Application of ubiquitination modification of protein in cell inflammatory response and hypercoagulable state related rheumatism

Abstract

The invention discloses application of ubiquitination modification of protein in rheumatism related to cell inflammatory response and hypercoagulable state, and belongs to the technical field of rheumatism molecular biology. Ubiquitin-binding enzyme UBE2A is highly expressed in RA patients, and an NF-kappa B signal channel is activated by mediating ubiquitination modification of NEMO, so that inflammatory response and hypercoagulable state are promoted. Bioinformatics analysis, machine learning screening, a cell co-culture model and clinical sample verification are comprehensively utilized to prove that UBE2A is a key molecule in the RA pathogenesis process, and it is prompted that UBE2A can be used as a new target for RA diagnosis / treatment. Therefore, UBE2A and related pathways thereof can be used for preparing medicines for treating rheumatoid arthritis, especially for regulating inflammation and abnormal blood coagulation.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology of rheumatic diseases, specifically relating to the application of ubiquitination modification of a protein in rheumatic diseases related to cellular inflammatory response and hypercoagulable state. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial tissue hyperplasia and chronic inflammation. Research suggests that immune genetics, environment, and infection play a dominant role in the pathogenesis of RA, but the exact mechanisms are not yet fully understood. The key to RA treatment lies in early detection and control of disease progression. Currently used medications include nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids, and biologics. Although these drugs can alleviate clinical symptoms to some extent, long-term, high-dose use can easily lead to adverse reactions such as liver and kidney damage and gastrointestinal disorders, resulting in poor patient tolerance and unsatisfactory long-term efficacy. Therefore, in-depth research into the pathogenesis of RA and the search for new potential therapeutic targets and effective drugs has significant practical value and social benefits.

[0003] An immune inflammatory response occurs throughout the entire pathogenesis of rheumatoid arthritis (RA), and persistent inflammatory stimulation can lead to joint swelling, pain, deformity, and loss of function. The synovial tissue is the core site of RA inflammation, with fibroblast-like synoviocytes (FLS) being key effector cells that promote synovial inflammation and cartilage and bone damage. Activated FLS not only release large amounts of inflammatory factors but also exhibit invasive behavior similar to tumor cells, exacerbating the condition. Studies show that a characteristic pathological product—pannus—forms during RA disease, composed of newly formed microvessels, proliferating and hypertrophic FLS, and inflammatory cells. Pannus, with its tumor-like characteristics, is the main pathological basis for joint destruction and cartilage damage. Progressive angiogenesis is often accompanied by intravascular coagulation and microcirculatory thrombosis. Research indicates that rats with a collagen-induced arthritis blood stasis syndrome model exhibit abnormal blood rheology, coagulation dysfunction, and platelet and endothelial dysfunction, accompanied by an imbalance of inflammation and coagulation factors. Therefore, regulating the inflammatory response and hypercoagulable state is an important direction for RA treatment.

[0004] Post-translational modifications play a crucial role in the pathogenesis of synovitis. These modifications alter the structure and function of various proteins involved in synovial inflammation, leading to dysregulation of immune tolerance and the persistence of chronic synovitis in rheumatoid arthritis (RA). Studies have shown that ubiquitination, a key type of post-translational modification, also plays an important role in the pathogenesis of arthritis. However, the specific mechanisms regulating the ubiquitin-proteasome pathway in RA remain poorly understood. Therefore, exploring the pathogenesis of RA from the perspective of ubiquitination modifications is of great significance. Summary of the Invention

[0005] This invention reveals the key role of ubiquitin-binding enzyme UBE2A in RA through a comprehensive study involving bioinformatics, cell experiments, and clinical validation: it establishes that UBE2A activates the NF-κB signaling pathway by mediating NEMO ubiquitination modification, thereby promoting the inflammatory response and hypercoagulable state in RA.

[0006] This invention proposes the application of the ubiquitin-binding enzyme UBE2A in diagnostic formulations for rheumatic diseases associated with cellular inflammation and hypercoagulability. Increased expression of UBE2A and NEMO proteins in peripheral blood mononuclear cells (PBMCs), increased NEMO ubiquitination levels, activation of the NF-κB pathway, and elevated levels of pro-inflammatory and procoagulant cytokines promote the inflammatory response and hypercoagulable state in rheumatoid arthritis. Therefore, formulations that detect UBE2A and NEMO proteins in PBMCs can be used in the preparation of diagnostic formulations for rheumatic diseases associated with cellular inflammation and hypercoagulability.

[0007] This invention also proposes the application of ubiquitin-conjugating enzyme UBE2A in the treatment of rheumatic diseases associated with cellular inflammation and hypercoagulable states. Inhibiting the expression of ubiquitin-conjugating enzyme UBE2A can reduce NEMO ubiquitination, further inhibiting the inflammatory response and hypercoagulable state in rheumatoid arthritis. Therefore, preparations that inhibit the expression of ubiquitin-conjugating enzyme UBE2A can be used in the preparation of treatments for rheumatic diseases associated with cellular inflammation and hypercoagulable states.

[0008] This invention screened differentially expressed genes related to RA from the GEO dataset (GSE89408) using bioinformatics analysis, and identified UBE2A as the core gene using machine learning (including KNN, RF, and LASSO algorithms). Gene set enrichment analysis (GSEA) showed that the ubiquitination pathway was highly enriched in RA. A cell co-culture model (co-culture of RA patient PBMCs with fibroblast-like synovial cells (FLS)) confirmed the upregulation of UBE2A and NEMO expression, and enhanced NEMO ubiquitination. Furthermore, clinical sample validation revealed increased expression of UBE2A and NEMO proteins, increased NEMO ubiquitination levels, activation of the NF-κB pathway, and elevated levels of pro-inflammatory and procoagulant cytokines in peripheral blood mononuclear cells (PBMCs) of RA patients.

[0009] Therefore, this invention reveals the pathogenesis of RA inflammation and hypercoagulable state mediated by UBE2A, making it a new target for the treatment / diagnosis of RA. Attached Figure Description

[0010] Figure 1 This is a prediction of ubiquitination modification of RA.

[0011] Figure 2 Screening for ubiquitination-modified core gene UBE2A for machine learning.

[0012] Figure 3 Enrichment analysis of the GO and KEGG pathways for core genes of RA.

[0013] Figure 4 The expression of UBE2A, NEMO, and NEMO ubiquitination in co-cultured cells.

[0014] Figure 5 Clinical trial results: expression of UBE2A, NEMO, NEMO ubiquitination and NF-κB in RA patients.

[0015] Figure 6 Clinical trial results: Expression of inflammation and coagulation factors in RA patients. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommended conditions.

[0017] Example 1

[0018] Screening for RA ubiquitination modification of the core gene UBE2A

[0019] 1. Collection and preprocessing of potential target gene data for RA

[0020] The dataset GSE89408 was downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), and logarithms were normalized using the R package "limma". The expression values ​​were then normalized using the "Combat" algorithm in the R package "SVA" to eliminate batch effects. The dataset contains synovial biopsy samples from 152 RA patients and 28 healthy individuals. Differentially expressed genes (DEGs) were identified using the R package "limma" with the selection criteria of p < 0.05 and |logFC| > 1.5. Finally, volcano plots were generated from the DEGs, and heatmaps were created using the "ggplot2" package to visually display the distribution of upregulated and downregulated genes. Clinical symptom-related genes and human phenotype ontology databases for RA-BS were collected from the SoFDA platform using "blood stasis syndrome" and "chronic musculoskeletal pain" as keywords for data retrieval.

[0021] 2. Gene set enrichment analysis (GSEA)

[0022] GSAE analysis was performed on DEGs from the GSE89408 dataset. GSEA was mainly performed using the R packages “ClusterProfiler” and “enrichplot”. These tools identified and evaluated KEGG pathway enrichment associated with core gene expression data in both samples (p < 0.05, FDR < 0.25, considered statistically significant).

[0023] 3. Construction of machine learning diagnostic models and selection of core targets

[0024] The KNN, RF, and LASSO machine learning algorithms were applied to further screen for core genes related to RA. The k-nearest neighbor (KNN) algorithm is a supervised machine learning algorithm primarily used for classification purposes. It has been widely used in disease prediction. KNN is a supervised algorithm that predicts the classification of unlabeled data by considering the features and labels of the training data. The RF algorithm utilizes ensemble learning to generate decision trees to evaluate feature genes. LASSO is a machine learning technique combining variable selection and regularization, implemented and executed using the "glmnet" package to identify pivot genes in the training set, using 10-fold cross-validation for selection and lambda with least binomial bias as the optimal value. Based on the diagnostic efficacy evaluation of the three algorithms, the optimal one was selected for subsequent research and validation.

[0025] 4. Enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

[0026] Duplicates in DEGs from the GSE89408 dataset related to RA were removed, followed by cross-analysis with RA-BS related targets to identify common targets, and then bioinformatics enrichment analysis was performed. GO and KEGG enrichment analyses were conducted by uploading common core targets to Metascape with the parameter set to "H species," and p < 0.05 defined as significant enrichment. The final results were filtered according to pathological and pharmacological significance and visualized as bar charts and Sankey diagrams, respectively.

[0027] The results show that:

[0028] Figure 1 This study aimed to predict ubiquitination modifications in renal retinoic acid (RA). Differentially expressed genes (DEGs) were identified using limma analysis, with selection criteria of p < 0.05 and |logFC| > 1.5. A total of 1371 upregulated genes and 996 downregulated genes were obtained. Figure 1 A). GSEA analysis showed that DEGs were significantly enriched in the ubiquitination pathway between the RA group and the normal group, suggesting that this pathway is closely related to the progression of RA. Figure 1B). The dot plot shows the top 20 significantly enriched pathways, among which the ubiquitin hydrolysis pathway (GeneRatio = 0.62, p < 0.05) is the most significant. Figure 1 C); the ridge map further shows that the genes in this pathway are generally upregulated (C); Figure 1 D). Gene-pathway network analysis revealed that pivotal genes such as UBE2A, UBE2E2, and UBE2L3 are involved in the ubiquitin hydrolysis pathway, suggesting that they may play a key role in the pathogenesis of RA. Figure 1 E and 1F).

[0029] Figure 2 UBE2A, a core gene for ubiquitination modification, was screened for machine learning. Further screening of core genes was conducted using KNN, RF, and LASSO machine learning algorithms. The absolute residual plot showed that the KNN model had the smallest prediction error. Figure 2 A); The comparison plot of model prediction error distribution shows that the model constructed based on differentially expressed genes (DEGs) screened by LASSO has the highest predictive consistency. Figure 2 B). The core DEGs obtained from the model selection are as follows: KNN models include B2M, RMRP, etc.; LASSO models include RMRP, EFNB1, etc.; RF models include HBB, IL1B, etc. Figure 2 C). Diagnostic efficacy assessment showed that the KNN model performed best (AUC = 0.997), while the LASSO (AUC = 0.988) and RF models (AUC = 0.986) showed robust performance. Figure 2 D). By taking the intersection of differentially expressed genes of GSE89408, genes related to RA blood stasis syndrome (RA-BS), genes screened by the KNN model, and genes related to the ubiquitination pathway, UBE2A was finally identified as the core gene. Figure 2 E).

[0030] Figure 3Enrichment analysis of GO and KEGG pathways for core genes in RA was performed. GO analysis identified leukocyte cell-cell adhesion, leukocyte migration, and positive regulation of cell-cell adhesion in biological processes (BP); cytoplasmic vesicle lumen, endopeptidase complex, and external side of plasma membrane in cellular components (CC); and chemokine activity, chemokine binding, and chemokine receptor binding in molecular functions (MF). KEGG enrichment analysis identified inflammation-related signaling pathways such as NF-κB, PI3K / AKT, and Th17. Figure 3 ).

[0031] Example 2

[0032] Expression of UBE2A, NEMO, and NEMO ubiquitination in co-cultured cells

[0033] 1. Extraction of peripheral blood mononuclear cells (PBMCs) from RA patients

[0034] Collect 5 mL of venous blood from RA patients using an EDTA anticoagulant tube. Mix the anticoagulant blood with PBS at a 1:1 ratio and gently mix. Slowly drop the diluted sample onto the Ficoll-Paque separation solution in a 15 mL centrifuge tube and centrifuge at 3000 rpm for 35 min. After centrifugation, the cells will separate into layers, from top to bottom: plasma layer, PBMC layer, Ficoll-Paque PLUS, and erythrocyte layer. Gently aspirate the PBMCs in the middle with a dropper. Add 3 times the amount of PBS, mix well, centrifuge at 1000 rpm for 10 min, and discard the supernatant. Repeat twice, resuspend the cells in 1 mL of PBS, transfer to a 1.5 mL EP tube, and store for later use.

[0035] 2. Isolation, identification, preparation and culture of RA-FLS

[0036] Tissue was taken from the knee joint of patients undergoing RA surgery and soaked in 75% alcohol for about 2 minutes. The tissue blocks were cut into squares with sides of approximately 0.1 cm and placed in 2 mL of complete fibroblast culture medium to infiltrate the tissue blocks. The tissue blocks were then placed in a 5% CO2 cell culture incubator. Once the cells growing around the tissue blocks had fused together, the cells were digested with trypsin. After the cells had spread, 50 μL of primary antibody was placed on a waterproof membrane and incubated with secondary antibody (secondary antibody:PBS = 1:500) at room temperature in the dark for 2 hours. The cells were then washed with PBS 3 times for 5 minutes each time, stained with DAPI (DAPI:PBS = 1:1000) for 5 minutes, and washed with PBS 3 times for 5 minutes each time. One drop of Fluoromount-G was placed on each slide, and the side with cells was placed on top to identify the cells as P1 generation cells. The cells were then seeded into 6-well plates, with approximately 1 × 10⁶ cells per well. 5 Fresh culture medium containing different concentrations of puromycin (1ug / mL, 2ug / mL, 3ug / mL, 4ug / mL, 5ug / mL, 6ug / mL, 7ug / mL) was added to 24-well plates already coated with cells. The lowest concentration was selected, and the result showed that the concentration of puromycin used was 1ug / mL. The RA-FLS immortalized cell line was completed. After the cells fused and the purity reached more than 95%, it was used for subsequent experiments.

[0037] 3. Co-culture of RA-PBMCs and RA-FLS cells (hereinafter referred to as co-cultured cells)

[0038] Take the prepared RA-FLS cells, digest them with 0.25% trypsin, and then pipette them into single suspension cells. Centrifuge at 1000 rpm for 15 min, add DMEM medium, and mix well. Adjust the RA-FLS cell count to the required number for the experiment, and add them to the lower chamber of a Transwell chamber. Take the prepared RA-PBMC cells, centrifuge them, and add them to the upper chamber of a Transwell chamber. Co-culture the RA-PBMC cells with the RA-FLS cells, and collect the co-cultured RA-FLS cells for subsequent experimental detection.

[0039] 4. Western blot (WB) analysis

[0040] Cell samples were collected and lysed with 1 mL of RIPA lysis buffer. Centrifuged at 10000×g for 10 min. The supernatant containing total protein was collected. Subsequently, using an SDS-PAGE gel preparation kit, 5X SDS-PAGE protein loading buffer was added to the collected supernatant at a ratio of 1:4, and the sample was heated in a boiling water bath to fully denature the proteins. After the sample cooled to room temperature, the protein sample was directly loaded into the wells of the SDS-PAGE gel. Pre-cut filter paper and PVDF membranes of the same size as the gel strips were soaked in methanol for 2-3 min, then immersed in transfer buffer for 5 min. After soaking, the protein membrane was rinsed in Western blotting buffer. The membrane was diluted with primary antibody dilution buffer according to the manufacturer's instructions, and then incubated with secondary antibody (horseradish peroxidase labeled) at a ratio of 1:2×10⁴. After washing three times, the protein content was determined using an ECL chemiluminescence assay kit according to the manufacturer's instructions.

[0041] 5. Co-Immunoprecipitation (Co-IP)

[0042] Co-cultured RA-FLS cells were resuspended and lysed on ice with IP buffer containing 1% protease inhibitor. The supernatant was harvested by centrifugation at 15000 g for 10 min (4 °C). The supernatant was then incubated overnight at 4 °C with anti-NEMO antibody and protein A / G magnetic beads. The beads were then washed three times with IP buffer. The ubiquitination of NEMO in the boiled IP samples was determined by Western blotting using anti-Ub antibody.

[0043] Experimental results show that:

[0044] Figure 4 The expression levels of UBE2A, NEMO, and NEMO ubiquitination in co-cultured cells were measured. Western blotting results showed that the protein expression levels of UBE2A and NEMO were significantly increased in the co-cultured cell group. Figure 4 A–C); COIP combined with WB detection revealed an increase in NEMO ubiquitination levels in the co-cultured cell group (A–C). Figure 4 (D and 4E).

[0045] Example 3

[0046] Observe the clinical relevance of UBE2A, NEMO, NEMO ubiquitination, NF-κB, inflammation, and coagulation cytokines.

[0047] In this embodiment, blood samples were collected from 30 patients with a confirmed diagnosis of RA and normal individuals. The concentrations of IL-6, TNF-α, IL-10, PAF, PAI-1, and PGI2 were measured using the corresponding ELISA kits according to the manufacturer's instructions.

[0048] Experimental results show that:

[0049] Figure 5 Clinical trial results: Expression of UBE2A, NEMO, NEMO ubiquitination, and NF-κB in RA patients. Western blotting showed that UBE2A and NEMO expression was elevated in the RA patient group compared to the control group. Figure 5 A and 5B); COIP combined with WB detection showed elevated NEMO ubiquitination levels in RA patients ( Figure 5 C and 5D). Western blotting also showed increased expression of p65 (nuclear), IKKα, p-IKKα, IKKβ, and p-IKKβ proteins, and decreased expression of p65 (plasma) protein in the RA patient group. Figure 5 E–5K).

[0050] Figure 6 Clinical trial results: Expression of inflammation and coagulation factors in RA patients. ELISA showed that IL-6, TNF-α, PAF, and PAI-1 levels were significantly elevated, while IL-10 and PGI2 levels were significantly decreased in the RA patient group. Figure 6 AF).

[0051] In summary, this invention comprehensively utilizes bioinformatics analysis, machine learning screening, cell co-culture models, and clinical sample validation to confirm that UBE2A is a key molecule in the pathogenesis of rheumatoid arthritis (RA) and suggests that it could serve as a novel target for RA diagnosis and treatment. Therefore, UBE2A and its related pathways can be used to prepare drugs for the treatment of rheumatoid arthritis, particularly those regulating inflammation and coagulation abnormalities.

Claims

1. The application of ubiquitination modification of a protein in rheumatic diseases associated with cellular inflammation and hypercoagulable state, characterized in that, The ubiquitin-binding enzyme UBE2A activates the NF-κB signaling pathway by mediating the ubiquitination of NEMO, thereby promoting the inflammatory response and hypercoagulable state in rheumatoid arthritis.

2. The application of a ubiquitin-binding enzyme, UBE2A, in a diagnostic agent for rheumatic diseases related to cellular inflammation and hypercoagulable states, characterized in that... Preparations for detecting UBE2A and NEMO proteins in peripheral blood mononuclear cells (PBMCs) can be used in the preparation of diagnostic agents for rheumatic diseases related to cellular inflammation and hypercoagulable states.

3. The application as described in claim 2, characterized in that, Increased expression of UBE2A and NEMO proteins, increased NEMO ubiquitination levels, activation of the NF-κB pathway, and elevated levels of pro-inflammatory and procoagulant cytokines promote the inflammatory response and hypercoagulable state in rheumatoid arthritis.

4. The application of a ubiquitin-binding enzyme, UBE2A, in a therapeutic agent for rheumatic diseases associated with cellular inflammation and hypercoagulable states, characterized in that... Preparations that inhibit the expression of ubiquitin-binding enzyme UBE2A can be used in the preparation of therapeutic agents for rheumatic diseases related to cellular inflammation and hypercoagulable states.

5. The application as described in claim 4, characterized in that, Inhibiting the expression of ubiquitin-binding enzyme UBE2A can reduce NEMO ubiquitination and further suppress the inflammatory response and hypercoagulable state of rheumatoid arthritis.

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