RNA composition for diagnosing or evaluating idiopathic inflammatory myopathy, kit and application

Abstract

The invention provides an RNA (Ribonucleic Acid) composition for diagnosing or evaluating idiopathic inflammatory myopathy, a kit and application. Wherein the RNA composition is prepared from hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1 (Diaphragmitis Factor A1). In patients with the idiopathic inflammatory myopathy, hsa-miR-590, hsa-miR-28, hsa-miR-409 and hsa-miR-146b can be obviously reduced, and DEFA1 can be obviously increased, so that the RNA composition can be used as a biomarker for diagnosing the idiopathic inflammatory myopathy or evaluating the treatment effect of the idiopathic inflammatory myopathy, has the advantages of high sensitivity, high specificity and high accuracy, and can be used for preparing a biomarker for treating the idiopathic inflammatory myopathy. And a reliable scientific basis and a personalized treatment idea can be provided for treatment and clinical diagnosis of idiopathic inflammatory myopathy.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to an RNA composition, a kit and applications for diagnosing or evaluating idiopathic inflammatory myopathy. Background Art

[0002] Idiopathic inflammatory myopathies (IIMs) are a group of rare and heterogeneous immune-mediated diseases characterized by a variety of muscle and non-muscle manifestations, including multiple clinically defined subtypes. Serological markers of IIMs include myositis-specific antibodies (MSA), such as anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibodies, anti-TIF1-γ antibodies, anti-Mi2 antibodies, and anti-tRNA synthetase antibodies. Among them, anti-MDA5-positive (MDA5-positive) + ) subtype is a rare but unique subtype of IIM, usually manifested by clinical muscle weakness and involvement of multiple organs (including skin, joints, lungs, heart and gastrointestinal tract). Interstitial lung disease (ILD) is a common complication associated with IIM and is associated with a higher mortality rate. It is estimated that the incidence of ILD in IIM patients varies by region, with 50%, 23% and 26% in Asia, North America and Europe, respectively, and is on the rise. Among them, the incidence of ILD in the anti-MDA5 positive subtype is significantly increased. Therefore, there is an urgent need for feasible and reliable biomarkers to combat MDA5-positive idiopathic inflammatory myopathy-associated interstitial lung disease (MDA5 + The purpose is to classify patients with MDA5-positive ILD to deepen our understanding of the pathogenesis of this subtype of disease, thereby achieving accurate diagnosis of clinically resistant MDA5-positive subtypes and identifying potential therapeutic targets. Summary of the Invention

[0003] The present invention provides an RNA composition and a kit for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, which have the advantages of high sensitivity, high specificity, and high accuracy, and can provide a reliable scientific basis and personalized treatment ideas for the treatment and clinical diagnosis of idiopathic inflammatory myopathy.

[0004] The present invention provides a use of the above-mentioned RNA composition or the above-mentioned kit in preparing a product for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

[0005] The present invention provides a system for diagnosing or evaluating idiopathic inflammatory myopathy and a computer-readable storage medium capable of implementing the system's functions. By combining reverse transcription with next-generation sequencing technology, the system can comprehensively and objectively reflect circulating RNA in body fluids. This overcomes the low sensitivity of high-throughput sequencing technology for detecting sncRNA and mRNA in EVs. It also addresses the problem of poor sequencing data quality caused by the large number of byproducts generated during EV RNA library construction, which in turn makes it difficult to obtain sufficient useful information for identifying specific biomarkers. The test results obtained using the above system or computer-readable storage medium are more realistic, comprehensive, and reliable.

[0006] The present invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, wherein the RNA composition comprises hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

[0007] The present invention provides a kit for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, wherein the kit comprises a reagent for detecting the above-mentioned RNA composition.

[0008] The kit as described above, wherein the reagents include: a reagent for extracting plasma extracellular vesicle RNA and a reagent for determining RNA expression.

[0009] The kit as described above, wherein the reagents for extracting plasma extracellular vesicle RNA include: at least one of ethylenediaminetetraacetic acid, exosome RNA extraction reagent, DNA lysis reagent, deoxyribonuclease I, ethanol, phosphate buffered saline, nucleic acid fluorescent dye, protease inhibitor, and RNase inhibitor.

[0010] The kit as described above, wherein the reagents for determining RNA expression include: T4 polynucleotide kinase, Escherichia coli Poly (A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template switching oligonucleotide, UMI molecular tag, DNA polymerase, Cas9 nuclease, sgRNA, and at least one of a DNA quantification reagent.

[0011] The present invention provides a use of the above-mentioned RNA composition or the above-mentioned kit in preparing a product for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

[0012] The use as described above, wherein the idiopathic inflammatory myopathy is anti-melanoma differentiation-related gene 5 antibody-positive idiopathic inflammatory myopathy with interstitial lung disease;

[0013] Optionally, in diagnosis or assessment, the concentration of each RNA in the RNA composition in plasma is measured to determine its expression level;

[0014] Optionally, the respective RNA is derived from extracellular vesicles of plasma.

[0015] The present invention provides a system for diagnosing or evaluating idiopathic inflammatory myopathy, comprising:

[0016] a sample obtaining module, which is used to provide a plasma sample from an individual to be tested;

[0017] a sample detection module for determining the expression level of the RNA composition in the plasma sample;

[0018] An analysis and evaluation module, which is used to perform a diagnosis or prognosis assessment on the individual to be tested based on the expression level of the determined RNA composition;

[0019] Among them, diagnosing the individual to be tested refers to determining whether the individual to be tested is an idiopathic inflammatory myopathy with interstitial lung disease patient who is anti-melanoma differentiation-related gene 5 antibody positive;

[0020] Among them, the prognostic evaluation of the individual to be tested refers to the evaluation of the prognosis of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-related gene 5 antibodies;

[0021] Among them, the RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

[0022] The present invention provides a computer-readable storage medium on which a computer program is stored, wherein the computer program is a computer program for realizing the functions of the system as described above.

[0023] The present invention provides a computer-readable storage medium having a computer program stored thereon, wherein when the computer program is executed by a processor, a computer-assisted diagnosis or evaluation method based on the above-mentioned RNA composition or the above-mentioned kit is implemented;

[0024] Optionally wherein the method of diagnosis or assessment comprises:

[0025] i) receiving test data of a plasma sample from an individual to be tested;

[0026] ii) comparing the test data with the reserved reference data using a preset machine learning model;

[0027] iii) Output diagnosis or evaluation results based on the comparison results.

[0028] The present invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic efficacy of idiopathic inflammatory myopathy. The RNA composition comprises hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. In patients with idiopathic inflammatory myopathy, hsa-miR-590, hsa-miR-28, hsa-miR-409, and hsa-miR-146b are significantly downregulated, while DEFA1 is significantly upregulated. Therefore, the RNA composition can be used as a biomarker for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic efficacy of idiopathic inflammatory myopathy. It has the advantages of high sensitivity, high specificity, and high accuracy, and can provide a reliable scientific basis and personalized treatment ideas for the treatment and clinical diagnosis of idiopathic inflammatory myopathy.

[0029] This invention was funded by the 2023 Chinese Academy of Medical Sciences Medical and Health Science and Technology Innovation Project-Major Collaborative Innovation Project "Research on the role and mechanism of innate immune nucleic acid recognition in systemic autoimmune diseases" (Grant No.: 2023-I2M-2-005). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a flowchart of EV RNA sequencing in Example 3 of the present invention;

[0031] Figure 2 This is a heat map of differentially expressed RNA between the MDA5 group and the HC group in Example 4 of the present invention;

[0032] Figure 3 This is the volcano plot of differentially expressed RNA between the MDA5 group and the HC group in Example 4 of the present invention;

[0033] Figure 4 This is a graph showing the RNA marker expression levels in the MDA5 group and the HC group in Example 4 of the present invention;

[0034] Figure 5 The confusion matrix diagram of the training set and the confusion matrix diagram of the validation set in Example 4 of the present invention;

[0035] Figure 6 The support vector machine classifier in MDA5 in Example 4 of the present invention + Performance results graph in IIM-ILD diagnosis;

[0036] Figure 7 This is a graph of the AUC value of the ROC curve of the support vector machine classifier in Example 4 of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0038] Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune diseases with diverse clinical manifestations, therapeutic responses, and prognoses. Muscle weakness is typically the hallmark clinical presentation, and other organs may also be affected, such as the skin, joints, lungs, heart, or gastrointestinal tract. Based on clinical, histopathological, and serological features, IIMs can be divided into different subtypes. These subtypes exhibit distinct organ manifestations, therapeutic responses, and prognoses, implying distinct pathophysiological mechanisms within each subtype.

[0039] Currently, the discovery and validation of biomarkers associated with IIMs is limited by the heterogeneity of clinical manifestations and disease progression among IIM subtypes. Existing clinical symptoms, such as Gottron's nodules, Gottron's sign, purpura, muscle weakness, and muscle atrophy, are not consistent across all patients. Furthermore, imaging features vary significantly across IIM subtypes, often lacking specific findings. Studies have shown that myositis-specific autoantibodies (MSA) are present in up to 60% of IIM patients. MSA is closely associated with diverse clinical phenotypes, aiding in the diagnosis of IIMs and important for predicting organ manifestations and prognosis. However, methods for detecting MSA, such as immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and dot immunoassay (DIA), have limitations. Relying solely on MSA is insufficient for accurate diagnosis and requires combination with other diagnostic methods. Furthermore, the diagnostic challenges of IIMs lie not only in the low specificity and sensitivity of commonly used antibodies, clinical symptoms, and imaging techniques, but also in the low incidence of the disease, which further contributes to the problem of delayed diagnosis. Therefore, standardized, safe, simple, economical, and rapid diagnostic methods are urgently needed to diagnose idiopathic inflammatory myopathies or evaluate the therapeutic effects of idiopathic inflammatory myopathies, thereby deepening our understanding of the pathogenesis of the disease, enabling accurate diagnosis, and identifying potential therapeutic targets.

[0040] To solve the above problems, the first aspect of the present invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, wherein the RNA composition comprises hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

[0041] The RNA markers hsa-miR-590, hsa-miR-28, hsa-miR-409, and hsa-miR-146b in the RNA composition are microRNAs (miRNAs). MiRNAs consist of single-stranded RNA precursors (pre-miRNAs) with hairpin structures of approximately 70 bases, which are processed by Dicer to form mature miRNAs. The reference sequence of hsa-miR-590 can be found at NCBI NR_030321.1, the reference sequence of hsa-miR-28 can be found at NCBI NR_029502.1, the reference sequence of hsa-miR-409 can be found at NCBI NR_029975.1, and the reference sequence of hsa-miR-146b can be found at NCBI NR_030169.1.

[0042] Furthermore, the 5' and 3' arms of some pre-miRNAs can each produce a mature miRNA, and therefore are named "-5p" and "-3p", respectively;

[0043] The nucleotide sequence of hsa-miR-590-5p is:

[0044] 5'-GAGCUUAUUCAUAAAAGUGCAG-3' (SEQ ID NO: 11);

[0045] The nucleotide sequence of hsa-miR-590-3p is:

[0046] 5'-UAAUUUUAUGUAUAAGCUAGU-3' (SEQ ID NO: 12);

[0047] The nucleotide sequence of hsa-miR-28-5p is:

[0048] 5'-AAGGAGCUCACAGUCUAUUGAG-3' (SEQ ID NO: 13);

[0049] The nucleotide sequence of hsa-miR-28-3p is:

[0050] 5'-CACUAGAUUGUGAGCUCCUGGA-3' (SEQ ID NO: 14);

[0051] The nucleotide sequence of hsa-miR-409-5p is:

[0052] 5'-AGGUUACCCGAGCAACUUUGCAU-3' (SEQ ID NO: 15);

[0053] The nucleotide sequence of hsa-miR-409-3p is:

[0054] 5'-GAAUGUUGCUCGGUGAACCCCU-3' (SEQ ID NO: 16);

[0055] The nucleotide sequence of hsa-miR-146b-5p is:

[0056] 5'-UGAGAACUGAAUUCCAAUAGGCUG-3' (SEQ ID NO: 17);

[0057] The nucleotide sequence of hsa-miR-146b-3p is:

[0058] 5'-GCCCUGUGGACUCAGUUCUGGU-3' (SEQ ID NO: 18).

[0059] In addition, DEFA1 (Defensin Alpha 1) is a gene encoding human α-defensin 1, belongs to the defensin family, is mainly expressed in neutrophils, and has antibacterial activity; in the present invention, the RNA marker DEFA1 is a messenger RNA (mRNA) encoding human α-defensin 1, its reference sequence can be seen in NCBI NM_004084.3, and its nucleotide sequence is shown in SEQ ID NO:19.

[0060] The above RNA composition can be used as a biomarker for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, thereby providing a reliable scientific basis and personalized treatment ideas for the treatment and clinical diagnosis of idiopathic inflammatory myopathy. It is also particularly suitable for anti-MDA5-positive idiopathic inflammatory myopathy-associated interstitial lung disease (MDA5 + IIM-ILD). Specifically, in MDA5 +In IIM-ILD patients, hsa-miR-590, hsa-miR-28, hsa-miR-409 and hsa-miR-146b were significantly downregulated, while DEFA1 was significantly upregulated. + The biomarker of IIM-ILD has a sensitivity of 93.75%, a specificity of 100%, an accuracy of 96.83%, and an AUC value of 0.966; in the validation set, its sensitivity was 95.65%, specificity was 94.74%, accuracy was 95.24%, and AUC value was 0.966. At the same time, the present invention has a large sample size and a perfect control setting, so the results are highly reliable, which can fully demonstrate that the identified marker is MDA5 + The above RNA composition can be used to detect potential MDA5 early. + Targeting IIM-ILD subtypes and conducting personalized intervention and treatment ultimately improves MDA5 + Cure rates of IIM-ILD subtypes.

[0061] A second aspect of the present invention provides a kit for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, wherein the kit comprises a reagent for detecting the above-mentioned RNA composition.

[0062] It is understandable that, when the above-mentioned RNA combination is known, those skilled in the art can also design primers based on the sequence of the above-mentioned RNA combination, and use polymerase chain reaction (PCR) combined with electrophoresis technology or real-time fluorescence quantitative PCR (qPCR) technology to detect the above-mentioned RNA combination to determine the expression of each RNA molecule in the RNA combination, and then use it to diagnose idiopathic inflammatory myopathy or evaluate the treatment effect of idiopathic inflammatory myopathy.

[0063] Furthermore, the above reagents include: reagents for extracting plasma extracellular vesicle RNA and reagents for determining RNA expression.

[0064] Specifically, the reagents used to extract plasma extracellular vesicle RNA may include: at least one of ethylenediaminetetraacetic acid, exosome RNA extraction reagent, DNA lysis reagent, deoxyribonuclease I, ethanol, phosphate buffered saline, nucleic acid fluorescent dye, protease inhibitor, and RNase inhibitor.

[0065] Reagents for determining RNA expression may include: T4 polynucleotide kinase, Escherichia coli Poly(A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template switching oligonucleotides, UMI molecular tags, DNA polymerase, Cas9 nuclease, sgRNA, and at least one of a DNA quantification reagent.

[0066] A third aspect of the present invention provides a use of the above-mentioned RNA composition or the above-mentioned kit in the preparation of a product for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

[0067] Specifically, the RNA composition or the kit can be used to prepare a product for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, including but not limited to a therapeutic drug. In particular, the idiopathic inflammatory myopathy can be anti-melanoma differentiation-associated gene 5 antibody-positive idiopathic inflammatory myopathy with interstitial lung disease (MDA5 + IIM-ILD). Experimental verification shows that the present invention is particularly suitable for diagnosing MDA5 + IIM-ILD or assessment of MDA5 + IIM-ILD therapeutic effect. Wherein, in diagnosis or assessment, the concentration of each RNA in the RNA composition in plasma can be detected to determine its expression level. Wherein, each RNA can be derived from extracellular vesicles in plasma.

[0068] Specifically, extracellular vesicles (EVs) are present in body fluids such as plasma, serum, urine, saliva, cerebrospinal fluid, and breast milk. They are a heterogeneous group of membrane-bound structures secreted by cells, encapsulated by a lipid bilayer, and incapable of self-replication (lacking a functional nucleus). As a source of self-antigens and immune complexes, EVs play a crucial role in the pathogenesis of autoimmune diseases. EVs have emerged as potential biomarkers in a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), systemic sclerosis (SSc), chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis (MG), and IIM. Because EVs are ubiquitous in body fluids and possess a lipid bilayer that effectively protects the encapsulated RNA from degradation by ribonucleases, they exhibit high stability and tissue specificity in body fluids. Therefore, the molecular cargo of EVs has the potential to help classify disease subtypes and elucidate biological mechanisms, potentially improving diagnostic accuracy, predicting prognosis, and monitoring treatment efficacy. However, plasma EV RNA is currently used as a marker for MDA5 +The research on IIM-ILD related biomarkers still has the problems of low sensitivity, low specificity and low accuracy, which need to be solved urgently. Therefore, in order to solve this problem, the present invention extracts EVs from plasma, further extracts EV RNA, and then uses the above kit to detect the concentration of each RNA in the above RNA composition in plasma to determine its expression level, and finally plays a role in diagnosing or monitoring MDA5 + The role of IIM-ILD has the advantages of high sensitivity, high specificity and high accuracy.

[0069] In addition, those skilled in the art can use PCR technology (such as real-time quantitative PCR technology) to detect the concentration of each RNA in the above-mentioned RNA composition to determine its expression level, or use sequencing methods to detect the concentration of each RNA in the above-mentioned RNA composition to determine its expression level.

[0070] A fourth aspect of the present invention provides a system for diagnosing or evaluating idiopathic inflammatory myopathy, comprising:

[0071] a sample obtaining module, which is used to provide a plasma sample from an individual to be tested;

[0072] a sample detection module for determining the expression level of the RNA composition in the plasma sample;

[0073] An analysis and evaluation module, which is used to perform a diagnosis or prognosis assessment on the individual to be tested based on the expression level of the determined RNA composition;

[0074] Among them, diagnosing the individual to be tested refers to determining whether the individual to be tested is an idiopathic inflammatory myopathy with interstitial lung disease patient who is anti-melanoma differentiation-related gene 5 antibody positive;

[0075] Among them, the prognostic evaluation of the individual to be tested refers to the evaluation of the prognosis of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-related gene 5 antibodies;

[0076] Among them, the RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

[0077] First, the sample acquisition module of the present invention is used to provide a plasma sample from an individual to be tested.

[0078] In one specific embodiment, peripheral venous blood can be collected from the individual to be tested and placed in a blood collection tube. The tube is then stored upright at room temperature (25°C). Upon use, the tube is opened, the peripheral blood is drawn into a centrifuge tube, and the tube is then balanced and centrifuged at 1300×g for 10 minutes at room temperature (25°C). The upper plasma layer is then drawn into a new centrifuge tube, which is then balanced and centrifuged at 2500×g for 15 minutes at room temperature (25°C). The supernatant is then aspirated to obtain a plasma sample. This plasma sample is platelet-poor plasma (PPP) and can be stored at -80°C for extended periods.

[0079] Secondly, the sample detection module of the present invention is used to determine the expression level of the RNA composition in the plasma sample.

[0080] Specifically, plasma samples can be removed from a -80°C freezer and immediately thawed at 37°C. Centrifuge at 2500×g for 15 minutes at 4°C to remove precipitated proteins. The supernatant is then used to isolate plasma EVs and extract EV RNA using the Qiagen exoRNeasy Midi kit to obtain an EV RNA sample. The EV RNA sample is then washed and eluted, incubated with DNase I at 37°C for 20 minutes to remove residual DNA, and purified and concentrated using the RNA Clean and Concentrator-5 kit to obtain a purified EV RNA sample. The purified EV RNA sample is dissolved in RNase-free ultrapure water for quality control. EV RNA samples are quantified using the Quant-iT RiboGreen RNA High Sensitivity Kit and integrity is assessed using an Agilent 2200 Bioanalyzer. Only EV RNA samples that pass both quantitative and qualitative quality control are used for subsequent EV RNA library construction and sequencing analysis. Qualified EV RNA samples were end-repaired and tailed using T4 polynucleotide kinase and Escherichia coli Poly(A) polymerase, and incubated at 37°C for 30 min to obtain a reaction mixture. The reaction mixture was purified and concentrated using the RNA Clean and Concentrator-5 kit to obtain a concentrated EV RNA sample.

[0081] Subsequently, the concentrated EV RNA sample was captured using the primer Oligo(dT)-UMI-N6-barcode-primer and subsequently reverse transcribed. The reverse transcription reaction system included SMARTScribe reverse transcriptase, SMARTScrib reverse transcriptase reaction buffer, dithiothreitol, dNTPs, RiboLock RNase inhibitor, template-switching oligonucleotide, PEG 8000, the primer Oligo(dT)-UMI-N6-barcode-primer, and the concentrated EV RNA sample. The reverse transcription reaction system included incubation at 42°C for 90 minutes and 70°C for 10 minutes. The resulting cDNA sample was stored at 4°C. The resulting cDNA sample was mixed with 2×SeqAmp CB PCR Buffer, PCR1 primers, and SeqAmp DNA polymerase and amplified by thermal cycling: denaturation at 98°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 30 seconds. The thermal cycled amplification product was purified and size-selected twice using Hieff NGS DNA selection beads. Prepare the CRISPR-Cas9 reaction system, mix thoroughly, and incubate in a 25°C metal bath for 10 minutes. Add the CRISPR-Cas9 reaction system to the selection beads to thoroughly resuspend them. Incubate the beads on a magnetic stand at room temperature (25°C) for 5 minutes to completely elute the DNA from the beads. The CRISPR-Cas9 reaction system containing DNA was then placed in a PCR instrument and incubated at 37°C for 60 minutes and 65°C for 5 minutes to remove abundant ribosomal and mitochondrial RNA, resulting in amplified and purified cDNA.

[0082] Next, the sequencing library amplification reaction system consisted of the purified cDNA sample, 2× SeqAmp CB PCR Buffer, PCR2 primers, and SeqAmp DNA polymerase. The amplification protocol included an initial denaturation at 94°C for 1 minute, followed by 17 cycles of denaturation at 98°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 30 seconds, followed by a final extension at 68°C for 2 minutes. The amplified product was purified and size-selected twice using Hieff NGS DNA selection beads, and the supernatant was aspirated to obtain the sequencing library. The sequencing library concentration was determined using the Qubit® dsDNA HS Assay Kit, which required a concentration >1 ng / μL. The nucleic acid fragment distribution of the sequencing library was assessed using an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Analysis Kit.

[0083] Finally, the analysis and evaluation module of the present invention is used to perform diagnosis or prognosis evaluation on the individual to be tested based on the expression level of the determined RNA composition.

[0084] The sequencing library can be placed on the Illumina NovaSeq 6000 platform for sequencing to detect the expression levels of hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. Through machine learning, 60% of the total samples are defined as the training set, and the remaining 40% are defined as the validation set. Boruta is used to select features for the training set and the validation set to find all relevant machine learning variables, and machine learning is continuously used to find the variables that can distinguish MDA5. + The support vector machine (SVM) classifier of IIM-ILD patients and healthy subjects, including the RNA combination of five RNA markers hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1, can be used as the diagnostic threshold of 0.5, and a value above 0.5 is considered MDA5. + IIM-ILD patients, if below 0.5, are non-MDA5 + IIM-ILD patients.

[0085] Among them, diagnosing the individual to be tested refers to determining whether the individual to be tested is a patient with idiopathic inflammatory myopathy with interstitial lung disease who is positive for anti-melanoma differentiation-related gene 5 antibodies.

[0086] For example, in one embodiment, a support vector machine model can be used to implement diagnosis or prognosis assessment of the individual to be tested, with 0.5 as the diagnostic threshold, and a value above 0.5 is MDA5. + In patients with IIM-ILD, if the value is less than 0.5, it is not MDA5 + IIM-ILD patients.

[0087] In addition, the prognostic evaluation of the individual to be tested refers to evaluating the prognostic status of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-related gene 5 antibodies.

[0088] For example, the expression level of MDA5 was assessed using RNA composition. + The prognosis of IIM-ILD patients is similar to that of healthy subjects, with no statistical difference, which proves that MDA5 + The prognosis of IIM-ILD patients is good; if the expression level is similar to that before treatment and there is no statistical difference, it proves that MDA5 + The prognosis of IIM-ILD patients is poor, and the corresponding treatment options are not suitable for these patients.

[0089] By combining reverse transcription with next-generation sequencing, the present invention overcomes the problem that most studies, which rely solely on reverse transcription combined with real-time quantitative PCR and microarray technology to assess specific dysregulated miRNAs or other types of RNA in body fluids as candidate markers, fail to comprehensively and objectively reflect circulating RNA in body fluids. By utilizing next-generation sequencing, the present invention also overcomes the low sensitivity of high-throughput sequencing technologies for detecting small non-coding RNAs (sncRNAs) and messenger RNAs (mRNAs) in EVs. Furthermore, the present invention, through the CRISPR-Cas9 reaction system, addresses the problem of poor sequencing data quality caused by the large number of byproducts generated during EV RNA library construction, which in turn makes it difficult to obtain sufficient useful information for identifying specific biomarkers.

[0090] According to the above-mentioned idiopathic inflammatory myopathy diagnosis or assessment system, the present invention further provides an idiopathic inflammatory myopathy diagnosis or assessment method, comprising:

[0091] obtaining a plasma sample from the individual to be tested;

[0092] determining the expression level of the RNA composition in the plasma sample;

[0093] Performing a diagnosis or prognosis assessment on the individual to be tested based on the expression level of the measured RNA composition;

[0094] Among them, diagnosing the individual to be tested refers to determining whether the individual to be tested is an idiopathic inflammatory myopathy with interstitial lung disease patient who is anti-melanoma differentiation-related gene 5 antibody positive;

[0095] Among them, the prognostic evaluation of the individual to be tested refers to the evaluation of the prognosis of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-related gene 5 antibodies;

[0096] Among them, the RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

[0097] A fifth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program is a computer program for implementing the functions of the system as described above.

[0098] A sixth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein when the computer program is executed by a processor, a computer-assisted diagnosis or assessment method based on the above-mentioned RNA composition or the above-mentioned kit is implemented; optionally, the diagnosis or assessment method comprises:

[0099] i) receiving test data of a plasma sample from an individual to be tested;

[0100] ii) comparing the test data with the reserved reference data using a preset machine learning model;

[0101] iii) Output diagnosis or evaluation results based on the comparison results.

[0102] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

[0103] Example 1: Collection and preparation of plasma samples

[0104] Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of autoimmune-mediated systemic connective tissue diseases characterized by skeletal muscle inflammatory cell infiltration and myofiber necrosis, degeneration, and regeneration. Clinically, they present as symmetrical weakness of the proximal muscles of the limbs, limb girdle muscles, neck muscles, and throat muscles, often affecting multiple organs. When the alveolar space and pulmonary interstitium are involved, interstitial lung disease (ILD) can develop. IIMs primarily include polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM).

[0105] Patients with idiopathic inflammatory myopathy and interstitial lung disease who were positive for anti-melanoma differentiation-associated gene 5 (MDA5) antibodies were enrolled in the study. + IIM-ILD) as the object to be detected. Among them, MDA5 + The inclusion criteria for IIM-ILD patients are as follows: (1) The diagnosis of PM or DM is based on the Bohan and Peter criteria; at the same time, since there is no recognized MDA5 + IIM diagnostic criteria, therefore MDA5 +IIM is a subtype of IIM, and its clinical diagnosis is based on the IIM classification criteria and the detection results of anti-MDA5 antibodies; (2) The diagnosis of ILD is based on respiratory symptoms, high-resolution computed tomography (HRCT) results, and pulmonary function test results; based on the confirmed diagnosis, ILD patients are divided into mild (>60%), moderate (40%-60%), and severe (<40%) according to the degree of decrease in the diffusion capacity for carbon monoxide (DLCO, % predicted value). Healthy people were used as the control group. Healthy people and enrolled patients (MDA5 + The general clinical characteristics of IIM-ILD can be seen in Table 1.

[0106] Table 1

[0107]

[0108] Peripheral venous blood samples were collected from 55 of the subjects and 51 controls. 2 ml of peripheral venous blood was collected from each sample and placed in EDTA-K2 blood collection tubes. The tubes were then stored upright at room temperature (25°C). The tubes were opened and 500 μl of peripheral blood was aspirated into 2 ml centrifuge tubes. After equalization, the tubes were centrifuged at 1300 × g for 10 minutes at room temperature (25°C). The supernatant plasma was aspirated into a new 2 ml centrifuge tube. After equalization, the tubes were centrifuged at 2500 × g for 15 minutes at room temperature (25°C). The supernatant was aspirated to obtain the plasma sample. This plasma sample, which is poor platelet plasma (PPP), can be stored at -80°C for extended periods.

[0109] Example 2: Preparation and Quality Control of Plasma Extracellular Vesicle RNA Samples

[0110] Plasma samples were removed from a -80°C freezer and immediately thawed at 37°C for 2 minutes. The samples were then centrifuged at 2500 × g for 15 minutes at 4°C to remove precipitated proteins. One ml of the supernatant was used to isolate extracellular vesicles (EVs) and extract EV RNA using the Qiagen exoRNeasy Midi kit to obtain EV RNA samples. The EV RNA samples were washed and eluted, and residual DNA was removed by incubation with DNase I (TaKaRa, Catalog No. 2270A) at 37°C for 20 minutes. The purified EV RNA samples were then purified and concentrated using the Zymo RNA Clean and Concentrator-5 kit (Cat. No. R1016).

[0111] Purified EV RNA samples were dissolved in 10 μl of RNase-free ultrapure water, and 2 μl was removed for quality control. EV RNA samples were quantified using the Quant-iT RiboGreen RNA High Sensitivity Kit and integrity was determined using an Agilent 2200 Bioanalyzer. 1.2–5.6 ng of EV RNA was extracted per mL of plasma, and the EV RNA distribution must conform to a standard length peak pattern. Only EV RNA samples that passed both quantitative and qualitative quality control were used for subsequent EV RNA library construction and sequencing analysis.

[0112] Example 3: EV RNA sequencing

[0113] This example establishes a simple, standardized, and highly sensitive cell-free RNA sequencing (cfRNA-seq) technology for EV RNA to construct a sequencing library for EV RNA and perform sequencing. The process involved can be seen in Figure 1The primers involved can be found in Table 2. In Table 2, the nucleotide sequence of primer TSO (SEQ ID NO: 1) is labeled with biotin at the 5' end, and the rG at the 19-21 bp of the nucleotide sequence represents riboguanine (RNA-G); the nucleotide sequence of primer Oligo(dT)-UMI-N6-barcode-primer (SEQ ID NO: 2) is labeled with biotin at the 5' end, and the 8N at the 21-28 bp of the nucleotide sequence represents the UMI sequence, and the 4N at the 29-32 bp represents the barcode. At the same time, in the nucleotide sequence, N indicates that the base of the deoxynucleotide at that location can be selected from any of adenine (A), guanine (G), cytosine (C) or thymine (T), and V indicates that the base of the deoxynucleotide at that location can be selected from any of A, G or C; the nucleotide sequence of primer 5'PCR1 (SEQ ID NO: 3) is labeled with biotin at the 5' end, and the nucleotide sequence at the 20-27 8N at the bp position represents the tag index, and in the nucleotide sequence, N indicates that the base of the deoxynucleotide at that position can be selected from any one of A, G, C or T, and * indicates phosphorothioate; the nucleotide sequence of primer 3'PCR1 (SEQ ID NO: 4) is labeled with Biotin at the 5' end, and 8N at the 20th to 27th bp of the nucleotide sequence represents the tag index, and in the nucleotide sequence, N indicates that the base of the deoxynucleotide at that position can be selected from any one of A, G, C or T, and * indicates phosphorothioate; in the nucleotide sequence of primer 5'PCR2 (SEQ ID NO: 5), * indicates phosphorothioate; in the nucleotide sequence of primer 3'PCR2 (SEQ ID NO: 6), * indicates phosphorothioate; in the nucleotide sequence of the sgRNA Pool library forward primer of the sgRNA DNA template (SEQ ID NO: 7), 20th to 39th bp of the nucleotide sequence are labeled with Biotin; The 20N at the bp position represents the specific target sequence in the sgRNA, and N in the nucleotide sequence indicates that the base of the deoxynucleotide at that position can be selected from any of A, G, C or T. The specific process is as follows:

[0114] (1) EV RNA sample pretreatment: Qualified EV RNA samples were end-repaired and tailed using T4 polynucleotide kinase (Cat. No. M0201) produced by NEB and Escherichia coli Poly(A) polymerase (Cat. No. M0276) produced by NEB. The reaction volume was 20 μL and incubated at 37°C for 30 min to obtain a reaction mixture. Subsequently, the reaction mixture was purified and concentrated using the RNA Clean and Concentrator-5 kit (Cat. No. R1016) produced by Zymo to obtain a concentrated EV RNA sample with a volume of 6 μL.

[0115] (2) Reverse transcription: The concentrated EV RNA sample was captured using the primer Oligo(dT)-UMI-N6-barcode-primer and then reverse transcribed. The reverse transcription reaction system included: 2 μL of SMARTScribe reverse transcriptase, 4 μL of SMARTScrib reverse transcriptase reaction buffer, 2 μL of 20 mM dithiothreitol, 2 μL of dNTPs, 0.5 μL of RiboLock RNase inhibitor, 0.5 μL of template-switching oligos (TSO), 2 μL of PEG 8000, 1 μL of 1 μM Oligo(dT)-UMI-N6-barcode-primer, and 6 μL of concentrated EV RNA sample; the reverse transcription reaction system included: incubation at 42°C for 90 min and incubation at 70°C for 10 min; finally, 20 μL of cDNA sample was obtained and stored at 4°C.

[0116] (3) Pre-amplification and purification of sequencing library: 20 μL of cDNA sample, 25 μL of 2×SeqAmp CB PCR Buffer, 1 μL of 5'PCR1, 1 μL of 3'PCR1, and 1 μL of SeqAmp DNA polymerase were mixed and subjected to thermal cycling amplification (cycle number 6): denaturation at 98°C for 15 s, annealing at 55°C for 15 s, and extension at 68°C for 30 s. The thermal cycling amplification product was purified and size-selected twice using Hieff NGS DNA selection beads (product number 12601ES56) manufactured by Yeasen, at a volume ratio of 1.8:1 between the selection beads and the DNA in the thermal cycling amplification product. Prepare a CRISPR-Cas9 reaction system and mix thoroughly, then incubate in a 25°C metal bath for 10 min. The CRISPR-Cas9 reaction system includes: 12 μL of NEBuffer 3.1, 1 μL of 300 ng of rRNA sgRNA, 1 μL of 40 ng of mtRNA sgRNA, 8.3 μL of nuclease-free water, and 10 μL of 1 μM Cas9 nuclease. A primer pair (sgRNA library forward primer for sgRNA DNA template and sgRNA library reverse primer for sgRNA DNA template) and a primer pair (5' universal primer for sgRNA DNA template and 3' universal primer for sgRNA DNA template) are used to amplify the cDNA sample in one step to obtain the sgRNA DNA template. T7 RNA polymerase (NEB, catalog number E2050) is then used to transcribe the cDNA in vitro to obtain RNA sgRNA and mtRNA sgRNA. Add 21.5 μL of CRISPR-Cas9 reaction system to the above-mentioned selection beads to thoroughly resuspend the selection beads, and place the selection beads on a magnetic stand and incubate at room temperature (25°C) for 5 min to completely elute the DNA from the magnetic beads. Then place the CRISPR-Cas9 reaction system containing DNA in a PCR instrument, incubate at 37°C for 60 min, incubate at 65°C for 5 min, and store at 4°C to remove high-abundance ribosomal RNA and mitochondrial RNA to obtain the amplified and purified cDNA sample.

[0117] (5) Amplification and purification of sequencing library: The amplification reaction system of the sequencing library includes: 20 μL of amplified and purified cDNA sample, 50 μL of 2×SeqAmp CB PCR Buffer, 2.5 μL of 5'PCR2 and 2.5 μL of 3'PCR2, 2 μL of SeqAmp DNA polymerase and 23 μL of nuclease-free water. The amplification procedure of the sequencing library includes: initial denaturation at 94°C for 1 min; denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 68°C for 30 s, 17 cycles; final extension at 68°C for 2 min. The amplified product was purified and size-selected twice using Hieff NGS DNA selection beads at a volume ratio of 1:1 between the selection beads and the DNA in the amplified product. 16 μL of the supernatant solution was aspirated to obtain the sequencing library.

[0118] (6) Sequencing: The concentration of the sequencing library was determined using the Qubit® dsDNA HS Assay Kit, which was required to be >1 ng / μL. The nucleic acid fragment distribution of the sequencing library was evaluated using an Agilent 2100 bioanalyzer and a high-sensitivity DNA analysis kit (catalog number 5067-4626) produced by Agilent. Sequencing was then performed on the Illumina NovaSeq6000 platform.

[0119] Table 2

[0120]

[0121] Example 4: Plasma EV RNA Combination in MDA5 + Effect of IIM-ILD in diagnosis

[0122] (1) Use limma47 software to find the differentially expressed RNAs between the test subjects (MDA5 group) and the control group (HC group) (P value < 0.05, difference fold > 1.0), and draw heat maps and volcano maps respectively. Figure 2 is the heat map of differentially expressed RNAs in the MDA5 and HC groups; Figure 3 Volcano plot of differentially expressed RNA in MDA5 group and HC group, red is MDA5 + Upregulated (UP) RNA in IIM-ILD patients, MDA5 in blue + Downregulated (DOWN) RNA in IIM-ILD patients, MDA5 in gray + There were no differentially expressed (Not) RNAs between IIM-ILD patients and healthy subjects.

[0123] (2) According to Figure 2 and Figure 3Based on the results, unsupervised hierarchical clustering and principal component analysis (PCA) were performed on these differentially expressed RNAs. Through machine learning, 60% of the total samples were defined as the training set, and the remaining 40% as the validation set. Boruta was used to select features in the training and validation sets to find all relevant machine learning variables. Machine learning was then continued to find the optimal performance that could distinguish MDA5 + The support vector machine (SVM) classifier of IIM-ILD patients and healthy subjects included an RNA panel consisting of five RNA markers: hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. Among them, hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1 were significantly different compared with the HC group, and were significantly different in MDA5 + In IIM-ILD patients, hsa-miR-590, hsa-miR-28, hsa-miR-409, and hsa-miR-146b were significantly downregulated, while DEFA1 was significantly upregulated. Figure 4 , Figure 4 * indicates significant difference, p < 0.05; ** indicates significant difference, p < 0.01; *** indicates significant difference, p < 0.001. Figure 4 The results showed that the above five RNA markers have good MDA5 + Diagnostic effect of IIM-ILD.

[0124] Secondly, the Wilcoxon rank sum test was used to analyze the Log2 difference folds and P values of the five RNA markers in the test subjects (abbreviated as MDA5 group) and the control group (abbreviated as HC group). See Table 3 for details.

[0125] Table 3

[0126]

[0127] As shown in Table 3, hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1 had large fold differences and extremely small P values, indicating that the above five RNA markers have good MDA5 + Diagnostic effect of IIM-ILD.

[0128] (3) The five-fold cross-validation algorithm is used to calculate the classifier of the RNA combination consisting of the above five RNA markers. The results are shown in Figure 2. Figure 5 and Figure 6 The bootstrap method was used for 100 iterations, and the receiver operating characteristic (ROC) curve was used to evaluate the area under the curve (AUC) of the five RNA marker classifiers. Figure 7 .

[0129] in, Figure 5 The confusion matrix of the training set (Training Set) and the confusion matrix of the validation set (Validation Set) are shown; Figure 6 For the support vector machine classifier in MDA5 + Performance (Metrics) results in IIM-ILD diagnosis. Sensitivity, specificity, and accuracy are marked on the graph. The calculation found that the classifier containing the above combination of 5 RNA markers can distinguish healthy subjects from MDA5 in the training set. + The sensitivity of IIM-ILD patients was 93.75%, the specificity was 100%, and the accuracy was 96.83%. + The sensitivity, specificity, and accuracy for IIM-ILD patients were 95.65%, 94.74%, and 95.24%, respectively. These results demonstrate that the combination of five RNA markers performed well in both the training and validation sets.

[0130] Figure 7 The figure shows the area under the curve (AUC) of the support vector machine classifier receiver operating characteristic (ROC) curve, where ROC stands for receiver operating characteristic (ROC) and AUC stands for area under the ROC curve (AUC). The results show that in the training set, the AUC value of the five-RNA marker combination was 0.966 (95% CI, 0.964-0.968); in the validation set, the AUC value of the five-RNA marker combination was 0.966 (95% CI, 0.963-0.968). Since AUC values range from 0.5 to 1, with AUC values closer to 1.0 indicating higher detection accuracy and AUC values of 0.5 indicating the lowest accuracy, these results demonstrate that the five-RNA marker combination performed well in both the training and validation sets.

[0131] (4) A system for diagnosing or evaluating idiopathic inflammatory myopathy is developed by the methods of Examples 1-4 above, which comprises: a sample acquisition module for providing a plasma sample from an individual to be tested, a sample detection module for determining the expression level of an RNA composition in the plasma sample, and an analysis and evaluation module for diagnosing or evaluating the prognosis of the individual to be tested based on the expression level of the determined RNA composition. The analysis and evaluation module can be used to distinguish MDA5 from + The support vector machine classifier model of IIM-ILD patients and healthy subjects was implemented, with 0.5 as the diagnostic threshold, and those above 0.5 were MDA5 + IIM-ILD patients, if below 0.5, are non-MDA5 + IIM-ILD patients.

[0132] In summary, the present invention selects MDA5 + A total of 106 plasma samples from IIM-ILD patients and healthy subjects were collected, including 51 healthy subjects and 55 MDA5 + IIM-ILD, by isolating and purifying EVs from plasma, extracting RNA from EVs and performing RNA high-throughput sequencing, the RNA expression levels in plasma EVs were detected. A total of 1602 differentially expressed RNAs were obtained through bioinformatics analysis. Finally, 5 RNAs were selected as MDA5 + The biomarker of IIM-ILD has a sensitivity of 93.75%, a specificity of 100%, an accuracy of 96.83%, and an AUC of 0.966 in the training set, and a sensitivity of 95.65%, a specificity of 94.74%, an accuracy of 95.24%, and an AUC of 0.966 in the validation set. As the role of EVs in IIMs has not been fully characterized, comprehensive research on EV-related RNAs in IIMs is still insufficient, especially for MDA5. + While the spectrum of EV-associated RNA in the plasma of IIM-ILD patients remains to be further explored, this present invention, through the development of a more traceable, stable, and standardized EV RNA sequencing technology, has identified a high-quality RNA composition that can serve as a biomarker for diagnosing clinical subtypes of idiopathic inflammatory myopathy. This EV RNA sequencing technology and RNA composition lay the foundation for further research and identification of new therapeutic or diagnostic targets, as well as for elucidating pathophysiological mechanisms and developing targeted treatment strategies, enabling clinicians to implement timely and appropriate preventive and treatment measures.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, characterized in that: The RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

2. A kit for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy, characterized in that: The kit includes reagents for detecting the RNA composition of claim 1.

3. The kit according to claim 2, wherein The reagents include: a reagent for extracting plasma extracellular vesicle RNA and a reagent for determining RNA expression.

4. The kit according to claim 3, wherein The reagent for extracting plasma extracellular vesicle RNA includes: at least one of ethylenediaminetetraacetic acid, exosome RNA extraction reagent, DNA lysis reagent, deoxyribonuclease I, ethanol, phosphate buffered saline solution, nucleic acid fluorescent dye, protease inhibitor, and RNase inhibitor.

5. The kit according to claim 3 or 4, characterized in that The reagent for determining RNA expression includes: T4 polynucleotide kinase, Escherichia coli Poly (A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template switching oligonucleotide, UMI molecular tag, DNA polymerase, Cas9 nuclease, sgRNA, and at least one of a DNA quantification reagent.

6. Use of the RNA composition according to claim 1 or the kit according to any one of claims 2 to 5 in the preparation of a product for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

7. The use according to claim 6, characterized in that The idiopathic inflammatory myopathy is an anti-melanoma differentiation-related gene 5 antibody-positive idiopathic inflammatory myopathy with interstitial lung disease; Optionally, in the diagnosis or the assessment, the concentration of each RNA in the RNA composition in plasma is detected to determine its expression level; Optionally, said individual RNAs are derived from extracellular vesicles of said plasma.

8. A system for diagnosing or evaluating idiopathic inflammatory myopathy, characterized in that: include: a sample obtaining module, which is used to provide a plasma sample from an individual to be tested; a sample detection module, which is used to determine the expression level of the RNA composition in the plasma sample; an analysis and evaluation module, which is used to perform a diagnosis or prognosis assessment on the individual to be tested based on the expression level of the determined RNA composition; Wherein, the diagnosis of the individual to be tested refers to determining whether the individual to be tested is a patient with idiopathic inflammatory myopathy with interstitial lung disease who is positive for anti-melanoma differentiation-related gene 5 antibodies; Wherein, the prognostic evaluation of the individual to be tested refers to evaluating the prognosis of the patient with idiopathic inflammatory myopathy and interstitial lung disease who is positive for the anti-melanoma differentiation-related gene 5 antibody; Wherein, the RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b and DEFA1.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that: The computer program is a computer program that realizes the functions of the system according to claim 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, it implements a computer-assisted diagnosis or assessment method based on the RNA composition of claim 1 or the kit of any one of claims 2 to 5, optionally wherein the diagnosis or assessment method comprises: i) receiving test data of a plasma sample from an individual to be tested; ii) comparing the test data with reserved reference data using a preset machine learning model; iii) Output diagnosis or evaluation results based on the comparison results.

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