Application of circulating micro nucleic acid in ischemic stroke diagnosis

Abstract

The invention provides a biomarker for indicating stroke, particularly ischemic stroke, and the biomarker comprises one or more of the following miRNAs (micro Ribonucleic Acid): hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p, or a combination comprising the four miRNAs (micro Ribonucleic Acid). The invention also provides a method for diagnosing stroke, especially ischemic stroke, by using the biomarker. The invention also provides a kit and a gene chip which contain the biomarker and are used for diagnosing stroke, especially ischemic stroke, and a preparation method of the kit and the gene chip.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and disease diagnostic reagent preparation. Specifically, the present invention relates to micronucleic acid biomarkers of ischemic stroke, and the use of the micronucleic acid biomarkers in preparing a kit or gene chip for diagnosing or detecting ischemic stroke. Background Art

[0002] In the past 20 years, stroke has been ranked as the second leading cause of death worldwide. Most strokes are ischemic strokes, which are caused by obstruction of blood supply to the brain due to embolism or other vascular diseases, resulting in insufficient supply of oxygen and nutrients, causing brain damage. The current diagnosis of ischemic stroke (IS) is mainly based on the use of computed tomography or magnetic resonance imaging after the patient is hospitalized. In the past decade, several studies have emerged suggesting other potential diagnostic approaches for different diseases. Micronucleic acids (miRNAs) have become one of the most promising types of biomarkers in these approaches. Their function is to regulate the degradation of mRNAs and the transcription and translation of their target genes. Various sources for extracting miRNAs have been proposed, such as plasma, serum, peripheral blood mononuclear cells (PBMCs), cerebrospinal fluid (CSF), saliva, and urine. Circulating miRNAs have become the focus because of their ease of detection, high accuracy, strong specificity, and good stability.

[0003] In recent years, the understanding of the role of miRNAs in stroke patients has greatly improved. Studies have shown that the expression levels of miRNAs are associated with the prognosis of stroke. Studies have also shown that specific circulating miRNAs can be used as a tool to predict stroke risk by combining multiple clinical risk factors such as age, sex, smoking status, blood pressure, and body mass index. Some of these miRNAs include miR-6124, miR-5196-5p, and miR-4292, whose expression levels in serum are associated with stroke risk.

[0004] Early prediction and diagnosis of stroke, especially ischemic stroke, is crucial to reduce the risk of permanent brain damage caused by delayed prevention and treatment. The art also needs a method, kit or gene chip for diagnosing stroke, especially ischemic stroke, especially accurately and quickly detecting or predicting stroke by the expression level of specific related miRNA. Summary of the invention

[0005] The present invention provides micronucleic acid (microRNA, or miRNA) biomarkers indicating stroke, particularly ischemic stroke. The present invention provides methods for diagnosing whether a subject has a stroke or is at risk of a stroke by detecting changes in the levels of the biomarker miRNA, as well as kits or gene chips for these methods.

[0006] In one aspect of the present invention, biomarkers for stroke, particularly ischemic stroke, are provided, wherein the markers include miRNAs associated with cell aging pathways and neuroinflammatory pathways. The biomarkers can be used to diagnose whether a subject has suffered from stroke, particularly ischemic stroke, or is at risk of developing stroke, particularly ischemic stroke.

[0007] In one aspect of the present invention, the miRNA related to the cellular aging pathway and the neuroinflammatory pathway in the marker is one or any combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p.

[0008] In one aspect of the present invention, the miRNA in the marker is a combination of hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p.

[0009] In one aspect of the invention, the miRNA in the marker is hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p, and one or any combination of the following mRNAs: hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p.

[0010] In one aspect of the present invention, the miRNA in the marker is a combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-124-3p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p.

[0011] In one aspect of the invention, the miRNA in the marker is a circulating miRNA. Circulating miRNA refers to miRNA present in the circulatory system of an animal such as a human body. In one aspect of the invention, the miRNA in the marker is miRNA present in plasma, serum, cerebrospinal fluid (CSF), saliva and urine.

[0012] The sequence information of miRNAs involved in the present invention can be obtained from websites or databases such as https: / / www.ncbi.nlm.nih.gov / or https: / / www.mirbase.org / .

[0013] The present invention provides a method for diagnosing whether a subject has a stroke, especially an ischemic stroke, or is at risk of a stroke, especially an ischemic stroke, by measuring the aforementioned biomarkers of stroke, especially ischemic stroke. The present invention also provides the use of a reagent for measuring the level of the aforementioned biomarkers of stroke, especially ischemic stroke, in the preparation of a kit or device for diagnosing stroke, especially ischemic stroke. In one aspect of the present invention, the reagent for measuring the level of the biomarker is used to measure the expression level of one or any combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p. In another aspect of the present invention, the miRNA of the biomarker is a combination of hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p. In yet another aspect of the present invention, the miRNA of the biomarker is one or any combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-124-3p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p.

[0014] In one aspect of the present invention, the method or the kit and device diagnose stroke, especially ischemic stroke, by the following steps: a. measuring the level of the miRNA marker in the subject sample; b. comparing the expression level of the miRNA marker in the subject sample with the level of the miRNA in the disease-free control sample. In another aspect of the present invention, when the level of the miRNA in the subject sample is significantly different from the level of the corresponding miRNA in the disease-free control sample, it indicates that the possibility of stroke, especially ischemic stroke, is greater. In another aspect of the present invention, when the level of the miRNA in the subject sample is compared with the level of the corresponding miRNA in the disease-free control sample, one or a combination of the following phenomena exists: 1. hesa-let-7e-5p is upregulated; 2. hsa-miR-17-5p is upregulated; 3. hsa-miR-185-5p is upregulated; or 4. hsa-miR-124-3p is downregulated, it indicates that the possibility of stroke, especially ischemic stroke, is greater. In one aspect of the present invention, the sample is a body fluid sample of the subject's circulatory system, such as plasma, serum, cerebrospinal fluid (CSF), saliva and urine. In one aspect of the present invention, the miRNA in the marker is a circulating miRNA. The aforementioned method or the kit and device also include a step and method of extracting circulating miRNA from body fluids.

[0015] The level of miRNA gene product in the sample can be measured using any technique suitable for detecting RNA expression levels in biological samples. Suitable techniques (e.g., Northern blot analysis, RT-PCR, in situ hybridization) for measuring RNA expression levels in biological samples (e.g., cells, tissues) are well known to those skilled in the art. In a specific embodiment, the level of at least one miRNA is detected using Northern blot analysis. For example, total cell RNA can be purified from cells as follows: homogenized in the presence of nucleic acid extraction buffer, followed by centrifugation. Precipitate nucleic acid, remove DNA by treating and precipitating with DNA enzymes. Then, according to standard techniques, separate RNA molecules by gel electrophoresis on an agarose gel and transfer to a nitrocellulose filter. Then, by heating, RNA is immobilized on a filter membrane. Use DNA or RNA probes that are appropriately labeled and complementary to the target RNA to detect and quantify specific RNA.

[0016] The relative number of miRNAs in cells can also be determined by reverse transcribing the miRNAs and subsequently amplifying the reverse transcribed transcripts by polymerase chain reaction (RT-PCR). Methods for quantitative and semi-quantitative RT-PCR and variations thereof are well known to those skilled in the art.

[0017] In addition, a library of a gene chip (i.e., a microarray) can also be constructed, which contains a set of oligonucleotide (e.g., oligodeoxynucleotide) probes specific for a set of miRNAs. By using the microarray, the expression levels of multiple miRNAs in a biological sample can be determined by reverse transcribing RNA to produce a set of target oligodeoxynucleotides, which are then hybridized with the probe oligodeoxynucleotides on the microarray to produce a hybridization or expression profile. The hybridization profile of the test sample is then compared with the control sample to determine the miRNA with an altered expression level in solid cancer cells. Gene chips, i.e., microarrays, can be prepared from gene-specific oligonucleotide probes generated from known miRNA sequences.

[0018] In one aspect of the present invention, a kit or device for diagnosing whether a subject has suffered from a stroke or is at risk of a stroke, especially an ischemic stroke, is also provided, which contains reagents for measuring the levels of the aforementioned markers of stroke, especially ischemic stroke. In one aspect of the present invention, the reagent for measuring the level of the biomarker is a reagent for measuring the expression level of one or any combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p. In another aspect of the present invention, the miRNA of the biomarker is a combination of hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p. In yet another aspect of the present invention, the miRNA of the biomarker is one or any combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-124-3p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p.

[0019] In one embodiment of the present invention, three of the markers are upregulated (hesa-let-7e-5p; hsa-miR-17-5p; hsa-miR-185-5p) and one is downregulated (hsa-miR-124-3p). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The tissue differential expression of 12 miRNAs is shown.

[0021] Figure 2Shown are the relative expression levels of 12 miRNAs obtained from the plasma of IS patients and healthy subjects (control).

[0022] Figure 3 Shown are the enriched Reactome pathways targeted by the four validated miRNAs.

[0023] Figure 4 Enriched KEGG pathways targeted by the four validated miRNAs are shown.

[0024] Figure 5A Showing the interaction between miRNAs and targeted mRNAs in cellular aging pathways.

[0025] Figure 5B Showing the interactions between miRNAs and targeted mRNAs in neuroinflammatory pathways.

[0026] Figure 6 ROC curves showing the effects of the four miRNAs alone or in combination in distinguishing IS from healthy subjects. DETAILED DESCRIPTION

[0027] The essential contents and beneficial effects of the present invention will be further described below in conjunction with embodiments, which are only used to illustrate the present invention rather than to limit the present invention.

[0028] Example 1 Materials and Methods

[0029] Tissue-specific expression analysis of 12 miRNAs

[0030] Tissue enrichment analysis of the collected miRNAs was performed using data obtained from the Human miRNA tissue atlas. Quantified normalized data were used to visualize the expression level of each miRNA in 31 tissues. Expression levels of multiple samples obtained from the same tissue were grouped as their mean. The relative expression level of each miRNA in each tissue was calculated as z-scores for visualization.

[0031] RNA extraction from plasma

[0032] Human plasma was prepared from peripheral blood. Briefly, blood samples were obtained using EDTA tubes according to standard procedures 24 h after stroke onset. The samples were immediately placed on ice and centrifuged at 1000 g for 15 min at 4 °C. Total RNA was extracted from 200 μl of plasma using the Universal Extraction Kit (GeneDotech, #GD-101) according to the manufacturer's instructions. Total RNA was eluted by adding 15 μl of nuclease-free water and stored at -80 °C. Twenty patients with IS and 20 healthy subjects were recruited in this study. The recruited ischemic patients were defined as acute focal neurological deficits with the presence of diffusion-weighted imaging-positive lesions on magnetic resonance imaging or new lesions on delayed CT scans. The collection time point was 24 h after stroke onset. The collection and use of specimens in this experiment were signed and confirmed by patients and healthy subjects. The design of this study was approved by the corresponding Ethics Review Committee of Shenzhen Second People's Hospital (No.: 20200601022-FS01). The consent form was approved by the Medical Ethics Committee of Shenzhen Second People's Hospital.

[0033] cDNA synthesis and real-time PCR

[0034] According to the manufacturer's instructions (GeneDotech, #GD-102), 5 μl of eluted RNA was reverse transcribed in a 20 μl reaction. In short, 5 microliters of RNA were placed in a final volume of 20 microliters of transcription master solution, incubated at 42°C for 1 hour, and then inactivated at 95°C for 5 minutes. According to the instructions of PCR master mix (Probe) (GeneDotech, #GD-105), cDNA was diluted and detected in a 10 μl PCR reaction. Probe-based real-time PCR was used to quantify miRNAs. Amplification was performed in a 96-well plate of the StepOne plus detection system (Applied Biosystems), and each sample was triplicate. Amplification curves were analyzed and Ct was determined using ABI SDS software. The gene expression levels of the selected miRNAs were expressed as ΔCt relative to the average Ct value of external references (including cel-miR-39, 54 and 238). The fold change was calculated relative to the healthy group.

[0035] Biological significance

[0036] The target genes of each miRNA identified from the analysis were retrieved by R package multiMiR (Version 1.4.0). Only valid miRNA-target gene interactions obtained from mirTarBase were included. Pathway enrichment analysis of these target genes was performed by R packages ReactomePA (Version 1.26.0) and clusterProfiler (Version 3.10.1) based on Reactome and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. Gene Ontology (GO) analysis was used to analyze the enrichment of biological processes. A pathway or biological process was considered significantly enriched if its FalseDiscovery Rate (FDR)-adjusted P value was less than 0.05. The microRNA Target Filter module of IngenuityPathway Analysis (IPA) was used to further investigate the miRNA-mRNA interactions and their associated pathways. The miRNA-mRNA interaction network was drawn using PathDesigner in IPA software.

[0037] Receiver operating characteristic analysis

[0038] Receiver operating characteristic curve (ROC curve) analysis was performed using plasma samples from 20 IS patients and 20 healthy subjects collected from the hospital. Logistic models were established based on the normalized expression levels of each miRNA and disease group. Rpackage "pROC" was used to calculate the AUC value of each model and visualize the ROC curve.

[0039] Statistical analysis

[0040] Statistical analysis was performed using GraphPad Prism 8.2 and SPSS 19.0 statistical software packages, and Student's t-test (two-tailed) was used for statistical analysis in qRT-PCR between two groups of data. P values ​​< 0.05 were considered statistically significant.

[0041] Example 2 Meta-Analysis

[0042] Data collection and analysis

[0043] Papers of relevant studies were collected on PubMed from January 1, 1994 to December 31, 2022. The following search terms were used to search for studies on PubMed: ("stroke") AND ("Blood" OR "Serum" OR "Plasma" OR "Circulat*" OR "Peripheral" OR "PBMC") AND ("miRNA" OR "microRNA" OR "miR"). The titles, abstracts, and full texts of the collected studies were then screened and selected according to the eligibility criteria.

[0044] Eligibility Criteria

[0045] Studies were included in this meta-analysis if they: (1) were primary studies; (2) were case-control studies; (3) analyzed circulating human miRNAs in IS patients; (4) reported the type of specimens used; (5) reported the sample size of the case and control groups; (6) reported the miRNA analysis methods; (7) reported the statistical significance of each miRNA and its dysregulation status; (8) did not perform irrelevant comparisons. Excluded studies were: (1) were review articles; (2) did not include case and control (stroke and healthy) samples; (3) analyzed miRNAs in animal models or cell lines other than human blood, plasma, or serum; (4) did not report the type of specimens used; (5) did not report the sample size of the case and control groups; (6) did not report the miRNA analysis methods; (7) did not report the statistical significance of each miRNA and its dysregulation status; (8) performed comparisons other than stroke and healthy controls.

[0046] Meta-analysis to identify differentially expressed miRNAs

[0047] Meta-analysis was performed for each miRNA differentially expressed in IS patients in more than one study. The names of miRNAs were standardized using the R package miRNAmeConverter (Version 1.10.0) according to the miRBase database (version 22.0). The R package metafor (Version 3.0.2) was used to calculate the effect size (θ) of each eligible miRNA in the study. i ), and log ORs were calculated independently using a random effects model.

[0048] Results were considered significant if the P value was less than 0.05, and miRNAs were considered up-regulated or down-regulated if the overall effect size was greater or less than 0, respectively.

[0049] A total of 823 articles were found on PMC using the search terms. After excluding literature studies that did not meet the eligibility criteria, 29 studies remained. Among these studies, 14 measured miRNA levels in patient serum, 7 measured miRNA in plasma, 6 measured whole blood, and 2 measured peripheral blood mononuclear cells.

[0050] Differentially expressed miRNAs in meta-analysis

[0051] Twelve miRNAs were identified as significantly dysregulated by meta-analysis with p < 0.05, of which eight were upregulated (hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p), while four were downregulated (hsa-miR-124-3p, hsa-miR-126-3p, hsa-miR-130a-3p, hsa-miR-221-3p).

[0052] Tissue-specific expression levels of 12 miRNAs

[0053] To investigate the relationship between tissue specificity and IS, the expression levels of the aforementioned 12 identified miRNAs in multiple tissues were investigated. hsa-miR-487b-3p, hsa-miR-9-5p, and hsa-miR-124-3p were specifically expressed in central nervous system (CNS) tissues, including arachnoid, brain, dura mater, and spinal cord, while hsa-let-7e-5p and hsa-miR-218-5p were also highly expressed in CNS tissues. On the other hand, hsa-miR-17-5p, hsa-miR-185-5p, and hsa-miR-451a were found to be highly expressed in veins ( Figure 1 ).

[0054] Example 3 Validation of miRNAs in plasma of IS patients using qRT-PCR

[0055] Plasma samples were used to perform qRT-PCR validation of the 12 miRNAs identified from the meta-analysis. The relative expression levels of each miRNA in IS (n = 20) and healthy (n = 20) samples were measured ( Figure 2). Compared with the healthy control group, the expression levels of four miRNAs were significantly dysregulated in IS patients, of which three were upregulated (hsa-let-7e-5p, Fold-change [FC] = 1.50, p = 0.011; hsa-miR-17-5p, FC = 1.42, p = 0.015; hsa-miR-185-5p, FC = 1.83, p = 0.0019) and one was downregulated (hsa-miR-124-3p, FC = 0.68, p = 0.044). The dysregulation status of these miRNAs was in the same direction as the meta-analysis results, further indicating that these four miRNAs are biomarkers for IS patients.

[0056] Example 4 Biological significance of 4 validated miRNAs

[0057] A total of 3201 unique target genes were identified from the above four validated miRNAs. Pathway enrichment analysis identified 310 Reactome pathways and 103 KEGG pathways that were significantly enriched. For the enriched Reactome pathways, many of them were involved in the response to oxygen and glucose depletion caused by IS, including the response to stress, and also involved in the cell senescence pathway, the regulation pathway of cell cycle progression, the VEGF signaling pathway, and the AKT signaling pathway ( Figure 3 ). Other pathways related to neuroinflammatory responses were also involved, such as FoxO-mediated transcription and Toll-like receptor (TLR) cascades. These pathways are involved in apoptosis, as well as mediating cell proliferation and angiogenesis to compensate for the lack of blood supply to vital organs. Similar observations were obtained for the enriched KEGG pathways ( Figure 4 ).

[0058] IPA analysis showed that the above four validated miRNAs target 221 mRNAs known to be associated with inflammatory response, cardiovascular or nervous system diseases. The study found that different mRNAs are often co-targeted by multiple miRNAs. The relevant pathways of all these 221 mRNAs include IS-related pathways, such as the cellular senescence pathway and the neuroinflammation pathway ( Figure 5A and Figure 5B). The target genes of the four miRNAs are involved in multiple steps of the two pathways shown in the figure. Genes with important roles ranging from activation and transduction of signaling cascades to direct cellular senescence are targets of these miRNAs. In particular, TGFBR2, which leads to the transcription of CDKN1A, is targeted by hsa-let-7e-5p and hsa-miR-17-5p, and CDK6, which inhibits retinoblastoma protein (pRB), is targeted by hsa-let-7e-5p, hsa-miR-124-3p, and hsa-miR-185-5p. In neuroinflammation, ligands released by damaged neurons and macrophages bind to receptors expressed on microglia, activating multiple signaling cascades. In particular, hsa-miR-124-3p targets multiple important genes for neuroinflammation, including GSK3B, CREB3L2, MAPK14, RELA, and BDNF. These genes interact directly or indirectly to mediate the production of pro-inflammatory or anti-inflammatory proteins, leading to further neuronal damage or neuronal growth and repair.

[0059] Example 5 Diagnostic value of 4 validated miRNAs

[0060] To investigate the diagnostic value of the identified miRNAs, ROC curve analysis cross-validation was performed on the four previously validated miRNAs in the plasma of all 40 samples collected from the hospital.

[0061] The analysis showed that hsa-miR-185-5p showed the highest diagnostic value (AUC = 0.788), followed by hsa-let-7e-5p (AUC = 0.751), hsa-miR-17-5p (AUC = 0.718) and hsa-miR-124-3p (AUC = 0.675). When these four miRNAs were combined, the AUC increased to 0.873 ( Figure 6 ).

[0062] in conclusion

[0063] The present invention provides miRNA biomarkers that can distinguish and define the occurrence of stroke, particularly ischemic stroke. The miRNA biomarkers for stroke provided by the present invention are one or more of hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p, or a combination of these four miRNAs, or further a combination of 12 miRNAs (hsa-let-7e-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-124-3p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p). The inventors of the present application have discovered for the first time the role of specific miRNAs in pathways associated with stroke, especially ischemic stroke, and have particularly discovered the association between these miRNAs or their target genes in pathways associated with stroke, and have thus provided the use of a combination of these miRNAs in the comprehensive and accurate identification of stroke, especially ischemic stroke. The biomarker combination discovered by the present invention can effectively and accurately identify stroke, and the present invention thus provides a method for distinguishing and identifying stroke.

[0064] The above is an explanation of the present invention and cannot be regarded as a limitation of the present invention. Unless otherwise noted, the practice of the present invention will use the conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc., and it is obvious that the present invention can also be realized in other ways except that it is particularly described in the above description and embodiments. Other aspects and improvements within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. According to the teachings of the present invention, many changes and variations are feasible, and therefore they are within the scope of the present invention.

Claims

1. Markers for diagnosing stroke, including microRNAs (miRNAs) related to cell aging pathways and neuroinflammatory pathways, The miRNAs related to the cell aging pathway and the neuroinflammatory pathway are a combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p.

2. The marker of claim 1, wherein the stroke is an ischemic stroke.

3. The marker according to claim 1 or 2, wherein the miRNA is hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p, and one or any combination of the following mRNAs: hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p.

4. The marker according to claim 1 or 2, wherein the miRNA is a combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-218-5p, hsa-miR-222-3p, hsa-miR-451a, hsa-miR-487b-3p, hsa-miR-9-5p, hsa-miR-126-3p, hsa-miR-130a-3p and hsa-miR-221-3p. The marker according to claim 1 , wherein the microRNA is a circulating microRNA.

6. A kit or device for diagnosing stroke, comprising a reagent for measuring the level of a marker according to any one of claims 1 to 5, wherein the marker comprises microRNAs (miRNAs) associated with a cell aging pathway and a neuroinflammatory pathway, and the kit or device comprises a reagent for measuring the level of miRNAs, The miRNAs related to the cell aging pathway and the neuroinflammatory pathway include a combination of the following miRNAs: hsa-let-7e-5p, hsa-miR-124-3p, hsa-miR-17-5p and hsa-miR-185-5p.

7. The kit or device according to claim 6, wherein the reagent for measuring the level of miRNA is a reagent for reverse transcription of the miRNA and / or a reagent for amplifying or identifying the reverse transcription product.

8. The kit or device according to claim 6, wherein the kit or device diagnoses stroke by the following steps: a. measuring the level of the marker in a sample from a subject; b. comparing the level of said marker in the subject's sample to the expression level of said marker in a disease-free control.

9. The kit or device of claim 6, wherein the microRNA is a circulating microRNA.

10. The kit or device according to claim 6, wherein the device is a gene chip.

Images