Reagent for detecting miRNA marker, kit and application

Abstract

The invention discloses a reagent for detecting miRNA markers, a kit and application, and belongs to the technical field of biological medicine. The miRNA marker is at least one of miR-21, miR-101, miR-126, miR-30c and miR-423-5p, and an upstream primer sequence for detecting the miRNA marker comprises SEQ ID NO: 1 to SEQ ID NO: 5. By developing the specific primer sequence aiming at the miRNA marker and jointly detecting multiple miRNAs, the physiological and pathological changes of cardiac muscle cells in the early stage of heart failure can be finally captured, early diagnosis and accurate evaluation are realized, the specificity and sensitivity of diagnosis are improved, the defect that the specificity of an existing diagnosis method in the early stage of diseases is insufficient is overcome, and the diagnosis accuracy is improved. The method has important theoretical significance and potential practical value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a reagent, a kit and an application thereof for detecting miRNA markers. Background Art

[0002] Heart failure is the terminal stage of heart disease, and its pathophysiology is complex, involving multifaceted molecular and cellular changes, including myocardial cell apoptosis, fibrosis, hypertrophy, and ventricular remodeling. Currently, the diagnosis of heart failure relies on clinical symptoms, signs, and cardiac function assessments, lacking highly specific biomarkers. Existing markers such as BNP and NT-proBNP, while serving as the clinical gold standard, are susceptible to interference from factors such as renal function, obesity, and age. Traditional methods such as cardiac ultrasound lack sensitivity for early-stage heart failure and struggle to distinguish between different pathophysiological stages (e.g., HFrEF vs. HFpEF). Although existing treatments (e.g., beta-blockers, ACE inhibitors) can alleviate symptoms, their efficacy is limited and they lack specific targets.

[0003] In recent years, miRNAs have been shown to participate in the progression of heart failure by regulating key genes. For example, miR-21 promotes myocardial fibrosis by activating the TGF-β pathway; miR-423-5p exacerbates myocardial cell apoptosis by inhibiting BCL2 / CASP3 signaling; miR-126 affects ventricular remodeling by regulating vascular endothelial function; miR-30c can inhibit myocardial cell apoptosis and fibrosis and alleviate ventricular remodeling by targeting TGFβRII and apoptosis signaling effectors; miR-101 inhibits myocardial fibrosis by targeting genes such as TGFβRI and Smad2 in the TGF-β signaling pathway, while regulating the expression of genes related to cardiac development and remodeling to protect the myocardium from myocardial remodeling.

[0004] MiRNAs hold great potential for detecting heart failure, but the current lack of efficient and specific primer sequences results in insufficient detection accuracy. Primer sequence design and optimization are crucial for accurately detecting miRNA expression levels and are crucial for improving the accuracy and specificity of heart failure diagnosis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a reagent, a kit and an application for detecting miRNA markers, so as to solve the technical problem that the existing reagents are not accurate enough in diagnosing early heart failure.

[0006] To achieve the above object, the technical solution adopted by the present invention is: providing a reagent for detecting miRNA markers, wherein the miRNA marker is at least one of miR-21, miR-101, miR-126, miR-30c and miR-423-5p.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the upstream primer sequences for detecting miRNA markers include SEQ ID NO: 1 to SEQ ID NO: 5.

[0008] The present invention also discloses the use of the above reagent for detecting miRNA markers in the preparation of a heart failure diagnosis product.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, heart failure includes chronic heart failure caused by congenital heart disease, coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, arrhythmia, myocarditis caused by various causes, and cardiomyopathy.

[0010] The present invention also discloses a kit comprising the above reagent and used for diagnosing heart failure.

[0011] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the kit is used to detect the levels of miRNA biomarkers from patients.

[0012] Furthermore, heart failure includes chronic heart failure caused by congenital heart disease, coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, arrhythmia, myocarditis caused by various causes, and cardiomyopathy.

[0013] The beneficial effects of the present invention are: 1. This paper designs highly specific primer sequences for heart failure-related miRNAs (miR-21, miR-101, miR-126, miR-30c, and miR-423-5p). These primers are designed to accurately detect the expression levels of target miRNAs.

[0014] 2. By developing these specific primers and employing a strategy of combined detection of multiple miRNAs, the present invention is expected to more effectively capture the physiological and pathological changes in cardiomyocytes that occur in the early stages of heart failure. This technical solution aims to provide a new detection tool for the early diagnosis and accurate assessment of heart failure, potentially enhancing diagnostic potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the expression of miR-21 in mouse plasma; Figure 2 is the expression of miR-101 in mouse plasma; Figure 3 is the expression of miR-126 in mouse plasma; Figure 4is the expression of miR-30c in mouse plasma; Figure 5 is the expression of miR-423-5p in mouse plasma; Figure 6 The echocardiographic results of mice after successful model establishment; Figure 7 The changes in ejection fraction of mice; Figure 8 Shortening score changes for mice; Figure 9 The change of left ventricular end-diastolic diameter in mice; Figure 10 The change of left ventricular end-systolic diameter in mice; Figure 11 is the ROC diagnostic curve of miRNA-21; Figure 12 is the ROC diagnostic curve of miRNA-101; Figure 13 is the ROC diagnostic curve of miRNA-126; Figure 14 is the ROC diagnostic curve of miRNA-30c; Figure 15 is the ROC diagnostic curve of miRNA-423-5p; Figure 16 is the correlation between miRNA-21 expression and BNP; Figure 17 is the correlation between miRNA-101 expression and BNP; Figure 18 is the correlation between miRNA-126 expression and BNP; Figure 19 is the correlation between miRNA-30c expression and BNP; Figure 20 is the correlation between miRNA-423-5p expression and BNP; Figure 21 is the correlation between miRNA-21 expression and NT-proBN; Figure 22 is the correlation between miRNA-101 expression and NT-proBNP; Figure 23 is the correlation between miRNA-126 expression and NT-proBNP; Figure 24 is the correlation between miRNA-30c expression and NT-proBNP; Figure 25 The correlation between miRNA-423-5p expression and NT-proBNP. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. In the examples, where specific conditions are not specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. It is obvious to those skilled in the art that various modifications are within the spirit and scope of the present invention as defined and determined by the appended claims, and all inventions and creations utilizing the present invention are protected.

[0017] Example 1. Screening of miRNA Through a systematic search and analysis of relevant literature from the past 10 years, 86 articles were selected from 3,702 articles in three databases for inclusion in the study. A meta-analysis of 226 miRNAs was performed, revealing 58 that were found to be elevated and 13 that were found to be decreased. A meta-analysis of these 226 miRNAs ultimately selected miR-21, miR-423-5p, miR-126, miR-30c, and miR-101 as miRNAs for further investigation. This analysis, encompassing multiple studies, comprehensively assessed the expression changes of these miRNAs in patients with heart failure and their association with the development and progression of heart failure, laying a solid theoretical foundation for subsequent experimental research.

[0018] 2. Establishment of a Doxorubicin-Induced Heart Failure Mouse Model Healthy male mice weighing 20-25g were randomly divided into a control group and a model group, with 10 mice in each group. The model group mice were injected intraperitoneally with doxorubicin (2 mg / kg, once a week for a total of five injections) to induce heart failure, while the control group mice were injected with an equal volume of saline. Models were established after the fifth injection.

[0019] 3. Sample Collection At 1, 3, 6, and 10 weeks after model establishment, prepare anticoagulant tubes (containing EDTA), 1.5 mL centrifuge tubes, and alcohol cotton balls. Mice were fasted for 12 hours and anesthetized by intraperitoneal injection. The mice were immobilized, their eyes disinfected with alcohol, and the neck was gently pressed with the thumb to cause the eyeball to protrude. The retroorbital venous plexus was quickly punctured with scissors, allowing blood to flow into the anticoagulant tube. Hemostasis was achieved by applying pressure with a dry cotton ball for 5 minutes. The tube was immediately inverted to mix thoroughly, and the tube was centrifuged at 3000-4000 rpm for 10 minutes. The upper layer of pale yellow plasma was aspirated with a pipette into a fresh tube, aliquoted, and stored frozen at -80°C for long-term storage. Blood samples were collected from the mice and used to measure plasma miRNA expression and levels of the heart failure markers BNP and NT-proBNP.

[0020] 4. Extraction of plasma small RNA (Accurate Biotechnology, AG21027) Take 200 μL of plasma sample and add 1 mL of Buffer RLS for small RNA (lysis buffer). Pipette repeatedly or shake vigorously for 5-10 minutes at room temperature. Add 200 μL of chloroform (or 1-bromo-3-chloropropane) to the centrifuge tube, cap the tube tightly, and shake vigorously for 15 seconds. Let it sit at room temperature for 2-3 minutes, then centrifuge at 12,000 g for 15 minutes at 4°C. At this point, separate the homogenate and transfer the upper aqueous phase to a fresh EP tube (avoid aspirating the intermediate protein layer). Add 0.47 volumes of anhydrous ethanol to the aqueous phase (e.g., 235 μL of ethanol for 500 μL of aqueous phase), mix thoroughly, and transfer to Small RNA Mini Columns (maximum capacity 700 μL; excess volume should be loaded in batches). Centrifuge at room temperature for 1 minute (12,000 rpm). Discard the column and collect the filtrate. Add 0.75 volumes of anhydrous ethanol to the filtrate (e.g., add 525 μL of ethanol to 700 μL of filtrate), mix thoroughly, and transfer to a new adsorption column. Centrifuge at room temperature for 1 minute (12,000 rpm). Discard the filtrate and retain the adsorption column. Add 600 μL of Buffer RWA for small RNA (pre-mixed with anhydrous ethanol at a 2:3 ratio), let stand at room temperature for 2 minutes, centrifuge for 1 minute (12,000 rpm), and discard the filtrate. Add 650 μL of Buffer RWB (pre-mixed with anhydrous ethanol at a 3:7 ratio), repeat the wash cycle, centrifuge, and discard the filtrate. Transfer the adsorption column to a new RNase-Free Tube, add 10 μL of RNase-Free Water to the center of the column membrane, let stand at room temperature for 2 minutes, and centrifuge for 2 minutes (12,000 rpm) to elute the small RNA. Test the RNA concentration and quality, and store the product at -80°C until further use.

[0021] 5. miRNA cDNA chain synthesis (tailing method, Accurate Biotechnology, AG11716) To an EP tube, add 1.25 μL of miRNA RT Enzyme Mix, 5 μL of 2X miRNA RT Reaction Solution, and 20 ng–2 μg of total RNA (up to 8 μg can be added). Make up to 10 μL with RNase-free water. Avoid foaming during mixing. Before use, briefly centrifuge the miRNA RT Enzyme Mix to collect the solution at the bottom of the tube. Incubate at 37°C for 60 minutes, inactivate at 85°C for 5 minutes, and finally incubate at 4°C. The synthesized cDNA reaction solution can be stored at -20°C or used directly for fluorescence quantitative analysis.

[0022] 6. Real-time fluorescence quantitative detection of miRNA (Accurate Biotechnology, AG11733) For a 20μL reaction, add 10μL of 2X SYBR® Green Pro Taq HS Premix (Blue), ≤100ng of template (if pipetting is required, mix the template with 0.5μL of 40X Dilution Buffer before adding), 0.4μL of Primer F (10μM), 0.4μL of Primer R (10μM), and 0.4μL of ROX Reference Dye (4μM, which can be replaced with enzyme-free water if instrument calibration is not required). Make up to 20μL with RNase-free water. The reaction procedure is as follows: 1 cycle of pre-denaturation at 95°C for 30 seconds; 40 cycles of denaturation at 95°C for 5 seconds and annealing / extension at 60°C for 30 seconds. Finally, perform melting curve analysis.

[0023] The upstream primer sequences of miR-21, miR-101, miR-126, miR-30c and miR-423-5p used in the present invention for fluorescence quantitative PCR detection are shown in Table 1.

[0024] Table 1 Primer sequence information

[0025] Figure 1-Figure 5The expression of miR-21, miR-101, miR-126, miR-30c, and miR-423-5p in the plasma of control mice and mice with doxorubicin-induced heart failure at 1, 3, 6, and 10 weeks after successful model establishment is shown. In the doxorubicin-induced heart failure mouse model, plasma expression of miR-21 and miR-423-5p was upregulated over time, while miR-101, miR-126, and miR-30c were downregulated. This specific upregulation and downregulation trend, as well as its temporal pattern, reflects the dynamic changes in microRNA expression during heart failure progression. These changes are not only closely related to cardiomyocyte survival, vascular function, inflammatory response, and extracellular matrix remodeling, but also have potential diagnostic value. The changing trends in their expression levels can reflect the progression of heart failure and can serve as markers for early diagnosis and disease monitoring, providing new biomarkers for clinical diagnosis and facilitating early intervention and targeted treatment of heart failure.

[0026] Figure 6 These are the echocardiographic results of the control group mice and doxorubicin-induced heart failure mice at 1, 3, 6, and 10 weeks after successful model establishment. Figure 7-10 This is an analysis of various indicators in the echocardiographic results of the control group mice and doxorubicin-induced heart failure mice at 1, 3, 6, and 10 weeks after successful modeling. As time progressed, the ejection fraction (LVEF) and shortening fraction (LVFS) of the mice gradually decreased, and the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) of the mice gradually increased.

[0027] from Figure 11-Figure 15 It can be seen that compared with the control samples, the levels of miR-21 and miR-423-5p in the plasma of heart failure model mice increased, with AUCs of 0.9667 and 0.9697, respectively; the levels of miR-101, miR-126, and miR-30c in the plasma of heart failure model mice decreased, with AUCs of 0.9630, 0.9444, and 0.9744, respectively.

[0028] Figure 16-Figure 25 The results of comparison with the gold standard B-type natriuretic peptide (BNP) and N-terminal B-type natriuretic peptide precursor (NT-proBNP) for heart failure showed that changes in plasma miRNA expression were significantly correlated with changes in the levels of heart failure markers BNP and NT-proBNP.

Claims

1. A reagent for detecting miRNA markers, characterized in that The miRNA marker is at least one of miR-21, miR-101, miR-126, miR-30c and miR-423-5p.

2. The reagent for detecting miRNA markers according to claim 1, characterized in that The upstream primer sequences for detecting miRNA markers include SEQ ID NO: 1 to SEQ ID NO:

5.

3. Use of the reagent for detecting miRNA markers according to claim 1 in the preparation of a heart failure diagnostic product.

4. The use according to claim 3, characterized in that The heart failure includes congenital heart disease, coronary heart disease, acute myocardial infarction, rheumatic heart disease, hypertension, arrhythmia, myocarditis caused by various causes, and chronic heart failure caused by cardiomyopathy.

5. A kit for diagnosing heart failure, characterized in that: Contains the reagent according to claim 1 or 2.

6. The kit for diagnosing heart failure according to claim 5, characterized in that The kit is used to detect the level of miRNA biomarkers from a patient.

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