potential biomarkers for early diagnosis of acute stroke in humans. J Stroke Cerebrovasc Dis 2014; 23: 2607–2613.
21.
Tsai PC, Liao YC, Wang YS, Lin HF, Lin RT, Juo SH: Serum microRNA-21 and microRNA-221 as potential biomarkers for cerebrovascular disease. J Vasc Res 2013; 50: 346–354.
22.
The World Health Organization MONICA Project (monitoring trends and determinants in cardiovascular disease): a major international collaboration. WHO MONICA project principal investigators. J Clin Epidemiol 1988; 41: 105–114.
23.
Roldan-Valadez E, Gonzalez-Gutierrez O, Martinez-Lopez M: Diagnostic performance of PWI/DWI MRI parameters in discriminating hyperacute versus acute ischaemic stroke: finding the best thresholds. Clin Radiol 2012; 67: 250–257.
24.
Brott T, Adams HP, Jr., Olinger CP, Marler JR, Barsan WG, Biller J, Spilker J, Holleran R, Eberle R, Hertzberg V, et al: Measurements of acute cerebral infarction: a clinical examination scale. Stroke 1989; 20: 864–870.
25.
Li SH, Su SY, Liu JL: Differential Regulation of microRNAs in Patients with Ischemic Stroke. Curr Neurovasc Res 2015; 12: 214–221.
26.
Adams HP, Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE, 3rd: Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in acute stroke treatment. Stroke 1993; 24: 35–41.
27.
Crossland RE, Norden J, Bibby LA, Davis J, Dickinson AM: Evaluation of optimal extracellular vesicle small RNA isolation and qRT-PCR normalisation for serum and urine. J Immunol Methods 2016; 429: 39–49.
28.
Shi R, Sun YH, Zhang XH, Chiang VL: Poly(T) adaptor RT-PCR. Methods Mol Biol 2012; 822: 53–66.
29.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408.
30.
Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, Sprenger-Haussels M, Shaffer JM, Lader E, Skog J, Noerholm M: Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One 2015; 10:e0136133.
31.
Mirzaei H, Naseri G, Rezaee R, Mohammadi M, Banikazemi Z, Mirzaei HR, Salehi H, Peyvandi M, Pawelek JM, Sahebkar A: Curcumin: A new candidate for melanoma therapy? Int J cancer 2016; 139: 1683–1695.
32.
Barteneva NS, Fasler-Kan E, Bernimoulin M, Stern JN, Ponomarev ED, Duckett L, Vorobjev IA: Circulating microparticles: square the circle. BMC Cell Biol 2013; 14: 23.
33.
Khoury M, Alcayaga-Miranda F, Illanes SE, Figueroa FE: The Promising Potential of Menstrual Stem Cells for Antenatal Diagnosis and Cell Therapy. Front Immunol 2014; 5: 205.
34.
Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY: Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008; 18: 997–1006.
35.
Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M: Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 2011; 108: 5003–5008.
36.
Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C: Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005; 17: 879–887.
37.
Ji Q, Ji Y, Peng J, Zhou X, Chen X, Zhao H, Xu T, Chen L, Xu Y: Increased brain-specific MiR-9 and MiR-124 in the serum exosomes of acute ischemic stroke patients. PLoS One 2016; 11:e0163645.
38.
Wang F, Long G, Zhao C, Li H, Chaugai S, Wang Y, Chen C, Wang DW: Atherosclerosis-related circulating miRNAs as novel and sensitive predictors for acute myocardial infarction. PLoS One 2014; 9:e105734.
39.
Punga AR, Andersson M, Alimohammadi M, Punga T: Disease specific signature of circulating miR-150–5p and miR-21–5p in myasthenia gravis patients. J Neurol Sci 2015; 356: 90–96.
40.
Matsuzaki K, Fujita K, Jingushi K, Kawashima A, Ujike T, Nagahara A, Ueda Y, Tanigawa G, Yoshioka I, Ueda K, Hanayama R, Uemura M, Miyagawa Y, Tsujikawa K, Nonomura N: MiR-21–5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma. Oncotarget 2017; 8: 24668–24678.
41.
Kumarswamy R, Volkmann I, Thum T: Regulation and function of miRNA-21 in health and disease. RNA Biol 2011; 8: 706–713.
42.
Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, Zhang C: MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 2009; 284: 29514–29525.
43.
Zhou J, Zhang J: Identification of miRNA-21 and miRNA-24 in plasma as potential early stage markers of acute cerebral infarction. Mol Med Rep 2014; 10: 971–976.
44.
Xiang H, Tao X, Xia S, Qu J, Song H, Liu J, Shang D: Emodin alleviates sodium taurocholate-induced pancreatic acinar cell injury via microRNA-30a-5p-mediated inhibition of high-temperature requirement a/transforming growth factor beta 1 inflammatory signaling. Front Immunol 2017; 8: 1488.
45.
Fu X, Shen Y, Wang W, Li X: MiR-30a-5p ameliorates spinal cord injury-induced inflammatory responses and oxidative stress by targeting Neurod 1 through MAPK/ERK signalling. Clin Exp Pharmacol Physiol 2018; 45: 68–74.
46.
Fu LL, Wen X, Bao JK, Liu B: MicroRNA-modulated autophagic signaling networks in cancer. Int J Biochem Cell Biol 2012; 44: 733–736.
47.
Hill MD, Jackowski G, Bayer N, Lawrence M, Jaeschke R: Biochemical markers in acute ischemic stroke. CMAJ 2000; 162: 1139–1140.
48.
Jickling GC, Sharp FR: Blood biomarkers of ischemic stroke. Neurotherapeutics 2011; 8: 349–360.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.