A set of serum exosome tsrna markers for primary sjogren's syndrome and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a group of serum exosome tsRNA markers for diagnosing primary Sjogren's syndrome and application thereof. The serum exosome tsRNA marker is tRNA-Val-CAC-5' and / or tRNA-His-GTG-5'. The two serum exosome tsRNA markers have important significance for realizing early diagnosis, prediction and / or detection of primary Sjogren's syndrome, and simultaneously provide a research direction for developing a universal clinical primary Sjogren's syndrome detection product, such as a kit for clinical diagnosis of primary Sjogren's syndrome, which can be used for clinical auxiliary diagnosis of primary Sjogren's syndrome and preparation of a drug for treating primary Sjogren's syndrome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a set of serum exosomal tsRNA markers for primary Sjögren's syndrome and their applications. Background Technology

[0002] Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by chronic inflammatory damage to the lacrimal and salivary glands. Patients often present with typical symptoms such as dry mouth and dry eyes, and it can also affect multiple organs, including the lungs, kidneys, nervous system, and blood vessels. Epidemiological studies show that pSS primarily affects middle-aged women, has a prolonged course, and exhibits significant heterogeneity. Some patients may develop complications such as interstitial lung disease, renal tubular acidosis, or malignant lymphoma, severely impacting their quality of life and long-term prognosis.

[0003] Currently, the clinical diagnosis of pSS mainly relies on comprehensive assessment, including symptom scoring, autoantibody testing (such as anti-SSA / Ro and anti-SSB / La antibodies), tear and salivary secretion function tests, imaging examinations, and labial gland biopsy. Among these, labial gland biopsy, which obtains tissue from the submucosal minor salivary glands of the lower lip for pathological observation, is an important means of confirming pSS. However, this method has several limitations: first, it is significantly invasive, and the sampling process may cause bleeding, infection, or local nerve damage, resulting in low patient compliance; second, the results are greatly affected by the operator's experience, the sampling site, and the quality of the tissue, leading to insufficient diagnostic consistency; third, pathological interpretation is subjective and often fails to provide reliable evidence in the early or mild stages of the disease. In addition, although serum autoantibody testing is widely used in clinical practice, its sensitivity and specificity are limited, and some early-stage patients may be negative for anti-SSA / SSB antibodies, easily leading to missed or misdiagnosis; salivary gland function tests and imaging assessments are also easily affected by drug use, environmental factors, and testing conditions, lacking stability and reproducibility. Overall, existing diagnostic methods rely on invasive sampling or single immunological markers, which cannot yet meet the clinical needs for early detection, non-invasive diagnosis, and dynamic monitoring of pSS.

[0004] tRNA-derived small RNAs (tsRNAs) are a novel class of small non-coding RNAs discovered in recent years, approximately 18–40 nt in length. They originate from specific cleavage sites of tRNA precursors or mature tRNAs and are mainly divided into tRNA halves and tRNA-derived fragments (tRFs). Due to the rich chemical modifications of tRNA molecules, tsRNAs are structurally stable, highly resistant to degradation, and exhibit significant tissue and time specificity. Studies have shown that tsRNAs participate in various physiological and pathological processes through mechanisms such as regulating protein translation, stress responses, and immune processes. Exosomes are nanoscale vesicles (approximately 30–150 nm in diameter) secreted by cells, rich in bioactive molecules such as RNA, proteins, and lipids. They can stably carry tsRNAs and mediate intercellular signal transduction. Compared to miRNAs, exosomal tsRNAs have higher structural stability and reproducible detection, possessing potential advantages as ideal biomarkers for liquid biopsies.

[0005] However, research on serum exosomal tsRNA in autoimmune diseases, especially primary Sjögren's syndrome (pSS), is still in its infancy. Given the complex immunopathological mechanisms and multi-system involvement of pSS, serum exosomal tsRNA holds promise as a non-invasive, sensitive, and specific molecular marker for the early diagnosis and monitoring of pSS, possessing significant scientific research value and promising clinical applications. Summary of the Invention

[0006] The purpose of this invention is to provide a set of serum exosomal tsRNA markers for primary Sjögren's syndrome and their applications, so as to achieve effective diagnosis of primary Sjögren's syndrome.

[0007] Therefore, the present invention provides the following technical solution.

[0008] The first aspect of the present invention provides a set of serum exosomal tsRNA markers for the diagnosis of primary Sjögren's syndrome, said serum exosomal tsRNA markers being tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′.

[0009] In some embodiments of the present invention, the serum exosome tsRNA marker is a combination of tRNA-Val-CAC-5′ and tRNA-His-GTG-5′.

[0010] In some embodiments of the present invention, the nucleotide sequence of the tRNA-Val-CAC-5′ is shown in SEQ ID NO: 1, and the nucleotide sequence of the tRNA-His-GTG-5′ is shown in SEQ ID NO: 2.

[0011] A second aspect of the invention provides the use of the serum exosomal tsRNA marker as described above in the preparation of formulations for the detection, screening and / or diagnosis of primary Sjögren's syndrome.

[0012] A third aspect of the invention provides the use of a reagent for detecting serum exosomal tsRNA marker levels as described above in the preparation of products for detecting, screening, and / or diagnosing primary Sjögren's syndrome.

[0013] In some embodiments of the present invention, the reagents include reagents suitable for at least one of the following methods: digital PCR, real-time quantitative PCR, or sequencing.

[0014] In some embodiments of the present invention, the product is a chip, a reagent kit, a test strip, or an analysis platform.

[0015] A fourth aspect of the present invention provides a kit for detecting primary Sjögren's syndrome, comprising reagents for detecting serum exosomal tsRNA markers as described above, optionally, the detection being a quantitative detection of tsRNA marker levels in the serum of a subject.

[0016] In some embodiments of the present invention, the reagents include reagents suitable for at least one of the following methods: digital PCR, real-time quantitative PCR, or sequencing.

[0017] In some embodiments of the present invention, the kit includes: (1) Primers capable of reverse transcription of tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 3-4, respectively; (2) Primer pairs capable of PCR amplification of cDNA corresponding to tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primer pairs are shown in SEQ ID NO: 5-8, respectively; (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

[0018] A fifth aspect of the invention provides a method for assessing whether a drug can treat primary Sjögren's syndrome, comprising using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ as serum exosomal tsRNA markers to assess the efficacy of the drug.

[0019] A sixth aspect of the invention provides a method for screening compounds capable of treating primary Sjögren's syndrome, comprising using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ as serum exosomal tsRNA markers to assess the efficacy of the compounds.

[0020] By employing the above technical solution, the present invention has at least the following advantages: (1) Compared with traditional protein markers and miRNA markers, tsRNA in serum exosomes has higher stability, which helps resist degradation by RNases in serum, and its quantification is accurate, which helps improve the sensitivity and specificity of diagnosis of related diseases.

[0021] (2) This invention investigated the diagnostic role of serum exosomal tsRNA in primary Sjögren's syndrome and explored its clinical value in the diagnosis and screening of primary Sjögren's syndrome. The serum exosomal tsRNA markers screened by this invention can be used to prepare diagnostic kits for primary Sjögren's syndrome, which can make the diagnosis of primary Sjögren's syndrome more convenient.

[0022] (3) This invention has for the first time confirmed a group of novel serum exosomal tsRNA markers, namely tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, which are the first confirmed serum exosomal tsRNA markers for the diagnosis of pSS, laying the foundation for in-depth research on tsRNA in pSS.

[0023] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0024] Figure 1 (A) Volcano plot of differential expression of exosomal tsRNAs; (B) Heatmap of differential expression of serum exosomal tsRNAs in the top 10 high / low expression groups in the primary Sjögren's syndrome group; Figure 2 Figure showing the difference in expression levels of five serum exosomal tsRNAs between patients with primary Sjögren's syndrome and healthy controls during the initial screening stage. Figure 3Figure showing the difference in expression levels of two serum exosomal tsRNAs between patients with primary Sjögren's syndrome and healthy controls during the validation phase. Figure 4 For validation phase—ROC curves of two serum exosomal tsRNAs in the primary Sjögren's syndrome group and the healthy control group. Detailed Implementation

[0025] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0026] Primary Sjögren's syndrome is a chronic, systemic autoimmune disease that seriously endangers patients' health and quality of life. This invention addresses this issue by providing a set of serum exosomal tsRNA biomarkers for diagnosing primary Sjögren's syndrome, wherein the serum exosomal tsRNA biomarker combination is tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′.

[0027] It should be noted that the sequence information of tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ in this invention is shown in the table below: It should be noted that the two serum exosomal tsRNAs mentioned above can serve not only as early diagnostic biomarkers for primary Sjögren's syndrome, but also as screening biomarkers and diagnostic products for the syndrome. They may also become targets for drug treatment of primary Sjögren's syndrome.

[0028] Either of these biomarkers can be used alone as a basis for the diagnosis of primary Sjögren's syndrome. However, since a single biomarker may have a certain possibility of misdiagnosis, in order to improve the accuracy and reliability of the diagnosis, a more preferred approach is to combine the two biomarkers as biomarkers for primary Sjögren's syndrome.

[0029] Preferably, combining tRNA-Val-CAC-5′ and tRNA-His-GTG-5′ can improve the sensitivity and specificity of diagnosis of primary Sjögren's syndrome. This combined strategy helps reduce the possibility of misdiagnosis and improves the diagnostic accuracy of primary Sjögren's syndrome, providing a more reliable basis for patient treatment and management.

[0030] Therefore, although a single biomarker has some diagnostic value, the combination of two biomarkers is still preferred as the basis for the diagnosis of primary Sjögren's syndrome to ensure the accuracy and reliability of the diagnostic results.

[0031] In another embodiment of the present invention, the use of the combination of the two serum exosomal tsRNA markers described above in the preparation of a formulation for detecting, screening and / or diagnosing primary Sjögren's syndrome is provided.

[0032] In another embodiment of the invention, a reagent for detecting the combination of the two serum exosomal tsRNA markers described above is provided for use in the preparation of products for detecting, screening and / or diagnosing primary Sjögren's syndrome.

[0033] Since the aforementioned biomarkers for primary Sjögren's syndrome may be a single biomarker or a combination of multiple biomarkers, the corresponding diagnostic kits or testing devices can also take many forms. For example, they may be diagnostic kits or testing devices targeting a single biomarker, i.e., products specifically designed to detect either tRNA-Val-CAC-5′ or tRNA-His-GTG-5′. More exemplaryly, they may also be combinations of diagnostic products containing multiple biomarkers, i.e., products that simultaneously detect tRNA-Val-CAC-5′ and tRNA-His-GTG-5′. Exemplary products include, but are not limited to, chips, kits, test strips, or analytical platforms.

[0034] This diverse design allows diagnostic products to have a wider range of applications and greater flexibility, enabling the selection of appropriate testing protocols based on specific clinical needs and diagnostic requirements. However, whether it's a single-marker diagnostic kit or a combination of multiple markers, it is expected to provide relatively reliable and accurate diagnostic results.

[0035] The reagents included in the specific kits of this invention can be customized according to the detection method for each biomarker. This means that for the two biomarkers, tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, specific methods and reagents suitable for their detection can be selected.

[0036] The reagents included in the specific kits of this invention can be tailored to the detection method for each serum exosomal tsRNA biomarker. This means that for the two different serum exosomal tsRNA biomarkers, tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, specific methods and reagents suitable for their detection can be selected.

[0037] Since both of these biomarkers are known substances, there are already extensive and reliable detection methods available in existing technologies. For example, molecular biology techniques, such as polymerase chain reaction (PCR) and in situ hybridization, can be used to detect the gene expression levels of these biomarkers.

[0038] Based on the extensive experience and mature methods of existing technologies, the most suitable method for detecting the aforementioned biomarkers can be selected by referring to existing detection techniques and reagents. Then, according to the selected detection method, corresponding diagnostic kits or detection devices can be designed in accordance with existing technical standards and procedures.

[0039] PCR technology can be used to detect transcribed RNA of biomarkers. The RNA or fragments to be detected are amplified by PCR, and then quantitatively analyzed using methods such as gel electrophoresis or real-time quantitative PCR.

[0040] For example, considering detection efficiency and convenience, the kit can be a PCR kit.

[0041] In one example, the PCR kit includes: (1) Primers capable of reverse transcription of tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 3-4, respectively: (2) Primer pairs capable of PCR amplification of cDNA corresponding to tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primer pairs are shown in SEQ ID NO: 5-8, respectively: (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

[0042] In another embodiment of the present invention, the application of the above-mentioned serum exosome tsRNA marker in primary Sjögren's syndrome for non-pathological diagnostic or therapeutic purposes is provided. Primary Sjögren's syndrome for non-pathological diagnostic or therapeutic purposes can be for scientific research, non-medical commercial testing or examination, etc.

[0043] For example, serum exosomal tsRNA markers in primary Sjögren's syndrome can be used to study disease mechanisms, pathophysiological processes, and potential therapeutic targets. By analyzing changes in these markers during disease progression, we can gain a deeper understanding of the disease's development and metastasis mechanisms, providing a more comprehensive understanding for its prevention and treatment.

[0044] Biomarkers also play a crucial role in drug development. They serve as indicators for drug efficacy evaluation, helping researchers assess the safety and effectiveness of new drugs. By monitoring the impact of drugs on biomarkers, potential drug toxicity and adverse reactions can be detected early, guiding the adjustment and optimization of drug dosage.

[0045] Biomarkers can also be used in individual health management and preventive medicine. By regularly monitoring specific biomarkers, changes in an individual's health status can be detected in a timely manner, the risk of disease can be predicted, and corresponding health management measures can be taken, such as adjusting lifestyle, dietary habits, or drug treatment, to maintain health and slow disease progression.

[0046] Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these biomarkers, the extent to which different lifestyles affect health can be assessed, and individuals can be guided to take appropriate lifestyle interventions, such as weight loss, smoking cessation, and increased physical activity, thereby improving health status and reducing disease risk.

[0047] This invention also provides a method for assessing the status of primary Sjögren's syndrome in subjects. The key to this method is detecting the levels of tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ in the subject's serum exosomes.

[0048] First, the implementation of this method involves sample collection and processing. Extracting samples from the subjects' blood requires adherence to strict standard operating procedures to ensure sample quality and integrity.

[0049] Secondly, for the samples that have already been obtained, molecular biology techniques, such as PCR, can be used to accurately quantify the content level of a certain marker in the subject's tissues.

[0050] After obtaining the biomarker test results, they need to be compared and analyzed with relevant data from normal patients. Based on these comparisons, the subject's status of primary Sjögren's syndrome can be determined. If the level of a certain biomarker or combination of biomarkers is significantly higher than the normal range, it can be inferred that the subject has a high probability of having primary Sjögren's syndrome.

[0051] The reliability and accuracy of this method depend on the sensitivity and specificity of the selected detection method, as well as the determination of the reference range or threshold. Therefore, when implementing this method, it is necessary to strictly control experimental conditions and combine clinical experience and data analysis for the interpretation and diagnosis of results. Furthermore, the detection results of any single biomarker should be carefully evaluated and interpreted to avoid uncertainty in diagnostic results due to the potential for misinterpretation by a single biomarker.

[0052] This invention provides a method for pre-evaluating whether a drug can treat primary Sjögren's syndrome, which includes using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ biomarkers to assess the drug's efficacy for the purpose of precision medicine.

[0053] The implementation of this method requires starting with experimental design and sample collection. During the design phase of a study or clinical trial, the drug, treatment regimen, and evaluation indicators to be assessed must be clearly defined. Simultaneously, the selection criteria for subjects need to be determined, and appropriate biological samples, such as plasma, tissue, or cell samples, need to be collected for subsequent experimental analysis.

[0054] Once the biological sample is obtained, appropriate techniques and reagents can be used to detect the level of the selected biomarker. For example, PCR can accurately quantify the level of biomarkers in the subject's biological sample.

[0055] Next, the medication or treatment regimen needs to be administered to the subject and carried out according to the predetermined plan. During treatment, the subject's biological samples need to be monitored regularly, and the levels of the selected biomarkers need to be repeatedly tested. This allows for timely understanding of the drug's therapeutic effect and whether the expected treatment goals have been achieved.

[0056] Finally, based on the obtained test results, the therapeutic effect of the drug can be evaluated and determined. If the level of the selected biomarker changes significantly, showing a clear difference compared to before treatment, it can be inferred that the drug may have the potential to treat primary Sjögren's syndrome. Conversely, if the biomarker level does not change significantly or does not achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

[0057] This invention provides a method for screening compounds that can treat primary Sjögren's syndrome, which includes using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ as biomarkers for primary Sjögren's syndrome to evaluate the efficacy of the compounds in order to achieve the purpose of precision medicine.

[0058] First, the implementation of this method requires the establishment of a suitable experimental system and model. Cell lines or animal models of primary Sjögren's syndrome can be used to simulate the physiological processes of primary Sjögren's syndrome and to evaluate the effects of compounds on them.

[0059] Secondly, appropriate biomarkers should be selected to evaluate the efficacy of compounds. tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′, as biomarkers for primary Sjögren's syndrome, can reflect the biological processes and pathological state of primary Sjögren's syndrome. Therefore, they can be used as biomarkers to screen compounds for efficacy in evaluating their intervention effects on primary Sjögren's syndrome.

[0060] Next, a compound screening experiment is conducted. The compounds to be screened are added to cells or animals, and their effects on tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ in primary Sjögren's syndrome are observed. Appropriate techniques and reagents, such as molecular biology techniques, can be used to detect the levels of these markers. Based on the detection results, the potential therapeutic effect of the compounds on primary Sjögren's syndrome can be preliminarily assessed.

[0061] After obtaining preliminary screening results, further validation and confirmation experiments can be conducted. By using more refined and rigorous experimental design and procedures, the effects of compounds on biomarker levels can be verified, and their therapeutic efficacy for primary Sjögren's syndrome can be further evaluated.

[0062] Finally, based on the experimental results, further research and development will be conducted on compounds with potential therapeutic effects. This includes further evaluation of their pharmacological properties, toxic side effects, pharmacokinetics, and other drug attributes, as well as preclinical and clinical trials.

[0063] The present invention will further demonstrate the selection principle, process and effect of the above-mentioned biomarkers through a specific embodiment, so as to enable those skilled in the art to understand the essence of the present invention.

[0064] This invention first isolates serum exosomes from sex- and age-matched patients with primary Sjögren's syndrome and healthy individuals undergoing physical examinations. RNA is extracted and analyzed using tsRNA high-throughput sequencing. Through preliminary screening with small samples and clinical validation with large samples, a set of serum exosome tsRNA biomarkers associated with primary Sjögren's syndrome are obtained. Based on this, a kit for the clinical diagnosis of primary Sjögren's syndrome is developed, which can be used for the auxiliary clinical diagnosis of primary Sjögren's syndrome.

[0065] The specific process for marker screening in this invention includes: (1) Screening analysis of differential expression profile of serum exosome tsRNA: Patients with primary Sjögren's syndrome and healthy patients matched for gender and age were screened, serum exosomes were isolated, and the expression profile of tsRNA in serum exosomes was analyzed by sequencing. Differentially expressed tsRNAs were screened and validated in multiple stages using clinical samples.

[0066] (3) Real-time quantitative PCR (RT-qPCR) was performed on the serum exosomal tsRNAs screened above to identify tsRNAs associated with primary Sjögren's syndrome.

[0067] (4) Develop a diagnostic kit for primary Sjögren's syndrome based on the serum exosomal tsRNAs screened above.

[0068] Example 1: Data Sources and Grouping The dataset used for biomarker screening and validation in this invention comprises three independent datasets: a sequencing set, a training set, and a validation set. All samples were obtained from patients with primary Sjögren's syndrome and healthy individuals undergoing physical examinations at the Rheumatology Department and Health Examination Center of Nanjing Drum Tower Hospital. All patients with primary Sjögren's syndrome were individually assessed and enrolled by two physicians with intermediate professional titles. All subjects or their legal guardians were fully informed and signed written consent forms. This invention has been approved by the Institutional Review Committee of Nanjing University School of Medicine Affiliated Drum Tower Hospital.

[0069] The specific inclusion and exclusion criteria are based on the classification criteria jointly published by the American College of Rheumatology and the European League Against Rheumatism (ACR / EULAR) in 2016.

[0070] This study included 149 patients with primary Sjögren's syndrome (pSS), comprising 80 patients in the sequencing phase (four pools of 20 samples each), 23 patients in the training phase, and 46 patients in the validation phase. All patients met the 2016 ACR / EULAR (American College of Rheumatology / European League Against Rheumatism) pSS classification criteria and excluded confounding factors such as other autoimmune diseases, lymphoma, hepatitis C, or a history of head and neck radiotherapy. Serum samples were also collected from 149 age- and sex-matched healthy individuals as a healthy control group (HC). All participants signed informed consent forms, and the study protocol was approved by the hospital's medical ethics committee.

[0071] All patients were divided into three groups: a sequencing set (80 vs. 80, i.e., 80 patients with primary Sjögren's syndrome and 80 healthy individuals); a training set (23 vs. 23, i.e., 23 patients with primary Sjögren's syndrome and 23 healthy individuals); and a validation set (46 vs. 46, i.e., 46 patients with primary Sjögren's syndrome and 46 healthy individuals).

[0072] Example 2: Experimental Methods and Data Analysis 1 Experimental Methods 1.1 Exosomes were extracted from 100 μL of serum using the Thermo Fisher Precipitation Kit (USA), and total RNA was further extracted from the exosomes using the Trizol (Invitrogen Life Technologies) method.

[0073] 1.2 RNA Quality Assurance: RNA purity and concentration were determined by agarose gel electrophoresis and qubit assay. The quality requirements were: integrity was assessed using an Agilent 2100 (RIN ≥ 7.0). Accurate quantification was performed using qubit assay, requiring an RNA concentration ≥ 50 ng / μL and a total volume of at least 20 μL (i.e., total volume ≥ 1 μg) to ensure the complexity and coverage of the standard library construction.

[0074] 1.3 High-throughput sequencing of tsRNA ① The above RNA was recovered by PAGE electrophoresis; ②tsRNAs contain numerous epigenetic modifications, which can interfere with the construction of small RNA sequencing libraries. RNA samples were treated with ALKB demethylase before library construction. Subsequently, next-generation sequencing and high-throughput gene expression database big data analysis were used to screen tsRNAs. ③ Remove duplicate, mismatched, or excessively low expression sequences, and select sequences with a fold difference > 10 and P Differential analysis was performed on tsRNAs with values ​​<0.01; 1.4 RT-qPCR Quantitative Detection of tsRNA ① Extract total RNA from serum exosomes using the Trizol method described in 1.1; ② The RNA sample was reverse transcribed using tsRNA stem-loop primers synthesized by GenScript to obtain cDNA; ③ Quantitative PCR reaction of cDNA was performed using tsRNA forward primers synthesized by GenScript. ④ Detect and compare the differences in tsRNA expression levels in serum exosome samples from patients with primary Sjögren's syndrome and healthy individuals undergoing physical examinations.

[0075] 2 Data Processing and Analysis All data were statistically analyzed using Excel, Graphpad Prism 8.0, and SPSS 24.0 software. Values ​​are expressed as mean ± SEMs. P A value <0.05 is considered statistically significant. When the samples between groups are normally distributed and have the same variance, a t-test or one-way ANOVA is used; when the samples between groups are not normally distributed, a U-test or nonparametric test is used.

[0076] Example 3: Screening of biomarkers Serum exosomes from 80 patients with primary Sjögren's syndrome and 80 healthy patients (4 mixed samples) were obtained from the sequencing dataset using the method described in Example 2. These samples were sent to BGI Genomics for RNA sequencing. After quality control processing, differential expression volcano plots and differential heatmaps were generated. The results are shown below. Figure 1 .

[0077] like Figure 1 As shown, a total of 164 tsRNAs were identified in this dataset. Among them, 24 were expressed only in serum EVs of pSS patients, and 50 tsRNAs were expressed only in the healthy group. The expression was defined as log2FC>1 or log2 FC<-1, and... P Using a threshold of <0.05, the sequencing data was filtered to identify differentially expressed tsRNAs, resulting in five differentially expressed tsRNAs: tRNA-Gly-GCC_CCA_end, tRNA-Val-AAC_5_end, tRNA-Gly-TCC_CCA_end, tRNA-Val-CAC_5_end, and tRNA-His-GTG_5_end.

[0078] The five differentially expressed tsRNAs were then further validated using qRT-PCR in the training set (23 patients with primary Sjögren's syndrome and 23 healthy individuals). The specific procedures are described in Example 2. (1) Extraction of serum exosomes: Exosomes were extracted from 100 μL of training set serum sample using an exosome sedimentation kit, and total RNA was further extracted from the exosomes using the Trizol method.

[0079] (2) Absolute quantification analysis: Standards corresponding to the tsRNA were synthesized, a standard curve was plotted, and the CT values ​​of the samples were obtained by RT-qPCR detection. These values ​​were then converted into the absolute concentrations of the target tsRNAs using the standard curve, and a differential expression analysis graph was plotted. The results are shown in […]. Figure 2 .

[0080] like Figure 2 As shown, two tsRNAs with significantly different levels in the healthy group and the patient group were screened using the above method: tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, and their nucleotide sequences are shown in Table 1 as SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0081] Table 1 Nucleotide sequences of tRNA-Val-CAC-5′ and tRNA-His-GTG-5′ Example 4: Validation of the marker The two tsRNAs screened in Example 3, tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, were quantitatively analyzed and differentially expressed in serum samples from the validation set (46 patients with primary Sjögren's syndrome and 46 healthy individuals) using the method described in Example 2. Differential expression plots and receiver operating characteristic (ROC) curves were plotted. The results are shown in [Figure 2]. Figure 3 and 4 .

[0082] like Figure 3 As shown, by further validating the two differentially expressed tsRNAs selected from the test set, tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, in the validation set samples, it was found that their expression levels still showed significant differences.

[0083] like Figure 4 As shown, further analysis of the receiver operating characteristic (ROC) curves of tRNA-Val-CAC-5′ and tRNA-His-GTG-5′ for the diagnosis of primary Sjögren's syndrome showed that both tsRNAs have good diagnostic value for primary Sjögren's syndrome, and the area under the ROC curve for combined diagnosis can reach 0.7703.

[0084] Example 5: Detection kit for primary Sjögren's syndrome This embodiment provides a detection kit for primary Sjögren's syndrome. The kit is designed to detect the levels of two tsRNAs, tRNA-Val-CAC-5′ and tRNA-His-GTG-5′, in the serum exosomes of subjects, thereby achieving accurate diagnosis of primary Sjögren's syndrome.

[0085] The kit consists of: (1) Primers capable of reverse transcription of tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, the nucleotide sequences of which are shown in Table 2: Table 2 Reverse transcription primers (2) Primer pairs capable of PCR amplification of cDNA corresponding to tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, the nucleotide sequences of which are shown in Table 3: Table 3 PCR amplification primers (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A set of serum exosomal tsRNA markers for the diagnosis of primary Sjögren's syndrome, characterized in that, The serum exosomal tsRNA markers are tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′.

2. The serum exosome tsRNA marker according to claim 1, characterized in that, The serum exosomal tsRNA markers are a combination of tRNA-Val-CAC-5′ and tRNA-His-GTG-5′.

3. The serum exosome tsRNA marker according to claim 1 or 2, characterized in that, The nucleotide sequence of the tRNA-Val-CAC-5′ is shown in SEQ ID NO: 1, and the nucleotide sequence of the tRNA-His-GTG-5′ is shown in SEQ ID NO:

2.

4. The use of the serum exosomal tsRNA marker according to any one of claims 1-3 in the preparation of formulations for the detection, screening and / or diagnosis of primary Sjögren's syndrome.

5. The use of a reagent for detecting the level of serum exosomal tsRNA markers as described in any one of claims 1-3 in the preparation of products for detecting, screening and / or diagnosing primary Sjögren's syndrome.

6. The application according to claim 5, characterized in that, The reagents include those suitable for at least one of the following methods: digital PCR, real-time quantitative PCR, or sequencing. The products mentioned are chips, reagent kits, test strips, or analysis platforms.

7. A kit for detecting primary Sjögren's syndrome, characterized in that, Includes reagents for detecting serum exosomal tsRNA markers as described in any one of claims 1-3, wherein the detection is optionally a quantitative detection of tsRNA marker levels in serum exosomals of a subject; The reagents include those suitable for at least one of the following methods: digital PCR, real-time quantitative PCR, or sequencing.

8. The reagent kit according to claim 7, characterized in that, The kit includes: (1) Primers capable of reverse transcription of tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 3-4, respectively; (2) Primer pairs capable of PCR amplification of cDNA corresponding to tRNA-Val-CAC-5′ or tRNA-His-GTG-5′, wherein the nucleotide sequences of the primer pairs are shown in SEQ ID NO: 5-8, respectively; (3) dNTP / AMV reverse transcriptase and buffer, MgCl2, DEPC water and Taq DNA polymerase.

9. A method for assessing whether a drug can treat primary Sjögren's syndrome, characterized in that, This includes using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ as serum exosomal tsRNA markers to assess the efficacy of the drug.

10. A method for screening compounds capable of treating primary Sjögren's syndrome, characterized in that, This includes using tRNA-Val-CAC-5′ and / or tRNA-His-GTG-5′ as serum exosomal tsRNA markers to evaluate the efficacy of compounds.

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