Composition for diagnosing myocardial infarction and pharmaceutical composition for preventing or treating myocardial infarction

Abstract

The present invention relates to a composition for diagnosing myocardial infarction, a diagnostic kit comprising same, a method for providing information for diagnosing myocardial infarction, and a pharmaceutical composition for preventing or treating myocardial infarction. The composition, kit, and method of the present invention use exosome markers miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p, and miR-7225-5p, and thus are expected to be effective in diagnosis for the prevention of myocardial infarction and useful in developing therapeutic substances.

Description

Composition for diagnosing myocardial infarction and pharmaceutical composition for preventing or treating myocardial infarction

[0001] The present invention relates to a composition for diagnosing myocardial infarction, a diagnostic kit, and a method for providing information for diagnosing myocardial infarction. Furthermore, the present invention relates to a pharmaceutical composition for preventing or treating myocardial infarction.

[0002] Cardiovascular disease (CVD) is a leading cause of death in industrialized countries worldwide. It has also been a leading cause of death in Korea since the 1970s, and it is one of the fastest-growing diseases among the top ten causes of death in Koreans. The incidence of coronary artery disease, a leading cardiovascular disease, increased tenfold in less than 20 years, from 2.2 per 100,000 people in 1983 to 21.9 in 2001. Today, the incidence of patients with arteriosclerosis-related diseases is nearly comparable to that of cancer. The market size is estimated at hundreds of billions of won.

[0003] These cardiovascular diseases include myocardial infarction, angina, atherosclerosis, hypertension, heart failure, aneurysms, stroke, and arrhythmia. Coronary artery disease, a major cardiovascular disease, is usually caused by arteriosclerosis, which blocks or narrows the coronary arteries that supply blood to the heart. Myocardial infarction occurs when a coronary artery is completely blocked by arteriosclerosis, resulting in the death of heart muscle tissue. Angina, on the other hand, is chest pressure or pain caused by a narrowed coronary artery.

[0004] Traditionally, cardiovascular disease could only be diagnosed using physical methods when the disease was already advanced, limiting early diagnosis and prediction. Conventional diagnosis involves X-rays and ultrasound imaging of the heart and coronary arteries using contrast-enhanced equipment, but these methods can only be used after the disease has developed.

[0005] To predict cardiovascular disease, basic lipid-related tests (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides) and hsCRP, an inflammation test, are performed. However, these blood tests themselves do not directly reflect the individual's arteriosclerosis status, and can be confounding factors in the presence of underlying infectious or inflammatory vascular diseases. For this reason, the role of existing blood markers in confirming predictions through additional radiological tests, such as ultrasound, computed tomography, and magnetic resonance imaging, or through invasive testing, is very limited.

[0006] Because cardiovascular diseases often appear suddenly and without symptoms, they are closely linked to human life and functional limitations. Therefore, predicting and diagnosing advanced cardiovascular disease early on is crucial to saving lives and improving quality of life. However, the multiple tests required increase medical costs, and screening tests often face limitations in their universal application. Therefore, the need for biomarkers that facilitate easy diagnosis is growing.

[0007] The technical problem to be achieved by the present invention is to provide a composition and kit for diagnosing myocardial infarction.

[0008] In addition, a technical task to be achieved by the present invention is to provide a method for providing information for diagnosing myocardial infarction.

[0009] In addition, the technical problem to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating myocardial infarction.

[0010] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0011] In order to achieve the above technical task, one embodiment of the present invention provides a composition for diagnosing myocardial infarction, which comprises a substance that specifically binds to at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p.

[0012] In an embodiment of the present invention, the miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p may be present in exosomes isolated from heart tissue.

[0013] In an embodiment of the present invention, the material may include one or more of a primer, a probe, and an antisense nucleotide.

[0014] In order to achieve the above technical task, another embodiment of the present invention provides a kit for diagnosing myocardial infarction comprising the composition.

[0015] In order to achieve the above technical task, another embodiment of the present invention provides an information providing method for diagnosing myocardial infarction, comprising the step of measuring the expression level of at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p in a biological sample isolated from an individual.

[0016] In an embodiment of the present invention, if the expression level is lower than that of a normal control group, it may be determined that the possibility of developing myocardial infarction is higher than that of the control group.

[0017] In an embodiment of the present invention, the biological sample may be selected from the group consisting of tissue, cell, blood, serum, plasma, saliva, or urine.

[0018] In an embodiment of the present invention, the biological sample may be an exosome isolated from cardiac tissue of an individual.

[0019] In order to achieve the above technical task, another embodiment of the present invention provides a pharmaceutical composition for preventing or treating myocardial infarction comprising miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p or miR-7225-5p.

[0020] In an embodiment of the present invention, the composition may reduce cell death of cardiomyocytes or fibroblasts of cardiac tissue.

[0021] The present invention relates to a composition for diagnosing myocardial infarction, a diagnostic kit including the same, a method for providing information for diagnosing myocardial infarction, and a pharmaceutical composition for preventing or treating myocardial infarction. The composition, kit, and method of the present invention comprise exosomal markers miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, By utilizing miR-5107-5p and miR-7225-5p, it is expected that they will be effective in diagnosing and developing therapeutic substances for preventing myocardial infarction.

[0022] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0023] Figure 1 shows the experimental outline and characterization of exosomes extracted from cardiac tissue. (1A) Schematic representation of the experimental procedures used in the present study. (1B) Group images of hearts from myocardial infarction (MI) mouse models stained with or without triphenyltetrazolium chloride (TTC) to demonstrate morphological differences between groups. (1C) Transmission electron microscopy (TEM) images of isolated exosomes stained with phosphotungstic acid to demonstrate structural features. (1D) Immunoblot analysis of isolated exosomes using exosomal markers, including CD9, CD63, and CD81, along with an internal control. Group designations: Sham, control; MI-day 1, MI induced for 1 day; MI-day 3, MI induced for 3 days.

[0024] Figure 2 shows the comprehensive analysis results of exosomal RNA (exoRNA) sequencing data. (2A) Shows the distribution of the number of various read types for each group, including trimmed reads, nonadapter reads, short reads, and low-quality reads. (2B) Shows the reads (blue) remaining after removing ribosomal RNA (rRNA; red). (2C) Shows the read length distribution for each sample. (2D) Provides an analysis of the smRNA composition within each sample, which is classified into various types, such as miRNA (microRNA), piRNA (PIWI-interacting RNA), snoRNA (small nucleolar RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), siRNA (small interfering RNA), Y RNA, and scRNA (single-cell RNA).

[0025] Figure 3 shows the analysis of differentially expressed (DE) miRNAs using exosomal RNA (exoRNA) sequencing. (3A) Correlation Matrix: This panel provides a correlation matrix of all samples calculated using the Pearson coefficient based on normalized values. The correlation coefficient (r) ranges from -1 to 1, with values ​​closer to 1 indicating greater similarity between samples. (3B) Hierarchical Clustering: The left part of this panel shows hierarchical clustering of samples based on normalized expression normalization values, where samples with higher expression similarity are grouped together (distance metric = Euclidean distance, linkage method = full linkage) (left). The right part provides a heat map of two-way hierarchical clustering utilizing the Z-score of the normalized values, which are Log2-transformed for visualization. (3C) Quantitative Analysis of MicroRNAs (miRNAs): This section displays the number of mature miRNAs that are upregulated or downregulated based on the fold change (|FC| > 2) and p-value (p < 0.05) for each comparison pair. (3D) Smear Plot: This panel shows a smear plot representing the expression levels of miRNAs. The plot is designed to visually represent the distribution and changes in miRNA expression across samples.

[0026] Figures 4a, 4b, and 4c show comprehensive functional enrichment analyses of differentially expressed miRNAs. (4a and 4b) The top seven gene ontology (GO) terms are described, providing insights into the biological processes, cellular components, and molecular functions most affected by dysregulated miRNAs. (4c) The top seven pathways identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis are described, highlighting important pathways affected by altered mRNA expression across various groups.

[0027] Figure 5 illustrates the identification and selection of key candidate microRNAs (miRNAs). (5A) A schematic representation of the number of miRNAs showing increased (top) or decreased (bottom) expression in a cross-group comparison analysis. This visualization helps understand the overall distribution and directional trends of miRNA expression changes. (5B) A dotted line graph depicting the expression profiles of 20 miRNAs selectively selected across groups, allowing for comparison and detailed analysis of expression dynamics.

[0028] Figures 6 and 7 show the results of cytotoxicity assays using selected miRNA mimics in primary cardiomyocytes and fibroblasts. Immunofluorescence analysis confirmed the presence of specific markers (troponin-T in primary cardiomyocytes and vimentin in primary fibroblasts) in the isolated cells. Furthermore, the effect of miRNA mimic treatment on cell survival under hypoxic conditions was investigated. In the attached bar graphs, white and black bars represent the control (miRNA mimic negative control treatment) group, and gray bars represent the miRNA mimic-treated group. Statistical significance between the control and treatment groups was assessed using ANOVA. The p-value annotations indicate the significance level. *p < 0.05 and **p < 0.01 highlight significant differences in cytotoxicity responses. Error bars represent the standard deviation among five wells of the 96-well plate used for the cytotoxicity assay. N, normoxia; H, hypoxia; FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole; miR, microRNA.

[0029]

[0030] Hereinafter, the present invention will be described in detail.

[0031] The present invention relates to a composition for diagnosing myocardial infarction.

[0032] The composition of the present invention comprises a substance that specifically binds to at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p.

[0033] The above miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p are present in the sample derived from the diagnostic subject and may be present in exosomes isolated from heart tissue.

[0034] The sequences of the above miRNAs can be found in known genetic databases such as miRBASE (http: / www.mirbase.org / ) and the National Institutes of Health GenBank (NIH GenBank), and are summarized in Table 1.

[0035] Detection of exosomal miRNAs can typically be accomplished by extracting exosomes from a sample and detecting miRNAs within the extracted exosomes. Detection of exosomal miRNAs can be measured by hybridization and amplification reactions, but is not limited thereto and can be easily accomplished using various techniques known in the art.

[0036] The above diagnostic subjects are animals that currently have myocardial infarction, animals that have experienced myocardial infarction, or animals for which information for predicting or diagnosing the onset of myocardial infarction is desired, and the animals may be mammals including humans.

[0037] The above sample is separated from the diagnostic subject, and may be, for example, tissue, cell, blood, serum, plasma, saliva or urine, and specifically, may be exosomes separated from heart tissue of an individual separated from the diagnostic subject, but is not limited thereto.

[0038] The above material may comprise one or more of a primer, a probe, and an antisense nucleotide.

[0039] Detection of miRNAs using primers can be performed by amplifying a gene sequence using an amplification method such as PCR and then confirming whether the gene is amplified using a method known in the art.

[0040] A primer is a short nucleic acid sequence with a free 3' hydroxyl group at the terminal end, capable of forming base pairs with a complementary template and serving as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In the present invention, the presence of myocardial infarction can be diagnosed by performing PCR amplification using sense and antisense primers that specifically bind to the miRNA to determine the expression level. PCR conditions and the lengths of the sense and antisense primers can be modified based on those known in the art.

[0041] A probe is a labeled nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several dozen bases. Probes can be produced in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, etc. In the present invention, the presence of myocardial infarction can be diagnosed by hybridizing a probe complementary to the miRNA to determine the expression level. The selection of an appropriate probe and hybridization conditions can be modified based on those known in the art.

[0042] Such primers or probes can be appropriately designed by those skilled in the art based on known sequences.

[0043] For example, primers or probes can be chemically synthesized using phosphoramidite solid support methods or other well-known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0044] The composition of the present invention is a substance for use in predicting or diagnosing myocardial infarction in a patient suspected of having myocardial infarction, and is treated on a sample isolated from the subject of diagnosis to determine the expression level of at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p in the sample. It can be measured and used to predict or diagnose myocardial infarction in suspected patients.

[0045] For example, if the expression level of at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p, and miR-7225-5p in a sample of a patient with suspected myocardial infarction is compared with the expression level of a normal control, and the expression level of the patient with suspected myocardial infarction is lower than the expression level of the normal control, It can be judged that the risk of developing myocardial infarction is higher compared to the normal control group.

[0046] The present invention relates to a kit for diagnosing myocardial infarction comprising the above composition.

[0047] The above kit may include not only a preparation for measuring the expression level of the above miRNA, but also tools, reagents, etc. commonly used in the art suitable for use as a kit for diagnosing myocardial infarction.

[0048] Examples of the above tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, etc. When the labeling substance is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. The carrier may be a soluble carrier or an insoluble carrier. An example of a soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of an insoluble carrier may be a polymer such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharides, magnetic microparticles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

[0049] For example, the diagnostic kit of the present invention may be a kit containing the essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker miRNA, the RT-PCR kit may include a test tube or other appropriate container, a reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC water, sterile water, and the like.

[0050] The present invention relates to a method for providing information for diagnosing myocardial infarction.

[0051] The method of the present invention comprises a step of measuring the expression level of at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p in a biological sample isolated from an individual.

[0052] The above measurement may be performed by treating the sample with a substance that specifically binds to the miRNA.

[0053] The substance that specifically binds to the above miRNA may be within the range exemplified above.

[0054] The above samples and diagnostic subjects are as described above.

[0055] Methods for measuring the concentration of the above miRNA in a sample include, but are not limited to, reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), real-time reverse transcriptase polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chips.

[0056] The method of the present invention may further include a step of comparing the expression level with the expression level of a control group.

[0057] When the expression level of the miRNA in the sample of a patient suspected of myocardial infarction is compared with the expression level of a normal control group, if the expression level of the miRNA in the patient suspected of myocardial infarction is lower than that of the normal control group, the patient can be judged to have a high possibility of developing myocardial infarction. In addition, if the expression level of the miRNA is measured to be higher or not statistically significantly different from that of the normal control group (e.g., p<0.05), information can be provided that can determine that the patient suspected of myocardial infarction has a low possibility of developing myocardial infarction.

[0058] Additionally, by comparing the miRNA expression levels in samples from two patients suspected of having myocardial infarction, it can be determined that the patient with a lower expression level is more likely to develop myocardial infarction than the patient with a higher expression level.

[0059] In addition, when the expression level of miRNA in the samples of patients with suspected myocardial infarction is compared with the expression level of patients with myocardial infarction, if there is no statistically significant difference (P<0.05) between the expression level of patients with suspected myocardial infarction and the expression level of patients with myocardial infarction, it can be determined that the possibility of developing colon cancer is high.

[0060] The present invention relates to a pharmaceutical composition for preventing or treating myocardial infarction.

[0061] The composition of the present invention comprises miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p or miR-7225-5p.

[0062] The above miRNA may be derived from animals, including humans.

[0063] The above composition is excellent in preventing or treating myocardial infarction by reducing cell death of cardiomyocytes or fibroblasts in heart tissue.

[0064] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which is commonly used in the preparation of pharmaceuticals, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0065] The pharmaceutical composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention is not limited thereto and may be 0.01-2000 mg / kg (body weight) per day.

[0066] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined depending on the type of disease to which it is applied.

[0067] The pharmaceutical composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0068] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0069] Materials and Methods

[0070] 1. Animals

[0071] To establish the MI mouse model, 12-week-old male C57BL / 6 mice (23 ± 4 g; KOATECH, Pyeongtaek, South Korea) were used. After anesthesia with zoletil (30 mg / kg; Verbac, France) and xylazine (10 mg / kg; Bayer Korea, Ansan, South Korea), the mice were ventilated using a ventilator (Harvard Instruments, Holliston, MA, USA). The left anterior descending (LAD) artery was ligated with a 6-0 prolene suture (Ethicon, Diegem, Belgium). Subsequently, muscle and skin were sutured using a 4-0 prolene suture (Ethicon). The mice were divided into three groups based on the duration of suture closure after LAD artery ligation: sham, MI-1 day, and MI-3 day. To obtain cardiac tissue for analysis, mice were sacrificed on the indicated days, and simultaneous tissue collection was performed from all groups. To establish the MI animal model, 10 mice were selected from each group, and finally, 5 mice per group were randomly selected for further experiments.

[0072] 2. TTC (Triphenyltetrazolium Chloride) staining

[0073] Before TTC staining, isolated heart tissue was perfused repeatedly with 1x phosphate-buffered saline (PBS; Biosesang, Seongnam, Republic of Korea). Heart tissue from each experimental group was stained with TTC. For this purpose, the tissue was incubated in a 1% TTC solution (Sigma-Aldrich, St. Louis, MO, USA) containing TTC dissolved in 1x PBS. The tissue was incubated at 37°C for 1 hour, protected from light, to prevent photodegradation. After incubation, the tissue was fixed in a 4% paraformaldehyde solution (Biosesang, Seongnam, Republic of Korea) at 4°C for 4 hours. The tissue was then sectioned into 1-mm-thick sections for detailed examination. The stained and sectioned heart tissue was photographed using a digital camera (DIMIS M model, Anyang, Republic of Korea) to document the results of the TTC staining process. Infarcted tissue appears white, and viable tissue appears red. This photographic evidence is crucial for visualizing and analyzing the degree of infarction in the heart tissue.

[0074] 3. Exosome isolation

[0075] The heart tissue isolated from each group was cut into 1 mm pieces using a razor. 3Dissected into pieces. Subsequently, these tissue fragments were gently rinsed with cold 1x PBS in a cell strainer equipped with a 70 μm nylon mesh (SPL, Pocheon, South Korea). Five heart tissue fragments from the same group were pooled in a 50 mL tube. The heart tissues in each tube were incubated in serum-free medium supplemented with 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; Thermo Fisher Scientific, Grand Island, NY, USA) for 45 min at 37°C with gentle shaking (200 rpm). This step promotes the release of exosomes into the medium. After incubation, the tubes were centrifuged at 3000xg for 15 min at 4°C. This centrifugation step aimed to pellet intact cells and cell debris, leaving an exosome-rich supernatant. Exosomes were then isolated from the supernatant using the Exo2D EV Isolation Kit for RNA Analysis (EXOSOMEplus, Seoul, Korea) according to the manufacturer's instructions.

[0076] Briefly, Exo2D reagent was added to the supernatant at a 1:5 ratio, and the mixture was incubated at 4°C for 1 h. The mixture was then centrifuged at 3000×g for 30 min at 4°C. The resulting white pellet containing exosomes was resuspended in 1x PBS and stored at -80°C for future use. Total RNA was extracted from the purified exosomes using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL, USA) according to a standard protocol. This RNA served as the basis for subsequent RNA analysis and sequencing.

[0077] 4. Construction of mRNA library and miRNA sequencing

[0078] smRNA sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to Illumina's standard procedures.

[0079] This preparation included RNA fraction selection, adapter ligation, and sample amplification to construct a library suitable for high-throughput sequencing. The constructed smRNA library was then sequenced on the Illumina Hiseq 2500 Genome Analyzer platform. Sequencing parameters were set to achieve a read length of 50 base pairs (bp) using single-end sequencing. This approach was chosen to optimize the detection and analysis of smRNA species, particularly miRNAs. The library size range was determined to be between 145 and 160 bp, indicating successful library preparation and suitable for efficient miRNA sequencing. All processes related to smRNA library construction and sequencing were performed externally by MACROGEN Inc. (Seoul, Korea), ensuring high-quality and reliable sequencing data.

[0080] 5. Data Analysis of miRNA Sequencing

[0081] After smRNA sequencing, the raw sequence data underwent an initial filtering process. This step included quality-based filtering to isolate high-quality reads from the dataset. The processed reads were further refined by trimming adapter sequences and removing any reads aligned to rRNA sequences. This refinement ensured that the dataset for analysis consisted solely of relevant, high-quality miRNA sequences. The processed, high-quality reads were then classified and analyzed. Known miRNAs were identified using miRbase v22.1, a comprehensive miRNA sequence database. Other types of RNA sequences were classified using RNAcentral 14.0, a noncoding RNA sequence database. Furthermore, novel miRNA prediction was performed using Biomedicines miRDeep2, a tool specifically designed for novel miRNA discovery. Identification of differentially expressed miRNAs was a crucial step in this analysis. Statistical methods, including fold change calculations and the exact test function of edgeR (version 3.9), were used. Hierarchical clustering was also utilized to understand miRNA expression patterns under various conditions. The GO and KEGG databases played a crucial role in analyzing the functions and pathways of target genes identified in the DE miRNA analysis. These analyses provided insight into the biological significance of the observed miRNA expression patterns. All processes related to miRNA sequencing data analysis were performed by MACROGEN Inc., located in Seoul, South Korea, ensuring expert and thorough analysis of the sequencing data.

[0082] 6. Transmission electron microscope (TEM) analysis

[0083] Exosomes extracted from cardiac tissue were first prepared for TEM analysis by fixing them in a 0.1% paraformaldehyde solution (Biosesang, Seongnam, South Korea) for 30 minutes. This step is crucial for preserving the structural integrity of the exosomes during subsequent analysis. A 10 μL aliquot of each exosome sample was placed on a piece of Parafilm. Formvar / carbon-supported copper grids (200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA) were then suspended over each sample droplet for 7 minutes. This method allowed the exosomes to attach to the grid while providing sufficient support for detailed TEM examination. The grids with attached exosomes were then washed with three alternating drops of ultrapure water, each wash lasting 2 minutes. This step allowed the removal of residual fixative. Subsequently, the grids were stained with a 2% phosphotungstic acid solution (pH 7.0; Sigma-Aldrich, St. Louis, MO, USA) for 30 s to provide the contrast necessary for visualizing exosomes in TEM. After staining, the grids were air-dried overnight in a dark environment to prevent light-induced changes. Once dried, the exosomes were ready for visualization. The prepared samples were observed using a transmission electron microscope (JEM-F200; JEOL, Tokyo, Japan).

[0084] 7. Immunoblot analysis

[0085] Cells were lysed using RIPA buffer (Thermo Fisher Scientific, Rockford, IR, USA) supplemented with 1% phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA) and 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). This ensures effective lysis of cellular components while preserving protein integrity.

[0086] The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to quantify protein concentration in lysates, allowing for accurate measurement of protein amounts for uniform loading in gel electrophoresis. Proteins were then separated by electrophoresis on SDS-PAGE under reducing conditions. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF; Sigma-Aldrich, St. Louis, MO, USA) membranes for immunoblotting. To prevent nonspecific binding, the membrane was blocked for 1 h with 5% skim milk (BD Difco; Sparks, MD, USA) in Tris-butyl sulfate-based trisodium salt (TBS-T) buffer (10 mM Tris-HCl (Sigma-Aldrich, St. Louis, MO, USA), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO, USA), and 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA)). The membrane was then incubated overnight at 4°C with primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:1000 dilution. After primary antibody incubation, the membrane was washed three times and then incubated with HRP-conjugated anti-mouse IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) in blocking buffer for 2 h. After three additional washes to remove excess secondary antibody, the membrane was prepared for detection. Protein bands were visualized using an ECL kit (Western Blotting Protection Kit, GE Healthcare, Buckinghamshire, UK). Band intensities were quantified using ImageJ software (NIH, version 1.54h), providing a quantitative analysis of protein expression.

[0087] 8. Isolation of primary cardiomyocytes and fibroblasts from neonatal mouse hearts

[0088] Primary cardiomyocytes and fibroblasts were isolated from 1-day-old C57BL / 6 mice (KOATECH, Pyeongtaek, South Korea) using a primary cardiomyocyte isolation kit (Thermo Fisher Scientific, Rockford, IL, USA) and a primary fibroblast isolation kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's protocol.

[0089] 1-3mm neonatal heart 3Heart slices were dissected into pieces and initially placed separately in cold Hank's Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Grand Island, NY, USA). After washing twice with 0.5 mL of cold HBSS, the heart slices were subjected to enzymatic digestion. For primary cardiomyocyte isolation, each heart in a tube was treated with 0.5 mL of enzyme 1 (containing papain) and 0.01 mL of cardiomyocyte dissociation enzyme 2 (containing thermolysin) and incubated at 37°C for 30 min. For primary fibroblast isolation, each heart was treated with 0.2 mL of reconstituted MEF dissociation enzyme (containing papain) and incubated at 37°C for 25 min. After enzymatic digestion, the heart tissues were washed twice with 0.5 mL of cold HBSS. Then, the tissues were mechanically disrupted by pipetting up and down 25 times for primary cardiomyocytes and 20 times for primary fibroblasts in 0.5 mL of complete DMEM (Thermo Fisher Scientific, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% penicillin / streptomycin (Thermo Fisher Scientific, Grand Island, NY, USA). The resulting cell suspensions were combined, and cell concentration and viability were measured. Then, 4 × 10 cells were seeded in 96-well plates (SPL, Pocheon, South Korea) for transfection with miRNA mimics. 4 Cells were seeded at a density of 10 cells / well. For immunofluorescence analysis, cells were seeded at a density of 2 × 10 in 4-well cell culture slides (SPL, Pocheon, Republic of Korea). 5 Cells were seeded at 10 / well.

[0090] 9. Transfection using miRNA mimics

[0091] To prepare for transfection, primary cardiomyocytes and fibroblasts were seeded in 96-well plates. The following day, each well was transfected with 20 miRNA mimics at a concentration of 1 pmol / well using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. A miRNA mimic negative control was used for comparison. All miRNA mimics used in this study were supplied by Genolution Pharmaceuticals (Seoul, Korea), and specific details are provided in Table 1. Twenty-four hours after transfection, cells were transferred to serum-free medium (SFM) for the control group and exposed to normoxic or hypoxic conditions for an additional 24 hours. This setup was designed to simulate an in vitro environment similar to MI. For hypoxic treatment, cells were maintained at 37°C in an anaerobic atmosphere system (Technomart, Seoul, Korea) under 5% CO2, 5% H2, and 0.5% O2.

[0092]

[0093] 10. Cytotoxicity analysis

[0094] To assess cytotoxicity in cell cultures, the ToxiLight BioAssay Kit (Lonza, Walkersville, MD, USA) was used. This kit is a non-destructive cell lysis assay specifically designed to measure the release of adenylate kinase (AK) from damaged cells.

[0095] The assay utilized a bioluminescence reaction that correlates with the amount of AK released from lysed cells. For the assay, 0.02 mL of cell culture supernatant was transferred to a new 96-well plate. 0.1 mL of AK test reagent dissolved in assay buffer was added. The mixture was then incubated at room temperature for 5 minutes to allow the bioluminescence reaction to proceed. After incubation, bioluminescence intensity was measured using a GloMax Discover Microplate Reader (Promega, Madison, WI, USA).

[0096] 11. Immunofluorescence analysis

[0097] Cell culture slides containing primary cardiomyocytes and fibroblasts were fixed overnight at 4°C with 4% paraformaldehyde solution (Biosesang, Seongnam, South Korea). After fixation, antigen retrieval was performed at 95°C for 10 min using sodium citrate buffer (0.1 M; CureBio, Seoul, South Korea). The slides were then permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min to allow antibody access to intracellular structures. Subsequently, the slides were blocked with 2.5% normal horse serum (Vector Laboratories, Newark, CA, USA) for 1 h to minimize nonspecific antibody binding. After blocking, slides were incubated with primary antibodies: anti-cardiac troponin T antibody (1:200 dilution; Abcam, Cambridge, UK) for cardiomyocytes or anti-vimentin antibody (1:200 dilution; Abcam, Cambridge, UK) for fibroblasts, overnight at 4°C. After primary antibody incubation, slides were washed and incubated with the appropriate secondary antibodies: fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:500 dilution; Jackson Immunoresearch, West Grove, PA, USA) or rhodamine-conjugated secondary antibody (1:500 dilution; Millipore, Bedford, MA, USA). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; 1:5000 dilution; Thermo Fisher Scientific, Rockford, IL, USA) to facilitate the identification of cellular structures. Finally, slides were examined under an Olympus IX83 microscope (Evident, Tokyo, Japan) for detailed visualization of immunofluorescence staining.

[0098] 12. Statistical Analysis

[0099] Data were analyzed using a two-sample t-test using the Statistical Package for the Social Sciences (SPSS, version 14.0K) software, and results are expressed as the mean ± standard error of the mean (SEM). When the t-test indicated a significant overall treatment effect (p < 0.05), differences between groups were assessed using the least significant difference (LSD) test, with significance set at p < 0.05. The p-values ​​from the RNA sequencing analysis were automatically extracted using the comparative analysis algorithm edgeR.

[0100] result

[0101] 1. Establishment of an MI animal model and isolation of exosomes from cardiac tissue

[0102] ExoRNA sequencing was performed on exosomes extracted from cardiac tissue of an established MI mouse model using C57BL / 6 mice (Fig. 1A). Five mice per group were used for sequencing. To confirm MI induction in this model, the morphology of the heart in the MI model was compared with that of the sham group. The infarct area was clearly visible in the heart in the MI model (Fig. 1B). Furthermore, TTC staining was used to compare the infarct area in the heart. This is because TTC staining reacts with mitochondrial enzymes in living cells to form a red compound. Uniform staining was observed in the sham group hearts, while the MI group showed TTC-negative areas in the infarct area (Fig. 1B). The size of exosomes extracted from the cardiac tissue was confirmed using TEM analysis (Fig. 1C). Immunoblotting was used to confirm the presence of exosomal markers, including CD9, CD64, and CD81, with β-actin, α-tubulin, and GAPDH serving as internal controls (Fig. 1D).

[0103] 2. ExoRNA sequence analysis and data processing

[0104] ExoRNA sequencing was performed on exosomes extracted from cardiac tissue of C57BL / 6 mice in the MI model. Sequencing adapters were ligated to exoRNA from cardiac tissue, followed by reverse transcription (RT) and PCR to amplify cDNA pools (Figure 2A). These cDNA fragments were sequenced on an Illumina platform. Data processing included organizing reads into categories: trimmed reads (adapter sequence removed), nonadapter reads (no adapter sequence), short reads (<17 bp after adapter trimming), and low-quality reads (reads containing one or more bases from trimmed or non-adapter reads). The number of reads in each group is shown in Figure 2A. Ribosomal RNA (rRNA) removal was performed to mitigate the effect of enrichment, and the remaining read counts are shown in Figure 2B. The read length distributions for each group are shown in Figure 2C. Typically, transfer RNA (tRNA) was 70-90 nucleotides (nt), small nucleolar RNA (snoRNA) was about 90 nt, small nuclear RNA (snRNA) was 100-300 nt, miRNA was about 22 nt, and PIWI-interacting RNA (piRNA) was about 27 nt in length. The small RNA (smRNA) composition of each sample, which represents the proportion of smRNA types (e.g., miRNA, candidate miRNA, rRNA, tRNA, snRNA, snoRNA, etc.), is shown in Figure 2D.

[0105] 3. Differentially expressed (DE) miRNA analysis

[0106] To assess the similarity between samples, correlation analysis and hierarchical clustering analysis were performed. In correlation analysis, a value closer to 1 indicates greater similarity. The highest similarity was observed between the sham and MI-1day groups, followed by the MI-1day and MI-3day groups. The lowest similarity was observed between the sham and MI-3day groups (Figure 3A). Hierarchical clustering analysis further supported these results, showing low similarity between the sham / MI-1day and MI-3day groups combined with high similarity between the sham and MI-1day groups (Figure 3B, left). A heat map utilizing the Euclidean method and complete linkage of hierarchical clustering analysis clustered mature miRNAs and samples based on expression levels (normalized values). This clustering highlighted significant differences between at least one pair of overall comparison groups (Figure 3B, right). A total of 174 mature miRNAs meeting the criteria of fold change (|FC| > 2) and p-value (p < 0.05) were classified based on differential expression between groups (Fig. 3C). These results were visually represented as a smear plot of mean logCPM (X-axis) and log2 fold change (Y-axis) to examine transcripts showing notable expression differences (Fig. 3D).

[0107] To elucidate exoRNA function in cardiac tissue in MI animal models and identify enriched functional terms, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses on differentially expressed mRNAs of dysregulated miRNAs. These analyses were performed separately for miRNAs and showed increased and decreased expression between the MI-1day and sham / MI-3day and sham groups (Figures 4A, B, and C). GO analysis included three categories: biological process, cellular component, and molecular function. The top seven enriched results are shown in Figures 4A and B. In the MI-1day group, the most enriched biological process terms included organelle organization, molecular function regulation, and nervous system development. In contrast, the MI-3day group highlighted terms focused on cell development, protein localization, and cellular catabolism. The major cellular component terms in the MI-1day group were cell projection, neuronal projection, and vesicle, while those in the MI-3day group were neuron, Golgi apparatus, and endomembrane system. The molecular function terms common to both the MI-1day and MI-3day groups were ion binding, cation binding, and transition metal ion binding. These findings suggest that specific miRNAs are involved in the fundamental biological regulation of MI. In addition, KEGG pathway analysis highlighted key pathways showing significant differences between the MI and sham groups, including endocytosis, mitogen-activated protein kinase (MAPK) signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, and Ras signaling pathway (Fig. 4c).

[0108] 4. Selection of differentially expressed miRNAs

[0109] To determine which miRNAs to investigate in vitro from differentially expressed miRNAs between the groups, a diagram was initially used, charting both increased and decreased miRNAs (Figure 5A). The primary focus of this study was on miRNAs with decreased expression in the MI group, as this approach allowed for a broader range of miRNAs to be explored in vitro. Consequently, miRNAs already known to be associated with MI and those with very low expression levels were excluded, improving the selection of candidate miRNAs for in vitro analysis. Consequently, 20 of the 49 miRNAs demonstrating decreased expression in the MI group were selected for in vitro functional studies (Figure 5B).

[0110] 5. Effect of selected miRNAs on hypoxic stress-induced cell death

[0111] To simulate MI conditions in vitro, primary cardiomyocytes and fibroblasts were isolated from neonatal mouse hearts and subjected to hypoxic stress. These cell types were selected because they are most abundant in cardiac tissue. After isolation, the cells were characterized using specific cell markers, such as troponin-T for cardiomyocytes and vimentin for fibroblasts. The cells were then transfected with 20 selected miRNA mimics and exposed to hypoxic conditions to assess their effects on cell viability. These observations revealed that, with the exception of miR-1247-5p, 19 of the selected miRNAs significantly reduced hypoxic stress-induced cell death in primary cardiomyocytes. In primary fibroblasts, 12 miRNAs (miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, and miR-1247-5p) effectively attenuated cytotoxicity under hypoxic conditions (Figs. 6 and 7).

[0112]

Claims

1. A composition for diagnosing myocardial infarction, comprising a substance that specifically binds to at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p, and miR-7225-5p.

2. A composition for diagnosing myocardial infarction according to claim 1, wherein the miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p are present in exosomes isolated from heart tissue.

3. A composition for diagnosing myocardial infarction according to claim 1, wherein the material comprises at least one of a primer, a probe, and an antisense nucleotide.

4. A kit for diagnosing myocardial infarction comprising the composition of claim 1.

5. A method for providing information for diagnosing myocardial infarction, comprising a step of measuring the expression level of at least one of miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p and miR-7225-5p in a biological sample isolated from an individual.

6. A method for providing information for diagnosing myocardial infarction, wherein, in claim 5, if the expression level is lower than that of a normal control group, the possibility of developing myocardial infarction is determined to be higher than that of the control group.

7. A method for providing information for diagnosing myocardial infarction, wherein the biological sample according to claim 5 is selected from the group consisting of tissue, cell, blood, serum, plasma, saliva, or urine.

8. A method for providing information for diagnosing myocardial infarction, wherein the biological sample according to claim 5 is an exosome isolated from the heart tissue of an individual.

9. A pharmaceutical composition for preventing or treating myocardial infarction comprising miR-30c-1-3p, miR-149-3p, miR-206-3p, miR-486a-3p, miR-673-3p, miR-690-3p, miR-700-5p, miR-706, miR-744-5p, miR-871-3p, miR-874-5p, miR-1247-5p, miR-1306-5p, miR-3057-3p, miR-3086-5p, miR-3470a, miR-3470b, miR-3968, miR-5107-5p, or miR-7225-5p.

10. A pharmaceutical composition for preventing or treating myocardial infarction, wherein the composition reduces cell death of cardiomyocytes or fibroblasts of heart tissue according to claim 9.

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