CN116622829A - miRNA biomarker for detecting fulminant myocarditis and application thereof - Google Patents

Abstract

The invention provides a miRNA biomarker for detecting fulminant myocarditis and application thereof, belonging to the technical fields of molecular diagnosis and molecular biology. The research of the invention discovers that the relative expression level of a plurality of miRNAs in plasma exosomes is obviously related to the occurrence of the pediatric fulminant myocarditis, wherein 4 miRNA molecules, namely hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-miR-532-5p, have higher diagnostic value. Proved by researches, each of the 4 miRNAs can be independently used as a biomarker for the pediatric fulminant myocarditis, and the marker spectrum set of the miRNAs has higher diagnostic value, and a rapid and accurate diagnostic mode is provided for clinic after combined application, so that the diagnosis and prognosis evaluation of the pediatric fulminant myocarditis are more convenient and easy to implement, and the kit has good practical application value.

Description

miRNA biomarker for detecting fulminant myocarditis and application thereof

Technical Field

The invention belongs to the technical fields of molecular diagnosis and molecular biology, and particularly relates to a miRNA biomarker for detecting fulminant myocarditis and application thereof.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Myocarditis is an inflammatory disease of the heart muscle that can lead to cardiac dysfunction, including reduced systolic or diastolic function, cardiac arrhythmias, and the like. The fulminant myocarditis is the most serious type, has the characteristics of rapid onset, rapid development and the like, can cause serious change of blood flow dynamics, cardiogenic shock, multisystem dysfunction and the like, needs muscle strength or mechanical circulation support, and has relatively high death rate. The etiology of myocarditis identified today is heterogeneous, including viral, bacterial or fungal pathogen-mediated infections, cardiac inflammation associated with certain immune and autoimmune diseases, and myocardial secondary inflammatory responses due to drug toxicity or other diseases. However, the exact pathogenesis remains to be further investigated. Current diagnostic tests, such as serum cardiac biomarker (troponin T, troponin I, creatine kinase, brain isozymes, brain natriuretic peptide) detection, electrocardiography, echocardiography, and cardiovascular magnetic resonance are all non-specific. Endocardial biopsy is the gold standard for current myocarditis diagnosis, however its invasive nature and the impact of clinical settings make it rarely used.

Clinical manifestations of myocarditis heterogeneity, including no myocardial dysfunction to rapid progressive heart failure, and the difficulty in children accurately expressing their own discomfort, present a great challenge to clinicians in early diagnosis of pediatric fulminant myocarditis (Fulminant Myocarditis, FM). At present, myocarditis is treated by symptomatic treatment, and the death rate of children FM patients is high and the prognosis is poor. Therefore, there is a need to further explore the pathogenesis of myocarditis to find new specific noninvasive early diagnostic markers and to determine specific therapeutic targets.

miRNAs are a class of highly conserved endogenous single-stranded non-coding RNA molecules, 19-25 nucleotides in length. Their regulatory function was first described in 1993 in caenorhabditis elegans. miRNAs inhibit expression of a target gene by binding to the target gene to cut off the target gene or to suppress translation of the target gene. Some deregulated miRNAs are involved in the etiology and pathogenesis of a variety of diseases, exhibiting phase-dependent changes. For example, miR-21 (down-regulation) inhibits apoptosis by PDCD4 in CVB3 infected mice; miR-381 (down-regulated) reduces myocardial damage by targeting COX-2 and acts as an anti-inflammatory factor. Exosomes are 30-150 nm extracellular vesicles, consisting of nucleotides and proteins, secreted by specific types of cells, present in various body fluids. Unlike circulating miRNAs, exosomes are enriched in the circulatory system and protected from RNase degradation. Thus exosome miRNAs may have a greater potential as biomarkers for disease than circulating miRNAs. However, to date, there has been no report on the use of exosome mirnas for diagnosis of pediatric fulminant myocarditis.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a miRNA biomarker for detecting fulminant myocarditis and application thereof. According to the invention, the relative expression level of a plurality of miRNAs in the plasma exosome is obviously related to the occurrence of the pediatric fulminant myocarditis, wherein 4 miRNA molecules, namely hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-miR-532-5p, have higher diagnostic value. The 4 miRNAs can be independently used as biomarkers of the pediatric fulminant myocarditis, and the marker spectrum set has higher diagnostic value, and a rapid and accurate diagnostic mode is provided for clinic after combined application, so that the diagnosis and prognosis evaluation of the pediatric fulminant myocarditis are more convenient and feasible. Based on the above results, the present invention has been completed.

In order to achieve the above object, the present invention relates to the following technical solutions:

in a first aspect of the invention, there is provided a biomarker for the detection of pediatric fulminant myocarditis, the biomarker being selected from any one or more of the following mirnas:

hsa-miR-30e-5p、hsa-miR-146a-5p、hsa-miR-361-5p、hsa-miR-532-5p。

more specifically, the miRNA is the miRNA of the plasma exosome of the subject.

More specifically, the biomarkers for pediatric fulminant myocarditis are selected from the group consisting of hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-miR-532-5 p.

Specifically, the biomarker for detecting the pediatric fulminant myocarditis is specifically a biomarker for early diagnosis (or early-stage auxiliary diagnosis) and/or prognosis evaluation of the pediatric fulminant myocarditis.

In a second aspect, the invention provides the use of a substance for detecting the expression level of a biomarker as described above in the manufacture of a product for detecting pediatric fulminant myocarditis.

Wherein the substance includes, but is not limited to, a substance that detects the expression level of the above-described biomarker based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method.

Such products include, but are not limited to, devices (such as oligonucleotide probes or integration thereof, high throughput miRNA detection chips on chip substrates or detection substrates, and microfluidic detection chips), kits, and apparatus.

In a third aspect of the invention, there is provided a kit comprising:

one or more devices for detecting the above biomarkers.

In a fourth aspect of the invention, there is provided an apparatus comprising the kit described above.

In a fifth aspect of the invention, there is provided the use of the above kit and/or device for detecting pediatric fulminant myocarditis; in particular for early diagnosis (or early-stage auxiliary diagnosis) and/or prognosis evaluation of the primary myocarditis of children;

in a sixth aspect of the invention, there is provided a system for detecting pediatric fulminant myocarditis comprising:

i) An analysis unit comprising: a detection agent for determining the expression level of a biomarker selected from the above in a sample of a subject, and;

ii) an evaluation unit comprising a data processor, said data processor being tangibly embedded with an algorithm for comparing the quantity determined by said analysis unit with a reference, and being able to generate an output file comprising diagnostic results established on the basis of said comparison.

In a seventh aspect of the invention, there is provided a method for early diagnosis and/or prognosis evaluation of pediatric fulminant myocarditis, the method comprising: determining the presence or expression level of the above-mentioned biomarker in a biological sample from the subject, and comparing the expression level of the biomarker to a reference.

Wherein the biological sample is a plasma exosome.

The beneficial technical effects of one or more of the technical schemes are as follows:

according to the technical scheme, 4 plasma exosome miRNA molecules hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-532-5p screened by using a random forest model for the first time are obviously up-regulated in patients with the pediatric fulminant myocarditis, and ROC curve analysis further shows that the 4 plasma exosome miRNAs have considerable sensitivity and specificity in the aspect of diagnosing the pediatric fulminant myocarditis and have potential to become noninvasive diagnostic biomarkers, so that the 4 plasma exosome miRNAs have good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. FIG. 1 is a graph of plasma exosome characteristics from pediatric FM patients and HCs in the examples; (a) Plasma exosome markers CD63, CD81 and TSG101 from pediatric FM patients and HCs, while calnexin is negative; (b) Transmission Electron Microscopy (TEM) shows exosome morphology. Scale bar, 200mm; (c) NTA analysis showed particle sizes from exosomes of HCs and FM. HCs, healthy controls.

FIG. 2 is a graph showing the expression of miRNAs in pediatric FM patients and in HCs plasma exosomes in the examples; (a) The cluster heatmap shows the variation in miRNAs expression between the two groups (p <0.05, fc > 2). Red bands, high relative expression; blue bands, low relative expression; white bands, no change in gene expression. The shade of the color reflects the degree of increase or decrease in expression. (b) Volcanic images show that different expressed miRNAs have different p-values and FCs. Y-axis, p-value = 0.05 (-log 10 scaled); x-axis, fold change = 2 (log 2 scaled), red, up-regulated expression; blue, expression down-regulation; gray, no statistical difference in expression between the two groups. (c) Principal Component Analysis (PCA) between the two groups. (d) Venn diagram of miRNAs in circulating exosomes in adult and pediatric FM patients. Adults_Down, expressing down-regulated miRNAs in circulating exosomes of adult FM patients; child FM patient circulating exosomes express down-regulated mirnas; child FM patient circulating exosomes express down-regulated mirnas; adults_up, miRNAs that are up-regulated in circulating exosomes in adult FM patients.

FIG. 3 is a validation of miRNA expression and identification of biomarkers in the examples; (a) - (d) detecting the relative expression level of the selected miRNAs using real-time fluorescent quantitative PCR. The Mann-Whitney test was used to compare expression levels between the child heart set (n=15) and the healthy control set (n=15). * P <0.0001. Data are expressed as mean ± SD. (e) The relative expression levels of selected miRNAs in 15 pediatric FM patients. (f) Pearson correlation coefficient of 4 miRNAs with clinical serological index. ROC curve analysis of (g) - (j) miRNAs in FM diagnosis. (k) ROC curve analysis of the combined diagnosis of miRNAs.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

The skilled artisan understands variations of terms used in the present invention, such as "miRNA" and "miR", and which relate to short ribonucleic acid (RNA) molecules found in body fluids of eukaryotic cells and metazoan organisms. mirnas include human mirnas, mature single-stranded mirnas, precursor mirnas (pre-mirs), and variants thereof, which may be naturally occurring. In some cases, the term "miRNA" also includes primary miRNA transcripts (pri-mirnas) and duplex mirnas. Unless otherwise indicated, the designation of a particular miRNA when used in the present invention refers to a mature miRNA. miRNA precursors may consist of 25 to thousands of nucleotides, typically 40 to 130, 50 to 120, or 60 to 110 nucleotides. Typically, mature mirnas consist of 5 to 100 nucleotides, typically 10 to 50, 12 to 40, or 18 to 26 nucleotides. The term miRNA also includes the "guide" strand that ultimately enters the RNA-induced silencing complex (RNA-induced silencing complex, RISC) and the "passenger" strand that is complementary thereto.

Several miRNA sequences are known in the art, and it is understood that the database accession numbers for each miRNA shown below are human-derived mirnas. However, these database entries also provide database accession numbers for the respective mirnas, for example, from different sources: for example, any mammalian, reptile, or avian origin miRNA, such as, for example, those selected from laboratory animals (e.g., mice or rats), domestic animals (including, for example, guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, tortoises, snakes, or lizards), or primates (including chimpanzees, bonobos, and gorillas).

The term "miRNA combination" relates to a combination of mirnas of the invention. The amount of miRNA can be determined in a sample of a subject by techniques well known in the art. Depending on the nature of the sample, the amount may be determined by PCR-based techniques for quantifying the amount of polynucleotide or by other methods such as mass spectrometry or (next generation) sequencing, etc. The term "determining the amount of at least the miRNA in a miRNA combination" as used herein preferably relates to determining the amount of each miRNA in the combination separately to be able to compare the amount of each miRNA in the combination with a reference specific for said miRNA.

The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular point in time. The expression level can be measured/quantified/detected, for example, by the protein or mRNA expressed by the gene. Expression levels can be quantified, for example, as follows: the amount of the gene product of interest present in the sample is normalized with the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same individual at the same time or a portion of the same sample of the same size (weight, volume), or the amount of the gene product of interest/defined sample size (weight, volume, etc.) is determined. The expression level may be measured or detected by any method known in the art, such as methods for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or methods for indirect detection and measurement of a gene product of interest that typically work by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) that are specific for the gene product of interest. It is also known to the skilled person to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, multiplex ligation dependent probe amplification (Multiplex ligation-dependent probe amplification, MLPA) PCR).

The terms "index" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or for monitoring a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of a molecule including, but not limited to, a peptide, protein, and nucleic acid, or may be a change in the level or pattern of expression of such a molecule in a cell, or tissue, organ, or individual. The indicator may be the occurrence, development or presence of a disease in an individual or a sign of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The terms "down-regulating", "reducing" or "down-regulating" the level of an indicator refer to a decrease in the level of such an indicator in a sample as compared to a reference or reference sample. The terms "up-regulate", "raise" or "raise" of the level of an indicator refer to a higher level of such an indicator in a sample as compared to a reference or reference sample.

In principle, the reference amount can be calculated for a group or cohort of subjects specified in the present invention based on the mean or median of a given miRNA by applying standard statistical methods. In particular, the accuracy of a test, such as a method intended or not to determine an event, is best described by its recipient operating characteristics (receiver-operating characteristic, ROC) (see, inter alia, zweig 1993, clin. Chem. 39:561-577). ROC plots are plots of all sensitivity versus specificity pairs obtained from constantly changing decision thresholds over the entire range of data observed. The clinical manifestation of a diagnostic method depends on its accuracy, i.e. its ability to assign a subject correctly to a certain prognosis or diagnosis. ROC plots represent the overlap between the two distributions by plotting sensitivity versus 1-specificity over the complete threshold range suitable for discrimination. On the y-axis is a sensitivity or true positive score, which is defined as the ratio of the number of true positive test results to the sum of the number of true positive and false negative test results. This is also referred to as positive in the presence of a disease or condition. Which are calculated individually from the affected subgroups. On the x-axis is a false positive score or 1-specificity, which is defined as the ratio of the number of false positive results to the sum of the number of true negative and the number of false positive results. It is an index of specificity and is calculated entirely from unaffected subgroups. Since the true and false positive scores are calculated entirely separately, the ROC diagram is independent of the prevalence of events in the cohort by using test results from two different subgroups. Each point on the ROC diagram represents a sensitivity/-specificity pair corresponding to a particular decision threshold. The test with perfect discrimination (no overlap in the two results distributions) has ROC plots through the upper left corner with a true positive score of 1.0 or 100% (perfect sensitivity) and a false positive score of 0 (perfect specificity). The theoretical plot of the test without discrimination (the distribution of the two sets of results is the same) is a 45 ° diagonal from the lower left corner to the upper right corner. Most of the figures fall between these two extremes. If the ROC diagram falls completely below the 45 ° diagonal, this is easily corrected by reversing the "positive" criterion from "greater" to "less" and vice versa. Qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the test. Based on the expected confidence interval, a threshold can be derived from the ROC curve, allowing for diagnosis or prediction of a given event with an appropriate balance of sensitivity and specificity, respectively. Thus, the reference for the inventive method may preferably be generated by establishing the ROC for the group and deriving a threshold amount therefrom as described above. The ROC map allows deriving the appropriate threshold value, depending on the desired sensitivity and specificity of the diagnostic method. Preferably, the reference amount is within a range of values that represents at least 75% sensitivity and at least 45% specificity, or at least 80% sensitivity and at least 40% specificity, or at least 85% sensitivity and at least 33% specificity, or at least 90% sensitivity and at least 25% specificity.

If it is not known whether the donor has pediatric fulminant myocarditis, the reference amount as used in the present invention is preferably obtained from a subject sample obtained prior to treatment. The reference level may be a discrete number or may be a range of numbers. Obviously, the reference level or amount may vary between individual species of mirnas. Thus, preferably, the measurement system is calibrated with a sample or series of samples comprising a known amount of each specific miRNA. The skilled person will appreciate that in this case the amount of miRNA may preferably be expressed as Arbitrary Units (AU). Thus, preferably, the amount of miRNA is determined by comparing the signal obtained from the sample with the signal comprised in the calibration curve. The reference amount applicable to an individual subject may vary depending on a variety of physiological parameters (e.g., age or subpopulation). Thus, a suitable reference amount can be determined by the method of the invention from the reference sample to be analyzed together with the test sample (i.e. simultaneously or sequentially). Further, a threshold amount may be preferably used as the reference amount. The reference amount may preferably be obtained from a sample of a subject or group of subjects known to have pediatric fulminant myocarditis. The reference amount may also preferably be obtained from a sample of a subject or group of subjects known not to have pediatric fulminant myocarditis. It should be appreciated that the above amounts may vary due to statistical and measurement errors. Deviations, i.e. the decrease or increase in the amount of miRNA referred to in the present invention, are preferably statistically significant deviations, i.e. statistically significant decreases or statistically significant increases.

The term "kit" as used herein refers to a collection of the above components, preferably provided separately or in a single container. The container also preferably contains instructions for carrying out the method of the invention. The kit comprises the above components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, for example a user manual for adjusting the components (e.g. the concentration of the detection agent) and for interpreting the results of any assays regarding the diagnosis provided by the method of the invention. In particular, such a manual may include information for assigning the determined amount of gene product to the diagnostic type. Details are found elsewhere in this specification. Further, such a user manual may provide instructions for proper use of the kit components for determining the amount of the corresponding biomarker. The invention also relates to the use of said kit in any method according to the invention.

In one exemplary embodiment of the invention, a biomarker for the detection of pediatric fulminant myocarditis is provided, the biomarker being selected from any one or more of the following mirnas:

hsa-miR-30e-5p、hsa-miR-146a-5p、hsa-miR-361-5p、hsa-miR-532-5p。

in some embodiments of the invention, the miRNA is a subject plasma exosome miRNA.

In some embodiments of the invention, an increase in the expression level of hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-532-5p is indicative of a risk of developing a degenerative tissue condition or disease (particularly pediatric fulminant myocarditis); it also indicates that the individual suffers from an altered tissue state or disease (particularly pediatric fulminant myocarditis). In addition, elevated levels of the above-described miRNAs are indicative of the progression or stage of a tissue state or disease (e.g., pediatric fulminant myocarditis) in a subject. In particular, elevated levels of the above-described miRNAs are indicative of a worsening of a tissue state or disease (particularly pediatric fulminant myocarditis).

In some embodiments of the invention, the biomarkers for the detection of pediatric fulminant myocarditis are selected from the group consisting of hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-miR-532-5 p.

In some embodiments of the invention, the biomarkers described above for the detection of pediatric fulminant myocarditis are specifically biomarkers for early diagnosis (or early-assisted diagnosis) and/or prognosis evaluation of pediatric fulminant myocarditis.

In some embodiments of the invention, there is provided the use of a substance for detecting the expression level of a biomarker as described above for the manufacture of a product for detecting pediatric fulminant myocarditis.

In some embodiments of the invention, the substances include, but are not limited to, substances that detect the expression levels of the above-described biomarkers based on high throughput sequencing methods and/or based on quantitative PCR methods and/or based on probe hybridization methods.

In some embodiments of the invention, the products include, but are not limited to, devices (such as oligonucleotide probes or integration thereof, high throughput miRNA detection chips on chip substrates or detection substrates), kits, and apparatus.

In some embodiments of the invention, there is provided a kit comprising:

one or more devices for detecting the above biomarkers.

In some embodiments of the invention, there is provided an apparatus comprising the kit described above.

In some embodiments of the invention, there is provided the use of the above-described kit and/or device for detecting pediatric fulminant myocarditis; in particular for early diagnosis (or early-stage auxiliary diagnosis) and/or prognosis evaluation of the primary myocarditis of children;

in some embodiments of the invention, there is provided a system for detecting pediatric fulminant myocarditis comprising:

i) An analysis unit comprising: a detection agent for determining the expression level of a biomarker selected from the above in a sample of a subject, and

ii) an evaluation unit comprising a data processor, said data processor being tangibly embedded with an algorithm for comparing the quantity determined by said analysis unit with a reference, and being able to generate an output file comprising diagnostic results established on the basis of said comparison.

In some embodiments of the invention, there is provided a method for early diagnosis and/or recurrence monitoring of pediatric fulminant myocarditis, the method comprising: determining the presence or expression level of the above-mentioned biomarker in a biological sample from the subject, and comparing the expression level of the biomarker to a reference.

In some embodiments of the invention, the biological sample is a plasma exosome.

In some embodiments of the invention, the presence (particularly the amount) of at least one miRNA marker in the subject is compared to the presence (particularly the amount) of at least one miRNA marker in one or more references. In particular, the reference is a threshold value, a reference value or a reference sample.

In embodiments wherein reference is made to a threshold, an amount of at least one miRNA marker selected from hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p, and hsa-miR-532-5p equal to or greater than the threshold is indicative of the subject suffering from, having an increased risk of developing, or being exacerbated by pediatric fulminant myocarditis; however, an amount below the threshold is indicative of the subject having suffered from, having a reduced risk of developing, or having ameliorated a pediatric fulminant myocarditis. It will be appreciated that the above expression levels may differ due to statistical and measurement errors.

In some embodiments wherein the reference is a reference value that is representative of the absence of, presence of, or an increased or decreased risk of developing pediatric fulminant myocarditis.

In other embodiments of the invention, the reference sample is selected from a reference sample obtained from a healthy individual, a reference sample obtained from a diseased individual, a reference sample obtained from the same individual as the sample of interest at an earlier or later time point, and a reference sample representing a healthy individual either with or without or with an increased or decreased risk of developing pediatric fulminant myocarditis.

In some embodiments of the invention, the subject is a mammal, a human being is particularly preferred, and the human child is described.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions such as Sambrook et al, molecular cloning: the techniques and conditions described in the handbook or the molecular cloning laboratory Manual, or according to the manufacturer's recommendations.

Examples

1. Materials and methods

1. Basic information and sample collection of patients

We collected peripheral blood samples of 15 cases of fulminant myocarditis and 15 cases of children in healthy control group from Shandong province Hospital of China, and the sample collection time is 2021, 1, to 2022, 9, 28 days. All cases of fulminant myocarditis were clinically diagnosed according to the 2009 myocarditis cardiovascular magnetic resonance international consensus group voice, the 2013 European cardiology institute statement, the 2017 national cardiology institute expert consensus statement, the 2021 American heart Association "diagnosis and treatment science statement of pediatric myocarditis". The exclusion criteria were as follows: arrhythmia caused by non-fulminant myocarditis; other cardiovascular diseases such as congenital heart disease, dilated or hypertrophic cardiomyopathy, and heart valve disease; secondary to other immune diseases; and has been treated with glucocorticoids or immunosuppressants prior to specimen collection. The healthy control group was healthy volunteers whose age and sex matched with the cases of fulminant myocarditis. The basic conditions and serum and imaging examinations for both groups of patients are shown in tables 1-4, respectively. In addition, microarray detection was performed using 3 pairs of samples, and microarray data validation was performed using RT-qPCR for 15 pairs of samples (including 3 pairs of microarray detection). The study was approved by the institutional ethics committee, and parents of each patient (< 18 years) provided signed informed consent for study purposes.

TABLE 1 clinical characteristics of patients with pediatric fulminant myocarditis

* Heart rate is maintained by administration of isoprenaline.

ECMO, in vitro adventitial pulmonary oxygenation; FM, fulminant myocarditis; TP temporary pacemaker

TABLE 2 serological examination of pediatric FM patients

BNP-type B natriuretic peptide; HS-cTnT, hypersensitive troponin T; PCT procalcitonin; WBC, white blood cells; n (%) is the percentage of neutrophils.

TABLE 3 imaging examination of child FM patients

AT, atrial tachycardia; AVB: atrioventricular block; CMR, cardiac magnetic resonance; CRBBB, total right bundle branch block; ECG; FM, fulminant myocarditis; IVS, ventricular septum; LGE: late gadolinium enhancement; LV, left ventricle; LVEDD, left ventricular end-diastole inner diameter; LVEF, left ventricular ejection fraction; PAC, atrial extra systole; PE, pericardial effusion; PVC, ventricular extra-systole; vf: ventricular fibrillation; VT ventricular tachycardia.

TABLE 4 basic information of healthy control group

BNP-type B natriuretic peptide; ECG; HC, healthy control; HS-cTnT, hypersensitive troponin T; LVEDD, left ventricular end-diastole inner diameter; LVEF left ventricular ejection fraction

2. Separation of plasma exosomes

Peripheral blood samples were collected using EDTA tubes, and centrifuged at 1900g at 4 ℃ for 10 minutes within 2 hours after collection, and the obtained supernatant was centrifuged at 1600g at 4 ℃ for 10 minutes to obtain plasma.

Plasma samples were centrifuged at 3000g for 15 min at 4℃followed by dilution of the supernatant with 7 volumes of PBS, centrifugation at 13000g for 30min at 4℃and the obtained supernatant was filtered through a 0.22um filter, followed by centrifugation at 150000g for 4 hours at 4℃using an ultracentrifuge, discarding the supernatant, re-suspending the pellet with PBS, re-centrifuging at 150000g for 2 hours at 4℃and discarding the supernatant and re-suspending the pellet with 200ul PBS to obtain exosomes.

3. Identification of exosomes

The isolated exosomes described above were identified by the following method: western Blot (WB), transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA).

Protein extraction and WB extraction are as described in the prior art. Briefly, the exosome samples were lysed in RIPA buffer for 30min and then centrifuged at 1200rpm for 30min at 4 ℃. The concentration of exosome proteins was determined using BCA kit (Solarbio Science, china). 30 μg of exoproteins were electrophoresed on SDS-PAGE gels, followed by transfer of these protein bands onto polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% TBST-containing lipid-free milk for 2 hours at room temperature. The strips were then incubated with primary antibodies against CD63, CD81, TSG101 and calnexin (Abcam, USA) overnight at 4 ℃, the next day with secondary antibodies at room temperature for 2 hours. The PVDF membrane was then washed three times with TBST for 5 minutes each. Protein bands were visualized using a Tanon4600 automated chemiluminescent image analysis system (Tanon, china).

For TEM, 10. Mu.l of the exosome suspension was added to a 200 mesh carbon coated copper mesh and incubated for 20min at room temperature. After the copper mesh was fixed on 50 μl of 1% glutaraldehyde droplets for 5min and washed with ddH2O 2, the copper mesh was successively aligned with uranyl acetate solution and methylcellulose-uranyl acetate for 5min and dried under an incandescent lamp for 5min. Morphology of exosomes was observed and photographed using a transmission electron microscope (HT-7700, hitachi, japan).

In addition, NTA analysis was performed using ZetaView pmx120 (Particle MetrixGmbH, usa, germany) and its corresponding software (ZetaView 8.05.05SP2) according to manufacturer's instructions.

4.Small RNA microarray

Total RNA was extracted with TRIzol (Invitrogen, USA). The amount of RNA was measured using a NanoDrop ND-1000 spectrophotometer and the quality of RNA was measured using the bioanalyzer 2100.

For small RNA chip analysis, 100ng of total RNA was first dephosphorylated with 3 units of T4 polynucleotide kinase (T4 PNK) at 37℃for 40min, removing the (P) and (cP) chemical groups from the 3' end of the RNA, forming the 3-OH end. The reaction was terminated at 70℃for 5min, immediately cooled to 0℃and 7uL of DMSO was added, heated to 100℃for 3min to denature the RNA, immediately cooled to 0℃and finally 50mM pCp-Cy3 and 15 units of T4 RNA ligase were added to the reaction solution, and RNA end labeling was performed overnight in 28uL of the reaction solution at 16 ℃.

22.5uL of 2 Xhybridization buffer (Agilent) was mixed with the completed labeling reaction solution to a final volume of 45uL. The mixture was heated at 100 ℃ for 5min and then immediately cooled to 0 ℃.45uL of the labeled sample mixture was hybridized with the microarray at 55℃for 20 hours. The cells were washed with 6 XSSC containing 0.005% Triton X-102 for 10min at room temperature, followed by washing in 0.1 XSSC containing 0.005% Triton X-102 for 5min. Slides were scanned on an Agilent G2505C microarray scanner.

The acquired array images were analyzed using agilent feature extraction software (version 11.0.1.1). Log2 conversion and quantile normalization are carried out on the original intensity. After normalization, at least 5 probe signals with presence (P) or edge (M) QC markers were retained in 20 samples. Multiple probes from the same small RNA (miRNA/tsRNA/pre-miRNA/tRNA/snoRNA) were averaged and pooled to one RNA level. Differentially expressed miRNAs between the two groups were determined by screening Fold Change (FC) and statistical significance (p-value) thresholds.

5. Real-time quantitative PCR

4 miRNAs were selected as diagnostic biomarkers based on a random forest model, combining miRNA expression abundance, FC and P values simultaneously. cDNA was synthesized using Mir-X miRNA First-Strand Synthesis Kit (TAKARA, cat.No.63813). The resulting cDNA was further amplified using universal reverse primers and specific forward primers. The cycle parameters are: the cycle was performed at 95℃for 30s, followed by 95℃for 5s and 60℃for 30s for 40 cycles, followed by 65℃for 5s. The expression level of miRNA was analyzed by the delta Ct method. Cel-miR-39-3p was used as a constant control for quantification of exosome miRNAs. All reactions were repeated three times.

6. Data analysis

For the data obtained by real-time fluorescent quantitative PCR, a normalization test was performed using the Kolmogorov-Smirnov test. The non-normal distribution data was compared between the two groups using a non-parametric Mann-Whitney test. Pearson correlation analysis was used to calculate the correlation between exosome miRNAs and serological indicators (HS-TnT, BNP, ck-MB). Subject working characteristics (Receiver Operating Chamcteristic, ROC) curve analysis was used to assess the diagnostic potential of plasma exosome miRNAs to distinguish FM from HC groups. Statistical analysis was performed by IBM SPSS statistics 26.0 and graphpadprism version 8.0 software. A p value <0.05 is considered statistically significant.

2. Results

1. Identification of exosomes

Plasma exosomes are identified by their morphology, diameter distribution and enriched exosome protein markers. Transmission Electron Microscopy (TEM) images and nanoparticle follow-up analysis (NTA) showed that the isolated plasma exosomes were circular vesicles with diameters of about 50-150nm, median diameters of about 130nm, and clear membrane structures (fig. 1A, 1C). Western Blot (WB) analysis confirmed the expression of the known exosome positive biomarkers CD63, CD81, TSG101 and the negative marker Calnexin (fig. 1B). The above results are consistent with previously reported exosome characteristics. Taken together, these results indicate that exosomes were successfully isolated.

2. Expression profile of plasma exosome miRNAs of infant suffering from fulminant myocarditis

To identify miRNA expression profiles in FM pediatric plasma exosomes, 676 miRNAs were identified in 3 fulminant myocarditis children and 3 healthy volunteers by small RNA chip analysis. The change in expression of exosome miRNAs between FM and HC groups is shown by thermogram and volcanic plot (fig. 2A-B). There were 266 miRNAs in FM children that were differentially expressed from the control group (FC >2, p < 0.05), with 197 miRNAs up-regulated and 69 miRNAs down-regulated. Visualization of the relationship between the two groups of samples is given by the Principal Component Analysis (PCA) plot, indicating the presence of distinguishable miRNA expression profiles between the two groups (fig. 2C). The venn plot shows a comparison of miRNAs differentially expressed from circulating exosomes in adult FM, with only 21 miRNAs with the same expression trend in adult FM as in pediatric FM, indicating that the exosome miRNA expression profile in pediatric FM may be different from that in adult FM, but this may also be due to the small number of samples and large variation in expression between samples.

3. Identification of biomarkers

To verify the expression of miRNAs and explore whether plasma exosome miRNAs could have potential as children FM biomarkers, we utilized a random forest algorithm, combining expression abundance, FC and P values, finally selected 4 miRNA molecules: hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-miR-532-5p are verified by qRT-PCR in 15 FM pediatric patients and 15 healthy volunteers. The expression levels of these four miRNAs were all significantly elevated in FM children plasma exosomes (p < 0.05) compared to healthy volunteers (fig. 3A-D), consistent with microarray results. Of the 15 FM children tested, the relative expression levels of the 4 miRNA molecules were all different, with hsa-miR-146a-5p being most expressed in FM-09, FM-06, FM-012, FM-03 (FIG. 3E), notably, these 4 patients all died from heart failure and multiple organ failure, the most severe cases suggesting that hsa-miR-146a-5p may be associated with the severity of FM. And we calculate the correlation coefficient of 4 miRNAs and FM phenotype respectively, find that 4 miRNAs are positively correlated with HS-Tnt, BNP, CK-MB, wherein hsa-miR-146a-5p, hsa-miR-361-5p and HS-TnT are positively correlated (figure 3F), HS-TnT is an index for diagnosing FM relative specificity clinically at present, which suggests that the four miRNAs have potential as biomarkers for diagnosing FM children.

Subsequently we performed a subject work characteristic (ROC) curve analysis on4 miRNAs, respectively (fig. 3G-J)). The 4 miRNAs used for FM diagnosis showed relative accuracy in distinguishing FM and HC (area under the curve (AUC) =0.9600 [95% Confidence Interval (CI), 0.8968to 1.000],0.9867[95% CI,0.9552to 1.000],0.9822[95% CI,0.9427to 1.000], and 0.8978[95%CI,0.7622to 1.000 ]). And a combined diagnostic value analysis (auc=1 [95% CI,1.000to 1.000 ]) was performed on these four miRNAs (fig. 3K). These data indicate that exosome miRNAs from plasma sources provide FM children with good diagnostic capabilities, have excellent sensitivity and specificity, and the combined diagnosis is more accurate.

In summary, we screened 4 plasma exosome miRNA molecules using a random forest model: hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-532-5p are significantly up-regulated in the FM group. And the hsa-miR-146a-3p has the highest relative expression quantity in patients FM-09, FM-06, FM-012 and FM-03, and it is notable that the four patients die from heart failure and multi-organ failure and are the most serious cases. This suggests that our hsa-miR-146a-3p may be associated with clinical manifestation severity. And hsa-miR-146a-5p and hsa-miR-361-5p are in strong positive correlation with HS-TnT, which is an index for diagnosing the relative specificity of FM in clinic at present, so that four miRNAs have the potential of being used as biomarkers for diagnosing FM children. Further ROC curve analysis shows that AUCs of hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p and hsa-532-5p are 0.9600, 0.9867, 0.9822 and 0.8978 respectively, and the fact that exosome miRNAs have considerable sensitivity and specificity in the aspect of diagnosing FM is proved, so that the exosome miRNAs have potential to become noninvasive diagnostic biomarkers.

To our knowledge, this is the first study of developing circulating exosome miRNA expression profiles in the pediatric FM cohort, we have still found miRNA expression patterns different from adult myocarditis, and validated the possibility of plasma exosome mirnas as pediatric FM diagnostic markers.

It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A biomarker for the detection of pediatric fulminant myocarditis, wherein the biomarker is selected from any one or more of the following mirnas:

hsa-miR-30e-5p、hsa-miR-146a-5p、hsa-miR-361-5p、hsa-miR-532-5p。

2. the biomarker for the detection of pediatric fulminant myocarditis of claim 1, wherein the miRNA is a subject plasma exosome miRNA.

3. The biomarker for the detection of pediatric fulminant myocarditis of claim 1, wherein the biomarker is a group consisting of hsa-miR-30e-5p, hsa-miR-146a-5p, hsa-miR-361-5p, and hsa-miR-532-5 p.

4. A biomarker for the detection of pediatric fulminant myocarditis according to any of claims 1 to 3, wherein the biomarker is for early diagnosis and/or prognostic assessment of pediatric fulminant myocarditis.

5. Use of a substance that detects the expression level of a biomarker according to any of claims 1 to 4 in the manufacture of a product for the detection of pediatric fulminant myocarditis;

further, the substances include substances for detecting the expression level of the above-mentioned biomarkers based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method;

further, the products include devices, kits, and apparatuses.

6. A kit, comprising:

one or more devices for detecting the biomarker of any of claims 1-4.

7. An apparatus comprising the kit of claim 6.

8. Use of a kit and/or device according to claim 6 or 7 for the preparation of a product for the detection of pediatric fulminant myocarditis.

9. The use according to claim 8, wherein the detection of pediatric fulminant myocarditis, in particular early diagnosis and/or prognosis of pediatric fulminant myocarditis.

10. A system for detecting pediatric fulminant myocarditis comprising:

i) An analysis unit comprising: a detection agent for determining the expression level of a biomarker selected from the group consisting of the biomarkers of claims 1-4 in a sample of a subject, and

ii) an evaluation unit comprising a data processor, said data processor being tangibly embedded with an algorithm for comparing the quantity determined by said analysis unit with a reference, and being able to generate an output file comprising diagnostic results established on the basis of said comparison.