Use of reagents for detecting the expression level of PSIP1 gene or its encoded protein LEDGF / p75 in the preparation of products for evaluating the disease activity of systemic lupus erythematosus

Abstract

The application relates to application of a reagent for detecting expression levels of a PSIP1 gene or a coded protein LEDGF / p75 in preparation of a product for evaluating systemic lupus erythematosus disease activity, which makes up for the problems of insufficient sensitivity and specificity of existing evaluation indexes; the application also discloses a key role of the PSIP1 gene in a pathogenesis mechanism of systemic lupus erythematosus, and clearly shows the relationship between the PSIP1 gene in T cells and cell cycle arrest, cell aging and abnormal immune function, so as to provide a theoretical basis for target regulation of the PSIP1 to improve T cell function and delay or block disease progression; the application firstly proposes that the expression levels of the PSIP1 / LEDGF / p75 are detected to be used for evaluating systemic lupus erythematosus disease activity, auxiliary diagnosis, prognosis prediction and treatment effect monitoring, so as to provide a brand-new tool and direction for precise diagnosis, activity evaluation, prognosis judgment and future target treatment development of systemic lupus erythematosus.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the field of molecular biology detection and immunological application technology related to autoimmune diseases, and particularly to the application of reagents for detecting the expression level of the PSIP1 gene or its encoded protein LEDGF / p75 in the preparation of products for assessing the activity of systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology, characterized by the presence of autoantibodies, autoreactive B and T cells, and cytokine dysregulation, leading to inflammation and damage to multiple organs, posing a significant threat to human health. The clinical course of SLE is characterized by alternating periods of relapse and remission. Since disease activity directly impacts treatment decisions and prognostic assessment, accurately and objectively assessing SLE disease activity has been a key focus in both clinical and research fields.

[0003] Traditional methods for assessing SLE disease activity primarily rely on comprehensive clinical scoring systems, including the SLE Disease Activity Index (SLEDAI) and its modified version, the SLEDAI-2K score. These systems integrate clinical manifestations with laboratory test results to quantify disease activity. While these methods have been repeatedly validated in multicenter studies and have become one of the international standards for SLE activity assessment, they suffer from limitations such as high subjectivity and insufficient response to certain potential molecular changes. This has prompted numerous studies in the field to explore more objective and specific biomarkers.

[0004] In recent years, SLE disease activity has often been assessed using a combination of clinical scoring systems and relevant laboratory biomarkers. Traditional laboratory biomarkers include: 1) Anti-dsDNA antibodies: Elevated levels of anti-dsDNA antibodies are often associated with disease activity, especially in lupus nephritis, but their sensitivity and specificity vary considerably among different patients. 2) Complement levels (C3, C4): Complement depletion is considered an indirect indicator of immune complex-mediated inflammatory responses, but complement levels are influenced by multiple factors such as genetic background and infection. Although anti-dsDNA antibodies and complement levels can reflect disease activity in some patients, a single indicator is difficult to reliably and accurately reflect the overall disease status; the sensitivity and specificity of SLE activity markers such as anti-dsDNA antibodies and complement C3 / C4 are limited, and the clinical manifestations of some patients do not perfectly match the laboratory indicators, making it difficult to accurately and comprehensively reflect disease activity and organ damage.

[0005] To better assess SLE disease activity, current research has identified several new potential biomarkers, including: 1) Trend cell indices and inflammatory indices: Systemic immune inflammatory indices and related whole blood cell count-derived indicators (such as the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR)) are significantly correlated with traditional activity indicators such as SLEDAI. These indicators are convenient for clinical testing and may serve as auxiliary monitoring tools. However, these indicators essentially reflect the peripheral immune inflammatory state, and their mechanistic implications for the molecular state of immune cells remain relatively insufficient. 2) Association between T cell senescence and activity: Immune cell dysfunction, particularly T cell dysregulation and senescence, has been considered a key mechanism for changes in SLE activity and disease progression in recent years. T cell senescence-related biomarkers (such as CD57 and KLRG1) are significantly correlated with SLE activity, suggesting that changes in the intrinsic state of immune cells may reflect disease activity. This supports the potential value of T cell senescence indicators as biomarkers of disease activity, but no operational monitoring protocols targeting specific molecular mechanisms have yet been proposed. 3) PBMC Omics and Multi-Biomarker Combination Prediction: With the maturity of high-throughput omics technologies, researchers utilize PBMC proteomics and single-cell RNA-Seq data for systematic analysis to discover protein or gene combinations that may be related to disease activity. For example, one study used PBMC proteomics to screen for differentially expressed proteins between active and inactive SLE patients and further constructed multi-protein combinations to distinguish different activity states. The screened biomarker combinations can distinguish between active and inactive SLE states at the PBMC level, suggesting that changes in the intrinsic expression profile of PBMCs have potential application value in activity assessment. However, the universality and stability of specific biomarker combinations still need to be validated on a larger scale. 4) CD4 + Correlation between T cell gene expression and activity: Based on peripheral CD4 + T cell gene expression analysis has also identified some key genes that may be regulated by disease activity. These genes are related to immune responses, interferon pathways, and other factors, and show a correlation with clinical disease activity. Changes in the expression of these genes further support the correlation between molecular alterations in T cell subset states and disease activity, but have not yet evolved into mature techniques that can be used for clinical activity assessment.

[0006] Although the above studies have made some progress in exploring SLE activity-related indicators, there are still significant shortcomings: ① Most of them are correlation studies, and most biomarker studies only stay at the level of phenomenon correlation, lacking in-depth understanding of their role in the occurrence and development of the disease, lacking mechanistic explanations and molecular indicator schemes that can be directly used as clinical evaluation standards; ② Single indicators lack specificity and are difficult to comprehensively reflect the complex immune network changes in disease activity; ③ The elucidation of the internal molecular mechanisms of immune cells is lacking, and no monitoring strategy for directly linking activity to specific molecules has been proposed; ④ Some biomarkers are difficult to translate into clinical applications due to the complexity of detection methods, high cost, or poor stability.

[0007] Therefore, it is necessary to explore new molecular markers with mechanistic basis to improve the accuracy and specificity of SLE activity assessment. Summary of the Invention

[0008] To overcome at least one deficiency in existing technologies, this invention provides the application of reagents for detecting the expression level of the PSIP1 gene or its encoded protein LEDGF / p75 in the preparation of products for assessing the disease activity of systemic lupus erythematosus (SLE). This can be used for the assessment, monitoring, and related research of the disease status of SLE patients. Specifically, this invention includes: ① Providing a novel use for assessing the disease activity of the PSIP1 gene or its encoded protein LEDGF / p751, overcoming the shortcomings of existing assessment indicators in terms of sensitivity and specificity; ② Revealing the key role of the PSIP1 gene in the pathogenesis of SLE, clarifying the relationship between its reduction in T cells and cell cycle arrest, cellular senescence, and abnormal immune function; ③ Establishing an assessment method based on the expression level of PSIP1 or LEDGF / p751 to reflect the disease status of SLE, providing new molecular evidence for disease stratification management and efficacy monitoring; ④ Providing a theoretical basis and technical support for targeting and regulating PSIP1 to improve T cell function and delay or block SLE progression.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first aspect of the present invention is to provide the use of the PSIP1 gene, selected from at least one of the following uses: as a biomarker for assessing the activity of systemic lupus erythematosus (SLE); as a target in screening agents that regulate cell cycle and cell senescence; and as a target in screening drugs that delay or block the progression of SLE.

[0011] A second aspect of the invention is the use of a reagent for detecting the expression level of the PSIP1 gene or its encoded protein LEDGF / p75, selected from at least one of the following applications: in the preparation of formulations for screening cell cycle and cellular senescence regulators; in the preparation of auxiliary diagnostic products for systemic lupus erythematosus (SLE); in the preparation of products for assessing SLE activity; in the preparation of products for prognostic assessment of SLE; in the preparation of products for monitoring and evaluating the therapeutic effects of SLE treatment; in the preparation of products for differentiating clinical phenotypes of SLE; and in the screening of drugs for treating SLE.

[0012] The above products cover testing reagents, testing kits, and testing accessories.

[0013] Furthermore, the reagents for detecting the expression level of the PSIP1 gene include reagents for detecting PSIP1 mRNA levels and kits containing such reagents. Preferably, the kit for detecting PSIP1 mRNA levels includes a qRT-PCR kit.

[0014] Furthermore, the reagents for detecting the expression level of LEDGF / p75 protein include antibodies that specifically bind to LEDGF / p75 protein, reagents thereof, and kits containing such reagents. Preferably, the antibodies that specifically bind to LEDGF / p75 protein and their reagents include specific antibodies and their matching reagents used in Western blotting, immunofluorescence, flow cytometry, or ELISA detection.

[0015] Furthermore, in the application, the reagent for detecting the expression level of the PSIP1 gene or its encoded protein LEDGF / p75 is used in combination with a reagent for detecting the level of disease markers associated with systemic lupus erythematosus.

[0016] Furthermore, the disease markers associated with systemic lupus erythematosus include one or more of antinuclear antibodies (ANA), anti-double-stranded DNA antibodies (anti-dsDNA antibodies), anti-Smith antibodies, and C3 / C4 complement.

[0017] Furthermore, in this application, a mathematical model was established to demonstrate a negative correlation between the expression levels of the PSIP1 gene or LEDGF / p75 protein and the SLEDAI score. Specifically, through analysis of public databases and multiple batches of clinical samples (hundreds of cases in total), it was confirmed at both mRNA and protein levels that the expression of PSIP1 mRNA and LEDGF / p75 protein was significantly downregulated in SLE patients, and both were significantly negatively correlated with the SLEDAI score.

[0018] Furthermore, in the application described, a statistical association was established between the expression level of LEDGF / p75 protein and clinical symptoms and laboratory indicators.

[0019] Furthermore, the clinical symptoms include anemia, fever, and pericarditis; the laboratory indicators include red blood cell count, hemoglobin, erythrocyte sedimentation rate (ESR), and lupus anticoagulant normalized ratio (LA-NR). Specifically, patients with low LEDGF / p75 expression have more severe clinical manifestations (such as higher SLEDAI, higher LA-NR, anemia, elevated ESR, and a greater likelihood of pericarditis and fever).

[0020] Furthermore, the expression level of LEDGF / p75 protein increases as the disease improves, and it can be used to dynamically monitor treatment response.

[0021] Furthermore, in the application, the sample being detected includes a peripheral blood sample.

[0022] Furthermore, the peripheral blood sample includes peripheral blood mononuclear cells or T cells isolated therefrom.

[0023] Furthermore, in patients with systemic lupus erythematosus, the low expression of LEDGF / p75 protein leads to a decrease in the expression of downstream key cell cycle proteins CDK4 and CDK6, thereby triggering cell cycle arrest and cellular senescence.

[0024] Specifically, single-cell sequencing and protein validation showed that differential expression of LEDGF / p75 protein was mainly enriched in T cells, primarily in CD3+ cells. + In T cells, reduced LEDGF / p75 expression is functionally linked to SLE cell cycle arrest; PSIP1 expression is significantly correlated with CDK4 and CDK6, and these cell cycle molecules are also negatively correlated with SLEDAI scores. Decreased LEDGF / p75 protein levels are accompanied by decreased expression of downstream key cell cycle proteins CDK4 and CDK6. LEDGF / p75 maintains cell cycle progression by regulating CDK4 / 6; LEDGF / p75 deficiency is functionally linked to senescence induction. The absence of LEDGF / p75 impairs CDK4 / 6-mediated cell cycle progression, thereby inducing cell cycle arrest and promoting cellular senescence.

[0025] Furthermore, the agents that regulate cell cycle and cell senescence are agents that reverse cell senescence.

[0026] A third aspect of the present invention is to provide a system for predicting systemic lupus erythematosus disease using the PSIP1 gene or LEDGF / p75 protein, comprising:

[0027] The data acquisition module is used to acquire at least one of the expression level data of the PSIP1 gene and the expression level data of the LEDGF / p75 protein of the target subject.

[0028] The determination module is used to compare the expression level data of the PSIP1 gene obtained by the data acquisition module with a pre-established threshold; and / or, to compare the expression level data of the LEDGF / p75 protein obtained by the data acquisition module with a pre-established threshold to determine at least one of the following in the target subject's systemic lupus erythematosus: disease activity, disease clinical phenotype, and disease development stage.

[0029] Output module: Outputs the determination result of the determination module.

[0030] Furthermore, the target subjects are SLE patients who are suspected of having SLE, have been diagnosed with SLE and need to have their activity assessed, or are currently receiving treatment for SLE.

[0031] Furthermore, the data acquisition module uses reagents for detecting PSIP1 mRNA levels (such as qRT-PCR kits) to detect the expression level of PSIP1, and uses reagents for detecting LEDGF / p75 protein levels (such as specific antibodies and matching reagents used in Western Blot, immunofluorescence, flow cytometry, or ELISA detection, and kits containing them) to detect the expression level of LEDGF / p75 protein.

[0032] Furthermore, the system also includes a sample acquisition module for isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample of the target subject, extracting RNA to detect the expression level of PSIP1 mRNA; and / or for isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample of the target subject, lysing them to extract proteins to detect the expression level of LEDGF / p75 protein.

[0033] Furthermore, the establishment of the thresholds includes: collecting biological samples from healthy controls and SLE patients in the region, detecting the RNA and protein expression levels of LEDGF / p75, and establishing their normal reference range and pathological thresholds accordingly. Based on this, the constructed mathematical model and statistical correlation can be used to detect the samples of the subjects.

[0034] Furthermore, the LEDGF / p75 and SLEDAI score are specifically represented as follows: linear model Y = -9.30·X + 12.0 (where Y represents the SLEDAI score and X represents the expression level of LEDGF / p75).

[0035] Furthermore, based on the expression level of LEDGF / p75 protein, i.e. the ratio of protein gray value to internal control gray value (the ratio of LEDGF / p75 to β-actin), systemic lupus erythematosus (SLE) patients were divided into positive (ratio greater than or equal to 0.6) and negative (ratio less than or equal to 0.1).

[0036] Furthermore, thresholds can be determined in conjunction with other diagnostic reagents. Specifically, this includes detecting the expression levels of anti-dsDNA antibody and LEDGF / p75 protein in the serum of a sample comprising at least 100 SLE patients and 50 healthy controls. A joint predictive model is constructed using binary logistic regression analysis, and the optimal diagnostic cutoff value (i.e., the maximum Youden index) for the model's overall score is determined using an ROC curve. The discrimination criterion corresponding to this threshold is specifically defined by the following decision equation: 0.1007 × [anti-dsDNA] - 2.550 × [LEDGF / p75] > 1.7337 (where the anti-dsDNA concentration is in IU / mL, and the LEDGF / p75 is its ratio to the internal control gray value).

[0037] A fourth aspect of the present invention is to provide a method for assessing SLE disease status based on PSIP1 or LEDGF / p751 expression levels, which uses the system described in any of the third aspects of the present invention, and includes the steps of:

[0038] S1. Obtain at least one of the following: PSIP1 gene expression level data and LEDGF / p75 protein expression level data of the target subject;

[0039] S2. Compare PSIP1 expression level data with a pre-established threshold; and / or compare LEDGF / p75 protein expression level data with a pre-established threshold to determine the target subject's systemic lupus erythematosus disease activity, clinical phenotype, and disease progression.

[0040] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0041] (1) Novel Use of Molecular Markers: This invention is the first to propose using the expression level of the PSIP1 gene and its encoded protein LEDGF / p75 as a quantitative biomarker for directly assessing the activity of systemic lupus erythematosus (SLE). This use is completely different from any previously known function of this molecule (such as transcriptional coactivator, HIV integration cofactor, ophthalmic disease-related protein, etc.). The detection of the expression level of the PSIP1 gene and its encoded protein LEDGF / p75 is directly associated with the following clinical purposes: auxiliary diagnosis of SLE, graded assessment of SLE disease activity, prognostic assessment of SLE patients, and monitoring and efficacy evaluation of SLE treatment.

[0042] (2) Validation methods and correlation system for biomarkers: This invention utilizes public gene expression databases (GEO) and clinical sample cohorts for validation, constructing a complete chain of evidence for differential expression from PSIP1 mRNA levels (validated by bioinformatics analysis and qRT-PCR) to LEDGF / p75 protein levels (validated by Western blotting). This establishes a methodology for the negative correlation between PSIP1 expression levels or LEDGF / p75 expression levels and SLEDAI scores, thus providing an objective quantitative basis for assessing disease activity.

[0043] Based on the provided ROC curve analysis results, this invention demonstrates the significant advantages of the combined detection method by comparing the efficacy of single-indicator detection and combined-indicator detection. First, in distinguishing between diseased individuals and healthy controls, the combined detection of LEDGF / p75 and dsDNA shows superior diagnostic efficacy compared to either single indicator. Second, in assessing disease activity (using a SLEDAI score ≥6 as the standard for disease activity), the combined detection also exhibits excellent performance. Based on a local sample containing at least 100 SLE patients and 50 healthy controls, the expression levels of anti-dsDNA antibodies and LEDGF / p75 proteins in their serum were detected. A combined predictive model was constructed using binary logistic regression analysis, and the optimal diagnostic cutoff value (i.e., the maximum point of the Youden index) for the model's overall score was determined using ROC curve analysis. The aforementioned combined detection scheme of LEDGF / p75 and dsDNA can more accurately identify patients with high disease activity, providing a more effective tool for disease monitoring and treatment decisions.

[0044] Meanwhile, a statistical association between LEDGF / p75 expression status and specific clinical symptoms was established. Specifically, patients were divided into two groups based on their LEDGF / p75 expression levels: positive (the ratio of LEDGF / p75 gray value to the internal control gray value ≥ 0.6) and negative (the ratio of LEDGF / p75 gray value to the internal control gray value ≤ 0.1). Analysis using chi-square test or Fisher's exact test revealed that patients with negative LEDGF / p75 expression had a significantly higher incidence of specific clinical symptoms (such as anemia, fever, and pericarditis) than those with positive expression. Furthermore, some laboratory indicators (such as red blood cell count, hemoglobin, and erythrocyte sedimentation rate) showed significant differences between these patients and those with positive expression. These associations clarify the application value of LEDGF / p75 expression status in differentiating different clinical phenotypes (such as the presence or absence of specific symptoms).

[0045] All of the above demonstrates that LEDGF / p75 expression levels can be used for dynamic monitoring: by obtaining paired samples from the same patient at different disease activity stages, it was confirmed that LEDGF / p75 expression levels can dynamically change with disease activity, thus establishing its technical feasibility for longitudinal monitoring.

[0046] (3) Cell specificity and mechanism elucidation: Single-cell RNA sequencing analysis revealed that the differential expression of LEDGF / p75 in SLE was mainly located in T cell subsets; flow cytometry sorting combined with protein detection confirmed at the protein level that LEDGF / p75 was involved in CD3+ expression. + Enrichment of LEDGF / p75 in T cells and its differential expression in SLE patients. Further differential gene expression analysis and gene ontology enrichment analysis linked low LEDGF / p75 expression to cell cycle regulation pathway disorders (especially G1 / S phase transition). Experimental validation established a causal chain of "low LEDGF / p75 expression → decreased CDK4 / 6 expression → cell cycle arrest → cellular senescence (increased β-galactosidase activity)," directly linking the PSIP1 gene or its encoded protein LEDGF / p75 as a biomarker to immune cell senescence (one of the core pathological mechanisms of SLE).

[0047] In summary, this invention discloses for the first time a novel application of the PSIP1 gene and its encoded protein LEDGF / p75 in the diagnosis and treatment of systemic lupus erythematosus (SLE): the expression level of LEDGF / p75 in peripheral blood immune cells (especially T cells) of SLE patients is significantly negatively correlated with disease activity; that is, the lower the expression, the more active the disease. This indicates that the PSIP1 gene and its encoded protein LEDGF / p75 are not only excellent indicators of disease activity, but also participate in T cell senescence by regulating the cell cycle (affecting CDK4 / 6), and are directly related to the key pathological mechanisms of SLE. This invention is the first to propose using the detection of PSIP1 / LEDGF / p75 expression levels to assess SLE disease activity, assist in diagnosis, predict prognosis, and monitor treatment efficacy. It also reveals for the first time all the characteristics of LEDGF / p75 as a novel biomarker for SLE: correlation, specificity, dynamics, mechanism, and clinical applicability, providing a new tool and direction for the precise diagnosis, activity assessment, prognosis prediction, and future targeted therapy development of SLE. Attached Figure Description

[0048] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

[0049] Figure 1This is a schematic diagram illustrating the lack of correlation between anti-DFS70 antibody levels and LEDGF / p75 in peripheral blood PBMCs in one embodiment of the present invention.

[0050] Figure 2 This is a schematic diagram of the PSIP1 mRNA expression levels in SLE patients and healthy controls in GSE72509, GSE49454, and GSE61635 in one embodiment of the present invention;

[0051] Figure 3 This is a schematic diagram illustrating the correlation between PSIP1 mRNA expression levels and SLE disease activity in GSE49454 and GSE228066 in one embodiment of the present invention.

[0052] Figure 4 This is a schematic diagram of the PSIP1 mRNA expression levels in PBMCs of SLE patients and healthy controls in one embodiment of the present invention;

[0053] Figure 5 This is a schematic diagram of the expression levels of LEDGF / p75 protein in PBMCs of SLE patients and healthy controls in one embodiment of the present invention;

[0054] Figure 6 This is a schematic diagram illustrating the correlation between LEDGF / p75 protein abundance and SLEDAI in one embodiment of the present invention;

[0055] Figure 7 This is a schematic diagram illustrating the analysis of LEDGF / p75 protein expression levels and clinical parameters in one embodiment of the present invention;

[0056] Figure 8 This is a schematic diagram illustrating the paired sample analysis of LEDGF / p75 protein expression levels and SLE disease activity in one embodiment of the present invention;

[0057] Figure 9 This is an ROC curve of LEDGF / p75 and dsDNA combined detection to predict whether a person has the disease and the severity of the disease, according to one embodiment of the present invention.

[0058] Figure 10 This is a schematic diagram illustrating the expression characteristics of LEDGF / p75 in the peripheral immune compartment in one embodiment of the present invention; wherein, Part A: cell clustering in single-cell RNA sequencing analysis of PBMCs from SLE patients and healthy controls; Part B: major immune cell subsets based on annotation of typical marker genes; Part C: expression level of PSIP1 in PBMCs from SLE patients and healthy controls; Part D: differential expression analysis of PSIP1 in various immune cell subsets;

[0059] Figure 11In one embodiment of the present invention, the LEDGF / p75 protein in PBMCs is mainly located in CD3. + A schematic diagram of T cell expression;

[0060] Figure 12 This is a schematic diagram of transcriptomic analysis and key pathway enrichment related to LEDGF / p75 protein expression levels in SLE patients according to an embodiment of the present invention; wherein, Part A: hierarchical clustering heatmap of differentially expressed genes (DEGs) in SLE patients based on LEDGF / p75 protein level stratification; Part B: Volcano plot analysis of differentially expressed genes; Part C: Bubble diagram of gene ontology (GO) enrichment analysis; Part D: Expression changes of key cell cycle regulatory genes (such as CDC25B, CDKN1B, CDK6, etc.) in different LEDGF / p75 expression groups;

[0061] Figure 13 This is a schematic diagram illustrating the correlation between PSIP1 expression and CDK4 and CDK6 in one embodiment of the present invention;

[0062] Figure 14 This is a schematic diagram of senescence staining of PBMC cells from an SLE patient in one embodiment of the present invention. Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0064] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0065] Anti-DFS70 antibodies are products of the humoral immune response, specifically immunoglobulins produced by B cells / plasma cells in response to the DFS70 (i.e., LEDGF / p75) antigen. They are located in extracellular fluids such as peripheral blood serum, do not enter the cell nucleus, and participate in immune recognition only outside the cell. LEDGF / p75 protein is composed of… PSIP1 Gene-encoded nuclear functional proteins, located within the cell nucleus, directly participate in intracellular physiological activities such as chromatin remodeling, transcriptional regulation, and cell cycle progression. Their expression levels reflect the intrinsic molecular functional state of immune cells. In the inventors' previous research, they statistically analyzed the serum anti-DFS70 antibody levels and the LEDGF / p75 levels in peripheral blood PBMCs from 77 SLE patients affiliated with Renji Hospital of Shanghai Jiao Tong University School of Medicine. The results are as follows... Figure 1 As shown, there was no correlation between anti-DFS70 antibody levels and LEDGF / p75 levels in peripheral blood PBMCs (r=-0.07618, P=0.5102), indicating that the expression level of LEDGF / p75 cannot be inferred from the level of anti-DFS70 antibody.

[0066] The technical solution of the present invention will be described by way of example below in conjunction with the embodiments and accompanying drawings.

[0067] Example 1: PSIP1 and LEDGF / p75 are biomarkers for assessing the disease activity of systemic lupus erythematosus.

[0068] Systemic lupus erythematosus (SLE) is a complex autoimmune disease affecting multiple systems, caused by the combined effects of environmental and genetic factors. The interaction of these factors triggers an autoimmune response, leading to T cell activation, excessive B cell production of pathogenic autoantibodies, and cytokine dysregulation, resulting in tissue and organ damage. The presence of numerous autoantibodies is a major characteristic of SLE; antinuclear antibodies (ANA), anti-double-stranded DNA antibodies, and anti-Sm antibodies are commonly used biomarkers for SLE diagnosis. Indirect immunofluorescence assay (IFA) using human laryngeal carcinoma epithelial cells (HEp-2) as a substrate is the reference method for ANA detection. The International Antinuclear Antibody Fluorescence Karyotype Consensus Group has defined 30 fluorescence models, among which the dense fine speckle fluorescence model, known as the DFS (Dense Fine Speckles) karyotype, primarily targets the DFS70 protein, which is approximately 70 kDa in size. In early research, a Harvard University research team isolated a 75kDa protein from lens epithelial cells of cataract patients. This protein shared a significant homologous sequence with the liver cancer-derived growth factor (HDGF)-associated protein (HRP) family and was named lens epithelial-derived growth factor (LEDGF / p75). It was later confirmed that LEDGF / p75 shares the same amino acid sequence as DFS70. Although subsequent studies showed that LEDGF / p75 is not specifically present in lens epithelial cells, this name has been widely used and retained in the scientific community. LEDGF / p75 is also known as transcription cofactor PC4 and SFRS interacting protein 1 (PSIP1). A truncated variant of LEDGF / p75, measuring 52kDa, is called LEDGF / p52. The gene encoding both proteins is called PSIP1. LEDGF / p75 and LEDGF / p52 share the same amino acid sequence at their N-terminus, including a PWWP (proline-tryptophan-tryptophan-proline) domain, three charged regions (CR1, CR2, and CR3), a nuclear localization site (NLS), and two AT hook sequences. LEDGF / p75 has a human immunodeficiency virus integrase (HIV-IN) binding domain (IBD) at its C-terminus, while LEDGF / p52 has a unique amino acid sequence at its C-terminus. Studies have shown that LEDGF / p75 and LEDGF / p52 bind to histone H3K36 and splicing factors to regulate histone methylation, thereby modulating epigenetics.

[0069] This embodiment aims to clarify the value of the PSIP1 gene and its encoded protein LEDGF / p75 in the diagnosis and disease activity assessment of systemic lupus erythematosus (SLE), employing a strategy combining systematic bioinformatics and clinical validation. First, through integrated analysis of gene expression datasets from peripheral blood mononuclear cells (PBMCs) of multiple SLE patients and healthy controls in the Gene Expression Omnibus (GEO) database, the differences in PSIP1 mRNA expression levels between SLE patients and healthy controls were compared, and correlation analysis was performed with the SLEDAI score. Based on this, several healthy individuals and SLE patients were recruited, and changes in PSIP1 / LEDGF / p75 expression levels were validated at both the mRNA and protein levels using qRT-PCR and Western blot techniques, respectively, and their diagnostic efficacy and dynamic characteristics as biomarkers for monitoring disease activity were evaluated. Specific steps included:

[0070] (I) Bioinformatics Analysis;

[0071] (1) Bioinformatics analysis was performed on three datasets (GSE72509, GSE61635, and GSE121239) in the public database GEO to compare whether there was a difference in PSIP1 expression between SLE patients and healthy controls. The results are as follows: Figure 2 As shown in the figure. The results indicate that the PSIP1 mRNA level in peripheral blood mononuclear cells (PBMCs) of SLE patients was significantly lower than that in healthy donors.

[0072] (2) To establish its association with the clinical phenotype of the disease, Spearman correlation analysis was used to verify the statistical association between PSIP1 expression level and disease activity in an independent dataset containing SLEDAI score information. The results are as follows: Figure 3 As shown in the figure. Spearman correlation analysis revealed a significant negative correlation between PSIP1 expression and SLEDAI score (GSE49454: R²=0.0475, P=0.0043; GSE228066: R²=0.0503, P=0.0260). These results indicate that decreased PSIP1 expression is associated with increased disease activity, supporting its potential value as a biomarker for monitoring SLE progression.

[0073] (ii) Clinical validation of the expression levels of PSIP1 / LEDGF / p75;

[0074] (1) Specimen collection: Peripheral blood samples were collected from 56 healthy individuals and 71 SLE patients at Renji Hospital in Shanghai. Peripheral blood mononuclear cells (PBMCs) were isolated, and RNA was extracted to verify the mRNA expression level of PSIP1. In another group, peripheral blood samples were collected from 58 healthy individuals and 99 SLE patients. PBMCs were isolated, lysed, and proteins were extracted to verify the protein expression level of PSIP1.

[0075] (2) Preparation of peripheral blood mononuclear cells (PBMCs): 1) Take 5 mL of peripheral blood and add it to a 15 mL centrifuge tube. Centrifuge at 2000 rpm for 5 min. Aspirate the upper plasma layer and store it. Add 3 mL of 1×PBS buffer to the centrifuge tube and mix by pipetting. Take another centrifuge tube and add 3 mL of lymphocyte separation medium. Then, slowly and gently add the mixed blood cells to the tube and centrifuge at 2500 rpm for 30 min. 2) After centrifugation, the upper layer is diluted plasma, the middle layer is the separation medium, and the lower layer is blood cells. The white membrane layer between the upper plasma layer and the middle separation medium is the mononuclear cells (PBMCs). Take another centrifuge tube and add 3 mL of 1×PBS. Carefully aspirate the white membrane layer of PBMCs into the centrifuge tube, wash by pipetting, and centrifuge at 3500 rpm for 10 min. 3) Repeat the washing twice. 4) Discard the supernatant, resuspend in 3 mL of 1×PBS, centrifuge at 3000 rpm for 10 min, and discard the supernatant.

[0076] (3) Detection of PSIP1 mRNA;

[0077] ①RNA Extraction: 1) Add 1 mL of Trizol lysis buffer to the sample, mix by pipetting, incubate at room temperature for 10 minutes, add 0.2 mL of chloroform, seal the centrifuge tube tightly, and shake vigorously for 15 seconds. Incubate at room temperature for 3 minutes. 2) Centrifuge at 12000 g for 15 minutes at 4℃, transfer the upper aqueous phase to a new centrifuge tube, add 0.5 mL of isopropanol, seal the centrifuge tube tightly, invert and mix, and incubate at room temperature for 10 minutes. 3) Centrifuge at 12000 g for 10 minutes at 4℃, carefully discard the supernatant, add 1 mL of pre-chilled 75% ethanol, and invert and mix. 4) Centrifuge at 7500 g for 5 minutes at 4℃, carefully discard the supernatant and excess water on the tube wall, and dry at room temperature for 5-10 minutes (be careful not to over-dry, otherwise it will reduce the solubility of RNA). 5) Add 20 μL of RNase-Free Water and dissolve the RNA at room temperature (you can gently vortex or pipette to mix). 6) Use a Nanodrop spectrophotometer to detect RNA concentration and purity, loading 1 μL of each sample.

[0078] ②Reverse transcription: 1) Take 1 μg RNA from each sample, add 3 μL of 5*g DNA wiper Mix, and make up the volume to 15 μL with RNase-free water. Incubate at 42℃ for 2 min in a PCR instrument. 2) Add 5 μL of 4*HiScript IV qRT SuperMixa, mix well, and incubate at 37℃ for 15 min in a PCR instrument, then at 85℃ for 5 sec.

[0079] ③ Real-time quantitative PCR: 1) Gene sequences were retrieved from the NCBI (National Center for Biotechnology Information) website. Primer sequences were designed using Primer Premier 5 software, and primers were synthesized. 2) Primers were centrifuged at low speed and diluted to a working concentration of 10 μmol / L. The reaction system is shown in Table 1 below. 3) 2 μL of cDNA sample + 23 μL of the above system were added to each well of an eight-piece plate. The mixture was centrifuged at low speed and amplified in a quantitative PCR instrument at 62℃. 4) Experimental results are expressed as 2-ΔΔCT. Statistical analysis was performed using SPSS 2019 software and Graphpad Prism 9.7 software. -ΔΔCT =2 -（待测组目的基因平均CT值-待测组内参基因平均CT值） - （对照组目的基因平均值-对照组内参基因平均CT值）

[0080] Table 1 - PCR Reaction System

[0081]

[0082] ④ Analysis of qPCR experimental results: such as Figure 4 As shown, consistent with public datasets, qRT-PCR analysis revealed that PSIP1 mRNA expression in SLE patients was significantly lower than in healthy individuals (P<0.001), with the lowest expression levels observed in patients with high disease activity (SLEDAI>6).

[0083] (4) Detection of LEDGF / p75 protein;

[0084] ① Protein lysis: 1) Add 100 μL of strong RIPA lysis buffer + 1 μL of PMSF + 1 μL of protease phosphatase inhibitor to each PBMC tube and mix thoroughly by pipetting. 2) Lyse on ice for 30 min. 3) Fix the protein lysis buffer in an ice box and sonicate for 10 s, with a 30 s interval, repeating 5 times. 4) Centrifuge at 10000 g for 10 min, collect the supernatant, aliquot and store at -80℃.

[0085] ② BCA method for protein concentration determination: 1) Preparation of protein standards: Dissolve 1.2 mL of standard preparation solution and 30 mg of BSA thoroughly to prepare a 25 mg / mL protein standard solution, and then dilute it sequentially to 500, 400, 300, 200, 100, 50, and 25 μg / mL standards. 2) Preparation of BCA working solution: Prepare BCA working solution by mixing BCA reagent A and reagent B at a ratio of 50:1. 3) Protein concentration detection: Add 20 μL of standard or sample to each well, then add 200 μL of BCA working solution, mix well, and incubate at 37ºC for 30 minutes. 4) Read the absorbance values ​​using a microplate reader, plot a standard curve, and calculate the sample concentration based on the standard curve.

[0086] ③Western Blot: 1) Protein denaturation: Add 1 part 5× loading buffer to each sample at 5 sample volumes, and incubate in a PCR amplification instrument at 95℃ for 15 min. 2) Gel preparation: Place the gel preparation apparatus flat, take 2.2 mL of the lower gel solution and an equal volume of lower gel buffer, add 50 μL of modified coagulant and mix well. Take 1 mL of the upper gel solution and an equal volume of upper gel buffer, add 20 μL of modified coagulant and mix well. Gently shake to flatten the liquid surface, insert the comb, and let stand for 30 minutes. 3) SDS-PAGE electrophoresis: Install the vertical electrophoresis tank, add electrophoresis buffer between the two gel plates, remove the comb in the electrophoresis buffer, load 4 μL of marker sample and 20 μg of protein sample. Set the power supply to 80 V, and after the bands enter the lower gel, adjust the power supply to 120 V and electrophoresis for 60 minutes. 4) Transfer: Soak thick filter paper in transfer buffer and pre-cool at 4°C. Activate the PVDF membrane with methanol for 1 minute, then transfer it into transfer buffer. After removing the glass plate, remove the top layer of gel using a white skid and soak it in transfer buffer for 5 minutes. The transfer apparatus is assembled from bottom to top as follows: sponge → filter paper → gel → membrane → filter paper → sponge. After placing the transfer clamp in the tank, add sufficient transfer buffer and place the matching ice box in the tank. Cover the tank and plug in the power. Place the tank on ice to prepare for transfer. Set the power to 250 mA and the transfer time to 90 minutes. 5) Blocking: After transfer, add 20 mL of 5% BSA to cover the membrane and place it on a shaker for 1 hour. 6) Primary antibody incubation: Add 10 mL of 1:2000 rabbit anti-human LEDGF antibody (diluted with primary antibody dilution buffer) and place it on a shaker for 1 hour. 7) Recover the primary antibody, add 20 mL of 1×TBST and wash for 5 minutes. Repeat 3 times. 8) Secondary antibody incubation: Add 10 mL of HRP-labeled goat anti-rabbit IgG (diluted with blocking buffer) and incubate on a shaker for 1 h. 9) Recover the secondary antibody, wash with 20 mL of 1×TBST for 5 min, repeat 3 times. 10) Color development: Mix 200 μL of chemiluminescent color development solution A and 200 μL of solution B, cover the entire membrane, and image using a gel imaging system. 11) Antibody removal: Add 10 mL of antibody removal agent, incubate on a shaker for 5 min, recover the antibody removal agent, wash with 20 mL of 1×TBST for 5 min, repeat 3 times. 12) Blocking: Add 20 mL of 5% BSA to submerge the membrane, incubate on a shaker for 1 h. 13) Internal control antibody incubation: Add 10 mL of 1:5000 β-actin mouse monoclonal antibody (diluted with primary antibody dilution buffer), incubate on a shaker for 1 h. 14) Recover the internal control antibody, wash with 20 mL of 1×TBST for 5 min, repeat 3 times. 15) Secondary antibody incubation: Add 10 mL of HRP-labeled goat anti-mouse IgG (diluted with blocking buffer) and incubate on a shaker for 1 h. 16) Recover the secondary antibody, add 20 mL of 1×TBST and wash for 5 min, repeat 3 times.17) Color development: Mix 200 μL of chemiluminescent color development solution A and 200 μL of solution B, cover the entire membrane, and image using a gel imaging system. 18) Statistical analysis was performed using Chemidoc XRS+ software and Graphpad Prism 9.7 software.

[0087] ④ Western Blot analysis: Western blot analysis showed that LEDGF / p75 protein expression in SLE patients was significantly lower than that in healthy individuals (P<0.001), and the lowest expression level was observed in patients with high disease activity (SLEDAI>6). Figure 5 Furthermore, the abundance of LEDGF / p75 protein was significantly negatively correlated with SLEDAI (R²=0.0880, r=-0.391, P<0.0001). Figure 6 ).

[0088] Based on the above, normal reference ranges and pathological thresholds for SLE can be constructed. Specifically, this involves collecting biological samples from healthy controls and SLE patients in the region, detecting the RNA and protein expression levels of LEDGF / p75, and establishing normal reference ranges and pathological thresholds accordingly. The constructed mathematical model and statistical correlation can then be used to analyze the samples. For example, the relationship between LEDGF / p75 and the SLEDAI score can be represented by a linear model: Y = -9.30·X + 12.0 (where Y represents the SLEDAI score and X represents the expression level of LEDGF / p75), thus providing an objective quantitative basis for assessing disease activity.

[0089] In summary, the above experimental results strongly validate the downregulation of PSIP1 / LEDGF / p75 in SLE and support its potential clinical relevance as a biomarker associated with disease activity in multiple independent cohorts.

[0090] Example 2: PSIP1 / LEDGF / p75 are associated with key clinical features and disease progression in systemic lupus erythematosus.

[0091] To further clarify the clinical significance of LEDGF / p75 expression, this embodiment divides systemic lupus erythematosus (SLE) patients into positive (≥0.6) and negative (≤0.1) groups based on protein expression levels, i.e., the ratio of protein gray value to internal reference gray value (the ratio of LEDGF / p75 to β-actin).

[0092] (1) Clinical parameter analysis based on the protein expression levels of LEDGF / p75 showed that, for example Figure 7As shown in Table 2 below, LEDGF / p75 positive patients exhibited significantly higher red blood cell counts (4.04±0.71 vs 3.45±0.78 ×10^12 / L, P<0.010) and hemoglobin levels (119.90±22.13 vs 101±18.51 g / L, P=0.002), as well as significantly lower erythrocyte sedimentation rate (ESR) (24.43±22.88 vs 43.87±31.25 mm / h, P=0.044), lower disease activity (5.11±2.91 vs 13.03±10.53, P=0.003), lower pericarditis (0% vs 22.2%, P=0.042), and lower incidence of fever (5.6% vs 41.7%, P=0.010).

[0093] As shown in Table 2 below, further comparisons of LEDGF / p75 negative (≤0.1), weakly positive (≥0.1 ≤0.6), and positive subgroups (≥0.6) revealed significant differences in multiple hematological and clinical indicators, reinforcing the graded association between decreased LEDGF / p75 levels and more severe disease manifestations. These data strongly suggest that decreased LEDGF / p75 expression is associated with a more aggressive clinical phenotype in SLE.

[0094] Table 2 - Clinical and laboratory characteristics of SLE patients stratified by LEDGF / p75 expression

[0095]

[0096] Given the observed relationship between LEDGF / p75 expression and disease activity, the dynamic changes in LEDGF / p75 expression were then assessed to determine whether they might reflect disease progression. The results are as follows: Figure 8 As shown in the figure, a paired sample analysis of 18 SLE patients revealed a significant increase in LEDGF / p75 protein levels after clinical improvement. Specifically, LEDGF / p75 and the SLEDAI score exhibited a linear relationship: Y = -9.30·X + 12.0 (where Y represents the SLEDAI score and X represents the expression level of LEDGF / p75), thus providing an objective quantitative basis for assessing disease activity and consistent with fluctuations in disease activity. This observation suggests that continuous monitoring of LEDGF / p75 may provide valuable insights for evaluating treatment response and predicting disease outcomes.

[0097] (2) The protein expression level of LEDGF / p75 combined with the expression level of anti-dsDNA antibody can be adjusted to the ROC curve threshold for judging the negative and positive results of SLE, such as... Figure 9Specifically, based on a sample containing at least 100 SLE patients and 50 healthy controls, the expression levels of anti-dsDNA antibody and LEDGF / p75 protein in their serum were detected. A joint prediction model was constructed using binary logistic regression analysis, and the optimal diagnostic cutoff value (i.e., the maximum point of the Youden index) for the comprehensive score of this model was determined using ROC curves. The discrimination criterion corresponding to this threshold is specifically defined by the following decision equation: 0.09238 × [LEDGF / NC] + 1.016 × [anti-dsDNA] > -0.994 (where the anti-dsDNA concentration is in IU / mL, and the LEDGF / p75 is its ratio to the internal control gray value).

[0098] In summary, genetic, proteomic, and clinical relevance analyses indicate that LEDGF / p75 is a robust and clinically significant biomarker closely associated with disease severity and treatment response, supporting further evaluation in prospective longitudinal studies.

[0099] Example 3: PSIP1 / LEDGF / p75 preferentially expressed in T cells

[0100] To determine the cell distribution of PSIP1 / LEDGF / p75 in the peripheral immune compartment, this embodiment performed PBMC cell populations and validated the results at the protein level, specifically including:

[0101] (a) Detection of PSIP1 expression in PBMC cell populations;

[0102] This example analyzes a single-cell RNA sequencing dataset (GSE135779) containing 8 adult SLE patients and 6 matched healthy controls, totaling approximately 132,000 PBMCs. The results are as follows: Figure 10 As shown, dimensionality reduction and unsupervised clustering were performed, and major immune cell subsets, including CD4, were identified and annotated using typical marker genes. + and CD8 + T cells, B cells, monocytes, dendritic cells, NK cells, plasma cells, and megakaryocytes ( Figure 10 Parts A to B). In the entire PBMC dataset, PSIP1 expression was significantly reduced in SLE patients compared to healthy controls (…). Figure 10 Part C). Cell type analysis further revealed that this reduction was primarily localized in T cells and macrophages, with T cells exhibiting the most significant differential expression (part C). Figure 10 (Part D).

[0103] (ii) Given the core contribution of T cells to the pathogenesis of SLE, these observations were then validated at the protein level.

[0104] (1) Flow cytometry: The procedure for flow cytometry is described in the manufacturer's instructions. CD3 was isolated from 10 healthy controls, 10 LEDGF / p75-positive SLE patients, and 15 LEDGF / p75-negative SLE patients by flow cytometry. + T cells and CD3 - Non-T cells.

[0105] (2) Western blot: See Example 1 for the operation steps of Western blot.

[0106] (3) Verification results: such as Figure 11 As shown, the analysis results confirm that the LEDGF / p75 protein is mainly located in CD3. + LEDGF / p75 is expressed in T cells, and its level in LEDGF / p75-positive SLE patients is significantly higher than that in LEDGF / p75-negative patients and healthy controls. These results are consistent with scRNA-seq results and demonstrate that LEDGF / p75 is highly enriched in T cells, highlighting this cell population as a major contributor to its dysregulation in SLE.

[0107] Example 4: PSIP1 / LEDGF / p75 promotes cellular senescence in SLE by disrupting cell cycle regulation

[0108] Based on the verification in Example 3, it was found that the LEDGF / p75 protein is mainly located in CD3. + It is expressed in T cells, and its mechanism of action will be further investigated, including the following steps:

[0109] (a) Unsupervised hierarchical clustering;

[0110] To investigate the molecular consequences associated with altered LEDGF / p75 expression in SLE, RNA-Seq analysis was performed on PBMCs of eight SLE patients stratified according to LEDGF / p75 protein levels, and differentially expressed genes (DEGs) were subjected to unsupervised hierarchical clustering. The resulting heatmap showed a clear separation between the high and low LEDGF / p75 expression groups, indicating significant transcriptional differences. Figure 12 Part A).

[0111] (ii) Gene enrichment;

[0112] Gene Ontology (GO) enrichment analysis showed that DEGs were mainly enriched in pathways that regulate mitotic cell cycle checkpoints, DNA metabolism processes, and maintain cell cycle fidelity. Figure 12 (Parts B-C). Disruption of these processes is a recognized marker of cellular senescence, suggesting a functional link between reduced LEDGF / p75 and SLE cell cycle arrest.

[0113] (III) Volcano Map Analysis

[0114] Volcano plot analysis further identified several key DEGs with strong differential expression, including CDC25B, CDKN1B, CD3D, CDK6, and PSIP1 itself. Figure 12 (B~D parts). Many of these genes are key regulators of G1 / S and G2 / M transitions, and their dysregulation (especially the reduction of CDK6 and CDC25B) supports the possibility that LEDGF / p75 deficiency may drive cell cycle-related mechanisms of aging.

[0115] Based on the above findings, it is hypothesized that LEDGF / p75 may regulate cell cycle-related pathways in SLE. Mining multiple public datasets shows that PSIP1 expression is significantly associated with CDK4 and CDK6, and these cell cycle molecules are also negatively correlated with SLEDAI scores. Figure 13 This relationship was further validated in clinical samples. In a cohort of 33 SLE patients, decreased LEDGF / p75 protein levels were accompanied by reduced CDK4 and CDK6 expression, and a strong correlation was observed among the three proteins. Figure 13 These data collectively support the view that LEDGF / p75 maintains cell cycle progression by regulating CDK4 / 6.

[0116] (iv) Cellular senescence;

[0117] To determine whether decreased LEDGF / p75 expression functionally translates into increased cellular senescence, β-galactosidase activity in PBMCs from 12 SLE patients with low LEDGF / p75 expression, 5 SLE patients with high LEDGF / p75 expression, and 6 HDs was evaluated.

[0118] (1) The specific experimental steps are as follows: 1) Cell seeding and treatment: Seed cells in 24-well plates or well plates with coverslips and treat them. 2) Fixation: Discard the culture medium and gently rinse once with 1× PBS. Add 4% paraformaldehyde and fix at room temperature for 5-15 minutes. 3) Washing: Wash cells 3 times with 1× PBS, 3 minutes each time. 4) Preparation of staining working solution: Prepare fresh staining solution according to the kit instructions or formula (pH 6.0 staining solution containing X-gal). 5) Staining: Add an appropriate amount of staining solution to each well to ensure complete coverage of cells. Incubate overnight (12-16 hours) in a CO2-free incubator at 37°C. Note: Do not place in a CO2 incubator. 6) Termination and observation: Discard the staining solution and wash twice with PBS. 70% glycerol can be added for preservation. 7) Microscopic observation and counting: Observe under a regular optical microscope. Positive cells show a clear blue-green cytoplasm. Randomly select multiple fields of view (>100 cells / field of view) and calculate the percentage of positive cells.

[0119] (2) Results analysis: such as Figure 14 As shown, the experimental results indicate that the aging level in the LEDGF / p75 low expression group was significantly higher than that in patients with high LEDGF / p75 expression and healthy controls, suggesting a functional link between LEDGF / p75 deficiency and aging induction.

[0120] The results of this embodiment indicate that the loss of LEDGF / p75 impairs CDK4 / 6-mediated cell cycle progression and promotes cellular senescence, providing a mechanistic basis for its contribution to SLE immune dysregulation.

[0121] As can be seen from the above embodiments, this invention has for the first time explored and systematically verified a novel medical application of the known protein LEDGF / p75 as a biomarker of disease activity in SLE. It is not limited to discovering its expression differences, but also, through innovative multi-omics analysis and functional experiments, it abnormally localizes it to specific T cells and elucidates the molecular mechanism by which it promotes cell senescence by affecting the cell cycle. Thus, it constructs a complete knowledge system from "detection indicators" to "pathological links", providing a solid and unique technical foundation for the development of related diagnostic products and treatment strategies.

[0122] Based on the novel discovery that the expression level of PSIP1 / LEDGF / p75 in systemic lupus erythematosus (SLE) is closely related to disease activity, this invention provides a new use for it as a novel biomarker with broad clinical application prospects. Its advantages are specifically reflected in the following aspects: (1) It provides a novel, high-value biomarker that makes up for the shortcomings of existing biomarkers. This invention is the first to systematically demonstrate, in multiple independent cohorts (public databases and clinical samples), the expression level of PSIP1 / LEDGF / p75 in peripheral blood mononuclear cells (PBMCs) and T cells of SLE patients, which is significantly negatively correlated with the SLEDAI score. Its expression level decreases with increasing disease activity and rises again with disease remission, and its dynamic changes are highly synchronized with the disease process. From the mRNA level (public database, qRT-PCR) to the protein level (Western Blot), and then to the single-cell resolution (scRNA-seq) and functional level (SA-β-gal activity), it provides a comprehensive and multi-level solid chain of evidence, ensuring the reliability of the biomarker. PSIP1 / LEDGF / p75 can be used in combination with existing biomarkers to provide doctors with a more comprehensive and accurate tool for assessing disease activity, especially suitable for patients who do not respond atypically to traditional indicators, which helps to achieve individualized treatment management. (2) It goes beyond simple "correlation" and reveals the potential pathological mechanism, giving the biomarker a deeper biological meaning. This invention not only discovered expression differences, but also, through bioinformatics analysis and experimental verification, directly linked the low expression of PSIP1 / LEDGF / p75 to cell cycle arrest and cell senescence, a key mechanism of immune dysregulation in SLE. It was the first time that the dysregulation of PSIP1 / LEDGF / p75 in SLE mainly occurs in T cells, which provides a cellular basis for understanding the source and function of the biomarker, making it not only an indicator of "state" but also a possible link in "pathogenesis". The study found that its expression is positively correlated with the key cell cycle protein CDK4 / 6, suggesting that LEDGF / p75 may affect cell cycle progress by regulating CDK4 / 6. This provides a potential target for developing new therapies aimed at reversing T cell senescence, making the biomarker have both diagnostic and therapeutic guidance value. (3) It has clear clinical applicability and translational potential. The detection is based on peripheral blood (PBMCs or specific cell subpopulations), which is a non-invasive or minimally invasive sampling method, easy to obtain repeatedly, and convenient for dynamic monitoring. The techniques used in this invention, such as qRT-PCR, Western Blot, and flow cytometry, are routine techniques in clinical testing or research laboratories, and are easy to standardize and widely apply. In the future, they can be further developed into more convenient ELISA (enzyme-linked immunosorbent assay) or flow cytometry detection panels.(4) Guiding treatment and prognosis: By monitoring the changes in LEDGF / p75 levels before and after treatment, the treatment effect can be objectively evaluated, providing a basis for adjusting the treatment plan; low expression of LEDGF / p75 is associated with more severe clinical manifestations (such as anemia, increased erythrocyte sedimentation rate, pericarditis, and fever), which helps to identify high-risk patients and achieve early intervention; stratifying patients according to their LEDGF / p75 expression levels (such as negative, weakly positive, and positive) helps to distinguish different disease activity states and clinical phenotypes, enabling more refined patient management.

[0123] In summary, the PSIP1 / LEDGF / p75 biomarkers provided by this invention possess outstanding advantages such as strong innovation, a complete chain of evidence, in-depth mechanistic correlation, good clinical relevance, high feasibility of detection, and diverse application scenarios. It not only provides a powerful new tool for monitoring SLE disease activity, but more importantly, by linking it to the core pathological link of T-cell senescence, it opens up valuable new directions for understanding the immune aging mechanism of SLE and exploring new treatment strategies. Its application is expected to improve the accuracy of SLE diagnosis and treatment, improve patient prognosis, and has significant social benefits and market application prospects.

[0124] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of a reagent for detecting the expression level of LEDGF / p75 protein in the preparation of products for assessing the disease activity of systemic lupus erythematosus, characterized in that, The samples tested are peripheral blood mononuclear cells or T cells isolated from them.

2. The application according to claim 1, characterized in that, In the application, the reagent for detecting the expression level of LEDGF / p75 protein and the reagent for detecting the level of disease biomarkers associated with systemic lupus erythematosus are used in combination; wherein the disease biomarkers associated with systemic lupus erythematosus include one or more of antinuclear antibodies, anti-double-stranded DNA antibodies, anti-Smith antibodies, and C3 / C4 complement.

3. A system for predicting systemic lupus erythematosus using LEDGF / p75 protein, characterized in that, include: Sample collection module: used to isolate peripheral blood mononuclear cells from peripheral blood samples of the target subject, lyse and extract proteins to detect the expression level of LEDGF / p75 protein; Data acquisition module: used to obtain expression level data of LEDGF / p75 protein in the target subjects; The determination module is used to compare the expression level data of LEDGF / p75 protein obtained by the data acquisition module with a pre-established threshold to determine the disease activity of systemic lupus erythematosus in the target subject. Output module: Outputs the determination result of the determination module.

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