Rna compositions, kits and uses for idiopathic inflammatory myopathy diagnosis or assessment

Abstract

The application provides a RNA composition, a kit and application for idiopathic inflammatory myopathy diagnosis or evaluation. 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 down-regulated, and DEFA1 is significantly up-regulated, so that the RNA composition can be used as a biomarker for diagnosing idiopathic inflammatory myopathy or evaluating the treatment effect of idiopathic inflammatory myopathy, has the advantages of high sensitivity, high specificity and high accuracy, and can provide a reliable scientific basis and personalized treatment idea for the treatment and clinical diagnosis of idiopathic inflammatory myopathy.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more particularly to an RNA composition, kit, and application for the diagnosis or assessment of idiopathic inflammatory myopathy. Background Technology

[0002] Idiopathic inflammatory myopathies (IIM) are a rare and heterogeneous group of immune-mediated diseases characterized by a diversity of muscular and non-muscular manifestations, encompassing multiple clinically defined subtypes. Serological markers of IIM 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 these, anti-MDA5-positive (MDA5-positive) is particularly significant. + The anti-MDA5 subtype is a rare but unique subtype of IIM, typically presenting with clinical muscle weakness and involvement of multiple organs, including the skin, joints, lungs, heart, and gastrointestinal tract. Interstitial lung disease (ILD) is a common complication associated with IIM and is associated with high mortality. The estimated incidence of ILD in IIM patients varies regionally, with rates of 50%, 23%, and 26% in Asia, North America, and Europe, respectively, and is trending upward. The incidence of ILD is significantly higher in the anti-MDA5 positive subtype. Therefore, there is an urgent need for feasible and reliable biomarkers to combat anti-MDA5-positive idiopathic inflammatory myopathy-associated interstitial lung disease (MDA5). + Classifying patients with IIM-ILD will deepen our understanding of the pathogenesis of this subtype of disease, thereby enabling accurate clinical diagnosis of MDA5-positive subtypes and identifying potential therapeutic targets. Summary of the Invention

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

[0004] The present invention provides the use of the above-described RNA composition or the above-described kit in the preparation of products for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

[0005] This invention provides a diagnostic or assessment system for idiopathic inflammatory myopathy (EV) and a computer-readable storage medium that enables the system's functions. By combining reverse transcription with next-generation sequencing technology, it can comprehensively and objectively reflect circulating RNA in body fluids. This overcomes the low sensitivity of high-throughput sequencing technology in detecting sncRNA and mRNA in EVs, and also solves the problem of poor sequencing data quality caused by numerous byproducts generated during EV RNA library construction, which makes it difficult to obtain sufficient useful information for identifying specific biomarkers. The detection results obtained using the above system or computer-readable storage medium are more accurate, comprehensive, and reliable.

[0006] The present invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the treatment 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 treatment effect of idiopathic inflammatory myopathy, wherein the kit includes reagents for detecting the above-mentioned RNA composition.

[0008] The kit described above includes reagents for extracting extracellular vesicle RNA from plasma and reagents for determining RNA expression.

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

[0010] The kit described above includes at least one of the following reagents for determining RNA expression: T4 polynucleotide kinase, Escherichia coli Poly(A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template-converting oligonucleotides, UMI molecular tags, DNA polymerase, Cas9 nuclease, sgRNA, and DNA quantification reagents.

[0011] The present invention provides the use of the above-described RNA composition or the above-described kit in the preparation of products for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effect of idiopathic inflammatory myopathy.

[0012] As described above, idiopathic inflammatory myopathy is idiopathic inflammatory myopathy with interstitial lung disease that is positive for anti-melanoma differentiation-associated gene 5 antibody;

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

[0014] Optionally, each RNA is derived from extracellular vesicles in the blood plasma.

[0015] This invention provides a diagnostic or assessment system for idiopathic inflammatory myopathy, comprising:

[0016] The sample acquisition module is used to provide plasma samples from the individual to be tested;

[0017] The sample detection module is used to determine the expression level of RNA composition in plasma samples;

[0018] The analysis and evaluation module is used to diagnose or assess the prognosis of the individual being tested based on the expression level of the RNA composition being measured.

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

[0020] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-associated gene 5 antibody.

[0021] 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 having a computer program stored thereon, wherein the computer program is a computer program that implements the functions of the system 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, it implements a computer-aided diagnostic or evaluation method based on the above-described RNA composition or the above-described kit.

[0024] Optionally, the diagnostic or assessment methods include:

[0025] i) Receive test data from plasma samples from the individuals to be tested;

[0026] ii) The detection data is compared with the reserved reference data using a pre-set machine learning model;

[0027] iii) Output diagnostic or assessment results based on the comparison results.

[0028] This invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy (IIM) or evaluating the treatment efficacy of IIM. The RNA composition comprises hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. In patients with IIM, hsa-miR-590, hsa-miR-28, hsa-miR-409, and hsa-miR-146b are significantly downregulated, while DEFA1 is significantly upregulated. Therefore, this RNA composition can serve as a biomarker for diagnosing IIM or evaluating the treatment efficacy of IIM, exhibiting advantages of high sensitivity, high specificity, and high accuracy. It can provide reliable scientific evidence and personalized treatment strategies for the treatment and clinical diagnosis of IIM.

[0029] This invention was supported by the 2023 Major Collaborative Innovation Project of the Medical and Health Science and Technology Innovation Project of the Chinese Academy of Medical Sciences, entitled "Research on the role and mechanism of innate immune nucleic acid recognition in systemic autoimmune diseases" (Grant No.: 2023-I2M-2-005). Attached Figure Description

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

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

[0032] Figure 3 This is a volcano diagram of differentially expressed RNA in the MDA5 group and HC group in Example 4 of this 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 this invention.

[0034] Figure 5 This is a diagram showing the confusion matrix of the training set and the confusion matrix of the validation set in Embodiment 4 of the present invention;

[0035] Figure 6 The support vector machine classifier in Embodiment 4 of this invention is used in MDA5 + Performance results graph in IIM-ILD diagnostics;

[0036] Figure 7 This is a graph showing the AUC values ​​of the ROC curve of the support vector machine classifier in Embodiment 4 of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Idiopathic inflammatory myopathy (IIM) is a heterogeneous group of autoimmune diseases with varying clinical presentations, treatment responses, and prognoses. It is typically characterized by muscle weakness, but other organs may also be affected, such as the skin, joints, lungs, heart, or gastrointestinal tract. Based on clinical, histopathological, and serological characteristics, IIM can be divided into different subtypes, each with distinct organ manifestations, treatment responses, and prognoses, indicating a different pathophysiological mechanism within each subtype.

[0039] Currently, the discovery and validation of IIM-related biomarkers are limited by the heterogeneity of clinical manifestations and disease progression among IIM subtypes. Existing clinical symptoms such as Gottron's nodules, Gottron's sign, purplish-red spots, muscle weakness, and muscle atrophy are not consistent across all patients, and different IIM subtypes exhibit significant variations in imaging features, often lacking specificity. Studies have shown that up to 60% of IIM patients have myositis-specific autoantibodies (MSA). MSA is closely associated with different clinical phenotypes, aiding in the diagnosis of IIM and playing a crucial role in predicting organ manifestations and prognosis. However, methods for detecting MSA, such as immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and dot immunoassay (DIA), all have limitations. Relying solely on MSA is insufficient for accurate diagnosis and requires combination with other diagnostic methods. Furthermore, the diagnostic challenges of IIM 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 diagnostic delays. Therefore, there is an urgent need for standardized, safe, simple, economical, and rapid diagnostic methods to diagnose idiopathic inflammatory myopathy or assess the effectiveness of treatment for idiopathic inflammatory myopathy, in order to deepen our understanding of the disease's pathogenesis, thereby achieving accurate diagnosis and identifying potential therapeutic targets.

[0040] To address the aforementioned problems, the first aspect of the present invention provides an RNA composition for diagnosing idiopathic inflammatory myopathy or evaluating the treatment effect of idiopathic inflammatory myopathy, the RNA composition comprising 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 belong to microRNAs (miRNAs). miRNAs are mature miRNAs formed from single-stranded RNA precursors (pre-miRNAs) with a hairpin structure of approximately 70 bases, which are then processed by the Dicer enzyme. The reference sequences for hsa-miR-590, hsa-miR-28, hsa-miR-409, and hsa-miR-146b can be found in NCBI NR_030321.1, NCBI NR_029502.1, NCBI NR_029975.1, and NCBI NR_030169.1, respectively.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0060] The aforementioned RNA composition can serve as a biomarker for diagnosing idiopathic inflammatory myopathy or evaluating the treatment efficacy of idiopathic inflammatory myopathy, thereby providing a reliable scientific basis and personalized treatment approach 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. Experimental validation showed that, in the training set, the above RNA composition served as MDA5. + The biomarker for IIM-ILD showed a sensitivity of 93.75%, specificity of 100%, accuracy of 96.83%, and an AUC of 0.966. In the validation set, the sensitivity was 95.65%, specificity 94.74%, accuracy 95.24%, and AUC 0.966. Furthermore, this invention utilizes a large sample size and well-designed controls, resulting in highly reliable results that fully demonstrate the identified biomarker is MDA5. + Biomarkers for IIM-ILD. The above RNA composition can be used to detect potential MDA5 at an early stage. + Targeting the IIM-ILD subtype and implementing personalized interventions and treatments can ultimately improve MDA5 levels. + Cure rate for the IIM-ILD subtype.

[0061] A second aspect of the present invention provides a kit for diagnosing idiopathic inflammatory myopathy or evaluating the treatment effect of idiopathic inflammatory myopathy, the kit comprising reagents for detecting the above-described RNA composition.

[0062] It is understood that, given the above-mentioned RNA combination, 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 or real-time quantitative PCR (qPCR) to detect the above-mentioned RNA combination in order to determine the expression status 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 reagents mentioned above include: reagents for extracting extracellular vesicular RNA from plasma and reagents for determining RNA expression.

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

[0065] Reagents used to determine RNA expression may include at least one of the following: T4 polynucleotide kinase, Escherichia coli Poly(A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template-converting oligonucleotides, UMI molecular tags, DNA polymerase, Cas9 nuclease, sgRNA, and DNA quantitative reagents.

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

[0067] In detail, the aforementioned RNA composition or kit can be used to prepare products for diagnosing idiopathic inflammatory myopathy or evaluating the therapeutic effects of idiopathic inflammatory myopathy, including but not limited to therapeutic drugs. Specifically, the aforementioned idiopathic inflammatory myopathy can be idiopathic inflammatory myopathy with interstitial lung disease (MDA5) that is positive for anti-melanoma differentiation-associated gene 5 antibody. + IIM-ILD). Experimental verification has shown that this invention is particularly suitable for diagnosing MDA5. + IIM-ILD or assessment of MDA5 + The therapeutic efficacy of IIM-ILD. In diagnosis or evaluation, the concentration of individual RNAs in the plasma RNA composition can be measured to determine their expression levels. These RNAs can originate from extracellular vesicles in the plasma.

[0068] Specifically, extracellular vesicles (EVs) exist in bodily fluids such as plasma, serum, urine, saliva, cerebrospinal fluid, and breast milk. These are heterogeneous membrane structures secreted by cells, enclosed in a lipid bilayer, and unable to self-replicate (lacking a functional nucleus). EVs serve as a source of self-antigens and immune complexes, playing a crucial role in the pathogenesis of autoimmune diseases. EVs have become potential biomarkers for various 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 intraepithelial neoplasia (IIM). Because EVs are widely distributed in bodily fluids and possess a lipid bilayer that effectively protects the encapsulated RNA from degradation by ribonucleases, they exhibit high stability and tissue specificity in bodily fluids. Therefore, molecular loading of EVs holds potential for disease subtyping and elucidating biological mechanisms, potentially improving diagnostic accuracy, predicting prognosis, and monitoring treatment effectiveness. However, plasma EV RNA is currently being used as MDA5. +Research on biomarkers related to IIM-ILD still suffers from low sensitivity, low specificity, and low accuracy, which urgently need to be addressed. Therefore, this invention aims to solve this problem by extracting EVs from plasma, further extracting EV RNA, and then using the aforementioned kit to detect the concentration of each RNA in the aforementioned RNA composition in plasma to determine its expression level, ultimately achieving the diagnostic or monitoring function of MDA5. + The IIM-ILD has the advantages of high sensitivity, high specificity and high accuracy.

[0069] In addition, those skilled in the art can determine the expression level by detecting the concentration of each RNA in the above RNA composition using PCR technology (e.g., real-time quantitative PCR technology), or by using sequencing methods to detect the concentration of each RNA in the above RNA composition.

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

[0071] The sample acquisition module is used to provide plasma samples from the individual to be tested;

[0072] The sample detection module is used to determine the expression level of RNA composition in plasma samples;

[0073] The analysis and evaluation module is used to diagnose or assess the prognosis of the individual being tested based on the expression level of the RNA composition being measured.

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

[0075] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-associated gene 5 antibody.

[0076] 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 the individual to be tested.

[0078] In one specific embodiment, peripheral venous blood can be collected from the individual to be tested. The peripheral venous blood is placed in a blood collection tube and stored vertically at room temperature (25°C). When needed, the blood collection tube is opened, and the peripheral blood is drawn into a centrifuge tube. After balancing, the tube is centrifuged at 1300×g for 10 min at room temperature (25°C). The supernatant is then transferred to a new centrifuge tube, balanced, and centrifuged at 2500×g for 15 min at room temperature (25°C). The supernatant is then collected to obtain a plasma sample. This plasma sample is poorplatelet plasma (PPP) and can be stored for a long time at -80°C for future use.

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

[0080] Specifically, plasma samples can be removed from a -80°C freezer and immediately thawed at 37°C. They are then centrifuged at 2500×g for 15 min at 4°C to remove precipitated proteins. The supernatant is used to separate plasma EVs and extract EV RNA using the Qiagen exoRNeasy Midi kit, yielding EV RNA samples. The EV RNA samples are washed and eluted, then incubated with DNase I at 37°C for 20 min to remove residual DNA. The RNA is then purified and concentrated using the RNA Clean and Concentrator-5 kit to obtain purified EV RNA samples. The purified EV RNA samples are dissolved in RNase-free ultrapure water for quality control. Quantification of EV RNA samples is performed using the Quant-iT RiboGreen RNA High Sensitivity Kit, and integrity is checked using an Agilent 2200 Bioanalyzer. Only EV RNA samples that pass both quantitative and qualitative quality control are suitable for subsequent EV RNA library construction and sequencing analysis. End repair and tailing of qualified EV RNA samples were performed using T4 polynucleotide kinase and E. coli Poly(A) polymerase, followed by incubation at 37°C for 30 min to obtain a reaction mixture. The reaction mixture was then purified and concentrated using the RNA Clean and Concentrator-5 kit to obtain concentrated EV RNA samples.

[0081] Subsequently, the concentrated EV RNA sample was captured using primer Oligo(dT)-UMI-N6-barcode-primer, followed by reverse transcription. The reverse transcription reaction system included: SMARTScribe reverse transcriptase, SMARTScrib reverse transcriptase reaction buffer, dithiothreitol, dNTPs, RiboLock RNase inhibitor, template-converting oligonucleotides, PEG 8000, primer Oligo(dT)-UMI-N6-barcode-primer, and the concentrated EV RNA sample. The reverse transcription reaction consisted of incubation at 42°C for 90 min followed by incubation at 70°C for 10 min. The resulting cDNA sample was stored at 4°C. The obtained cDNA sample, 2×SeqAmp CB PCR Buffer, PCR1 primers, and SeqAmp DNA polymerase were mixed and subjected to thermal cycling amplification: 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 products were purified and size-selected twice using Hieff NGS DNA selection beads. The CRISPR-Cas9 reaction system was prepared and thoroughly mixed, then incubated in a 25°C metal bath for 10 min. The CRISPR-Cas9 reaction system was added to the selection beads to completely resuspend them, and the beads were incubated on a magnetic rack at room temperature (25°C) for 5 min 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 min, followed by incubation at 65°C for 5 min to remove high-abundance ribosomal RNA and mitochondrial RNA, yielding the amplified and purified cDNA sample.

[0082] Next, the amplification reaction system for the sequencing library included: amplified and purified cDNA sample, 2×SeqAmp CB PCR Buffer, PCR2 primers, and SeqAmp DNA polymerase. The amplification program for the sequencing library included: initial denaturation at 94℃ for 1 min; denaturation at 98℃ for 15 s, annealing at 55℃ for 30 s, extension at 68℃ for 30 s, for 17 cycles; and final extension at 68℃ for 2 min. The amplified products were purified and size-selected twice using Hieff NGS DNA selection beads, and the supernatant was collected to obtain the sequencing library. The concentration of the sequencing library was determined using the Qubit® dsDNA HS Assay Kit, and the concentration needed to be >1 ng / μL. The distribution of nucleic acid fragments in the sequencing library was evaluated using an Agilent 2100 bioanalyzer and a high-sensitivity DNA analysis kit.

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

[0084] The sequencing library can be sequenced on the Illumina NovaSeq 6000 platform to detect the expression levels of hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. Using machine learning, 60% of the total samples are defined as the training set, and the remaining 40% as the validation set. Boruta feature selection is used on both the training and validation sets to find all relevant machine learning variables. This process is repeated to identify variables that can distinguish MDA5. + Support vector machine (SVM) classifiers were used for IIM-ILD patients and healthy individuals. These classifiers consisted of an RNA combination comprising five RNA markers: hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. The model used a diagnostic threshold of 0.5; values ​​above 0.5 were classified as MDA5. + For IIM-ILD patients, a value below 0.5 indicates 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 and interstitial lung disease who is positive for anti-melanoma differentiation-associated gene 5 antibody.

[0086] For example, in one embodiment, a support vector machine model can be used to diagnose or assess the prognosis of the individual to be tested, with 0.5 as the diagnostic threshold and values ​​above 0.5 indicating MDA5. + For IIM-ILD patients, a value below 0.5 indicates they are not MDA5. + IIM-ILD patients.

[0087] In addition, prognostic assessment of the individuals to be tested refers to evaluating the prognosis of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-associated gene 5 antibody.

[0088] For example, assessing MDA5 expression levels using RNA compositions. + The prognosis of IIM-ILD patients is determined by whether their expression levels are similar to those of healthy individuals, with no statistically significant difference, thus proving that MDA5 is the cause of their prognosis. + Patients with IIM-ILD have a good prognosis; if the expression level is similar to that before treatment and there is no statistically significant difference, it proves that MDA5 + Patients with IIM-ILD have a poor prognosis, and the corresponding treatment plan is not suitable for them.

[0089] This invention overcomes the limitation of most studies that rely solely on reverse transcription combined with next-generation sequencing (NGS) technology to assess specific dysregulated miRNAs or other RNA types in body fluids as candidate markers, failing to comprehensively and objectively reflect circulating RNA in body fluids. Furthermore, by using NGS, this invention also overcomes the low sensitivity of high-throughput sequencing technologies for detecting small non-coding RNAs (sncRNAs) and messenger RNAs (mRNAs) in EVs. Moreover, the CRISPR-Cas9 reaction system addresses the issue of poor sequencing data quality caused by numerous byproducts generated during EV RNA library construction, which hinders the acquisition of sufficient useful information for identifying specific biomarkers.

[0090] Based on the above-described diagnostic or assessment system for idiopathic inflammatory myopathy, the present invention also provides a method for diagnosing or assessing idiopathic inflammatory myopathy, comprising:

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

[0092] Determine the expression level of RNA composition in plasma samples;

[0093] Based on the expression level of the RNA composition measured, a diagnosis or prognostic assessment is performed on the individual being tested.

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

[0095] Among them, prognostic assessment of the individuals to be tested refers to assessing the prognostic status of patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-associated gene 5 antibody.

[0096] 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 that implements 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 the computer program, when executed by a processor, implements a computer-aided diagnostic or evaluation method based on the aforementioned RNA composition or the aforementioned kit; optionally, the diagnostic or evaluation method includes:

[0099] i) Receive test data from plasma samples from the individuals to be tested;

[0100] ii) The detection data is compared with the reserved reference data using a pre-set machine learning model;

[0101] iii) Output diagnostic or assessment results based on the comparison results.

[0102] The technical solution of this application will be further explained below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or obtained through public channels.

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

[0104] Idiopathic inflammatory myopathies (IIM) are a group of heterogeneous, autoimmune-mediated systemic connective tissue diseases characterized by inflammatory cell infiltration and myofiber necrosis, degeneration, and regeneration in skeletal muscle. Clinically, they mainly manifest as symmetrical weakness of proximal muscles of the limbs, limb girdle muscles, neck muscles, and pharyngeal muscles. They often involve multiple organs, and when the alveoli and pulmonary interstitium are involved, they can cause interstitial lung disease (ILD). IIM mainly includes polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM).

[0105] Patients with idiopathic inflammatory myopathy and interstitial lung disease who are positive for anti-melanoma differentiation-associated gene 5 (MDA5) antibodies (MDA5) will be included in the study. + IIM-ILD) was used 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 currently no universally accepted MDA5... + IIM diagnostic criteria, therefore MDA5 +As a subtype of IIM, the clinical diagnosis of IIM 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; on the basis of a confirmed diagnosis, ILD patients are divided into mild (>60%), moderate (40%-60%) and severe (<40%) according to the degree of decrease in carbon monoxide diffusion capacity (DLCO, % predicted value). Healthy individuals are used as the control group. Healthy individuals and enrolled patients (MDA5) + The general clinical features of IIM-ILD are shown in Table 1.

[0106] Table 1

[0107]

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

[0109] Example 2: Preparation and quality control of extracellular vesicle RNA samples from plasma

[0110] Plasma samples were removed from a -80°C freezer and immediately thawed at 37°C for 2 min. They were then centrifuged at 2500×g for 15 min at 4°C to remove precipitated proteins. One ml of the supernatant was collected and used to separate plasma-derived extracellular vesicles (EVs) and extract EV RNA using the Qiagen exoRNeasy Midi kit, yielding EV RNA samples. The EV RNA samples were washed and eluted, then incubated at 37°C for 20 min with DNase I (TaKaRa, catalog number 2270A) to remove residual DNA. The purified EV RNA samples were then purified and concentrated using the Zymo RNA Clean and Concentrator-5 kit (catalog number R1016).

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

[0112] Example 3: EV RNA sequencing

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

[0114] (1) Pretreatment of EV RNA samples: Qualified EV RNA samples were end-repaired and tailed using T4 polynucleotide kinase (NEB, catalog number M0201) and E. coli Poly(A) polymerase (NEB, catalog number M0276). The reaction volume was 20 μL, and the mixture was incubated at 37°C for 30 min to obtain the reaction mixture. Subsequently, the reaction mixture was purified and concentrated using the RNA Clean and Concentrator-5 kit (Zymo, catalog number R1016) to obtain a concentrated EV RNA sample of 6 μL.

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

[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 and 1 μL of 3' PCR1, and 1 μL of SeqAmp DNA polymerase were mixed and subjected to thermal cycling amplification at 98℃ for 15 s, 55℃ for 15 s, and 68℃ for 30 s (cycle number 6). The thermally cycled amplification products were purified and size-selected twice using Hieff NGS DNA selection beads (Yeasen, catalog number 12601ES56) at a volume ratio of 1.8:1 of selection beads to DNA in the thermally cycled amplification products. Prepare the CRISPR-Cas9 reaction system and mix thoroughly. Incubate at 25°C in a metal bath for 10 min. The CRISPR-Cas9 reaction system includes: 12 μL NEBuffer 3.1, 1 μL (300 ng) rRNA sgRNA, 1 μL (40 ng) mtRNA sgRNA, 8.3 μL nuclease-free water, and 10 μL (1 μM) Cas9 nuclease. The cDNA sample is amplified in one step using primer pairs (forward primer and reverse primer for the sgRNA library of the sgRNA DNA template) and primer pairs (5' universal primer and 3' universal primer for the sgRNA DNA template) to obtain the sgRNA DNA template. Then, in vitro transcription is performed using T7 RNA polymerase (NEB, catalog number E2050) to obtain RNA sgRNA and mtRNA sgRNA. Add 21.5 μL of CRISPR-Cas9 reaction system to the selected beads to completely resuspend the selected beads, and incubate the selected beads at room temperature (25℃) for 5 min on a magnetic rack to completely wash the DNA off the magnetic beads. Then place the CRISPR-Cas9 reaction system containing DNA in a PCR instrument and incubate at 37℃ for 60 min, then at 65℃ for 5 min, and store at 4℃ to remove high-abundance ribosomal RNA and mitochondrial RNA, and obtain the amplified and purified cDNA sample.

[0117] (5) Amplification and purification of sequencing libraries: The amplification reaction system for sequencing libraries included: 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 program for sequencing libraries included: initial denaturation at 94℃ for 1 min; denaturation at 98℃ for 15 s, annealing at 55℃ for 30 s, extension at 68℃ for 30 s, for 17 cycles; and final extension at 68℃ for 2 min. Using Hieff NGS DNA selection beads, the amplification products were purified and size-selected twice at a 1:1 volume ratio of selection beads to DNA in the amplification products. 16 μL of supernatant was then collected to obtain the sequencing library.

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

[0119] Table 2

[0120]

[0121] Example 4: Plasma EV RNA conjugation in MDA5 + Effectiveness of IIM-ILD Diagnosis

[0122] (1) Differentially expressed RNAs (P < 0.05, fold change > 1.0) between the target group (MDA5 group) and the control group (HC group) were identified using limma47 software, and heatmaps and volcano plots were generated. Figure 2 Heatmap of differentially expressed RNAs in the MDA5 and HC groups; Figure 3 This is a volcano plot of differentially expressed RNAs in the MDA5 and HC groups, with red representing MDA5. + RNA upregulated (UP) in IIM-ILD patients, blue represents MDA5. + Downregulated RNA in IIM-ILD patients, gray represents MDA5. + RNA was not expressed differently in IIM-ILD patients and healthy individuals (Not).

[0123] (2) According to Figure 2 and Figure 3The results were analyzed using unsupervised hierarchical clustering and principal component analysis (PCA) on these differentially expressed RNAs. Machine learning was employed, defining 60% of the total samples as the training set and the remaining 40% as the validation set. Boruta feature selection was used on both the training and validation sets to identify all relevant machine learning variables. This process was repeated to find the optimal set capable of distinguishing MDA5. + Support vector machine (SVM) classifiers were used for IIM-ILD patients and healthy individuals, comprising an RNA combination consisting of five RNA markers: hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1. Among these, compared to the HC group, significant differences were observed in the markers hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1, and these differences were also observed 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. (See details...) Figure 4 , Figure 4 * indicates a significant difference (p < 0.05); ** indicates a significant difference (p < 0.01); *** indicates a significant difference (p < 0.001). Figure 4 The results showed that the above five RNA markers had good MDA5 activity. + IIM-ILD diagnostic efficacy.

[0124] Secondly, the Wilcoxon rank sum test was used to analyze the Log2 fold difference and P-value of the five RNA markers in the subjects to be tested (MDA5 group) and the control group (HC group), as detailed in Table 3.

[0125] Table 3

[0126]

[0127] As shown in Table 3, hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1 showed large fold differences and very small p-values, indicating that these five RNA markers have good MDA5 activity. + IIM-ILD diagnostic efficacy.

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

[0129] in, Figure 5 The confusion matrix for the training set and the confusion matrix for the validation set are shown in the diagram. Figure 6 For the support vector machine classifier in MDA5 + Performance (Metrics) Results for IIM-ILD Diagnosis. Sensitivity, specificity, and accuracy are labeled on the graph. Calculations showed that the classifier containing the above combination of 5 RNA markers was effective in distinguishing healthy individuals from MDA5 on the training set. + The sensitivity for IIM-ILD patients was 93.75%, the specificity was 100%, and the accuracy was 96.83%. For the validation set, it distinguished between healthy individuals and MDA5. + The sensitivity, specificity, and accuracy for IIM-ILD patients were 95.65%, 94.74%, and 95.24%, respectively. These results indicate that the combination of five RNA markers performed well in diagnosis on both the training and validation sets.

[0130] Figure 7 This is a graph showing the AUC values ​​of the ROC curve for the Support Vector Machine (SVM) classifier. ROC represents the Receiver Operating Characteristic Curve, and AUC represents the area under the curve. The results show that in the training set, the AUC value for the combination of five RNA markers is 0.966 (95% CI, 0.964–0.968); in the validation set, the AUC value is also 0.966 (95% CI, 0.963–0.968). Since the AUC value ranges between 0.5 and 1, and the closer the AUC is to 1.0, the higher the realism of the detection method, while an AUC of 0.5 indicates the lowest realism, these results demonstrate that the diagnostic performance of the combination of five RNA markers is good in both the training and validation sets.

[0131] (4) Using the methods described in Examples 1-4 above, a diagnostic or assessment system for idiopathic inflammatory myopathy was developed, comprising: a sample acquisition module for providing a plasma sample from the 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 assessment module for diagnosing or assessing the prognosis of the individual to be tested based on the measured expression level of the RNA composition. The analysis and assessment module can distinguish MDA5 as described in Example 4. + Support vector machine classifier models were implemented for IIM-ILD patients and healthy individuals, with a diagnostic threshold of 0.5; values ​​above 0.5 were classified as MDA5. + For IIM-ILD patients, a value below 0.5 indicates non-MDA5. + IIM-ILD patients.

[0132] In summary, this invention selects MDA5. + A total of 106 plasma samples were collected from IIM-ILD patients and healthy individuals, including 51 healthy individuals and 55 MDA5 patients. + IIM-ILD involved isolating and purifying EVs from plasma, extracting RNA from EVs, and performing high-throughput RNA sequencing to detect RNA expression levels in plasma EVs. Bioinformatics analysis yielded 1602 differentially expressed RNAs. Finally, feature selection and machine learning were used to select five RNAs as MDA5. + The biomarkers for IIM-ILD showed a sensitivity of 93.75%, specificity of 100%, accuracy of 96.83%, and AUC of 0.966 in the training set, and a sensitivity of 95.65%, specificity of 94.74%, accuracy of 95.24%, and AUC of 0.966 in the validation set. However, the role of EVs in IIM is not yet fully characterized, and comprehensive research on EV-related RNAs in IIM remains insufficient, especially regarding MDA5. + The EV-related RNA lineage in the plasma of IIM-ILD patients requires further exploration. Therefore, this invention establishes a more minimal, stable, and standardized EV RNA sequencing technology to identify high-quality RNA compositions as biomarkers for diagnosing clinical subtypes of idiopathic inflammatory myopathy. The aforementioned EV RNA sequencing technology and RNA compositions lay the foundation for further research and identification of new therapeutic or diagnostic targets, elucidation of pathophysiological mechanisms, and development of targeted treatment strategies, facilitating timely and appropriate prevention and treatment measures by clinicians.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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. The use of a reagent for detecting an RNA composition in the preparation of a product for diagnosing idiopathic inflammatory myopathy, characterized in that, The RNA composition comprises hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1; The idiopathic inflammatory myopathy is an idiopathic inflammatory myopathy with interstitial lung disease that is positive for anti-melanoma differentiation-associated gene 5 antibody. In the diagnosis, the concentration of the RNA composition in the plasma is detected to determine its expression level.

2. The application according to claim 1, characterized in that, The reagents include: reagents for extracting extracellular vesicular RNA from plasma and reagents for determining RNA expression.

3. The application according to claim 2, characterized in that, The reagents used for extracting extracellular vesicle RNA from plasma include at least one of the following: ethylenediaminetetraacetic acid, exosome RNA extraction reagent, DNA lysis reagent, deoxyribonuclease I, ethanol, phosphate buffer solution, protease inhibitor, and RNase inhibitor.

4. The application according to claim 2 or 3, characterized in that, The reagents used to determine RNA expression include at least one of the following: T4 polynucleotide kinase, Escherichia coli Poly(A) polymerase, reverse transcriptase, dithiothreitol, dNTPs, PEG 8000, template-converting oligonucleotides, UMI molecular tags, DNA polymerase, and DNA quantification reagents.

5. The application according to claim 1, characterized in that, The RNA is derived from extracellular vesicles in the plasma.

6. A diagnostic system for idiopathic inflammatory myopathy, characterized in that, include: The sample acquisition module is used to provide plasma samples from the individual to be tested; A sample detection module for determining the expression level of the RNA composition in the plasma sample; An analysis and evaluation module is used to diagnose the individual to be tested based on the expression level of the measured RNA composition; The diagnosis of the individual to be tested refers to determining whether the individual to be tested is a patient with idiopathic inflammatory myopathy and interstitial lung disease who is positive for anti-melanoma differentiation-associated gene 5 antibody. The RNA composition includes hsa-miR-590, hsa-miR-28, hsa-miR-409, hsa-miR-146b, and DEFA1.

7. A computer-readable storage medium having a computer program stored thereon, characterized in that, The computer program is a computer program that implements the functions of the system as described in claim 6.

8. 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-aided diagnostic method based on the reagent for detecting RNA compositions according to claim 1, wherein the diagnostic method includes: i) Receive test data from plasma samples from the individuals to be tested; ii) The detected data is compared with the reserved reference data using a preset machine learning model; iii) Output the diagnostic results based on the comparison results; The output diagnostic result refers to determining whether the individual to be tested is a patient with idiopathic inflammatory myopathy and interstitial lung disease who is positive for anti-melanoma differentiation-associated gene 5 antibody; In the diagnosis, the concentration of the RNA composition in the plasma is detected to determine its expression level.

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