Biomarkers for ulcerative colitis and its associated colorectal cancer and uses thereof

Abstract

The application discloses biomarkers of ulcerative colitis and its related colorectal cancer and application thereof, and belongs to the field of molecular biology technology and medicine. The application discloses the role of E3 ubiquitin ligase TRIM50 in ulcerative colitis and its related colorectal cancer. The key regulation role of TRIM50 in UC and UC-CRC is found and verified for the first time, and the potential of TRIM50 as a novel auxiliary detection biomarker and treatment target is determined. The expression level of TRIM50 can be used as a specific biomarker of UC and UC-CRC, and is applied to early screening, disease assessment or prognosis judgment of the disease; and targeted regulation of the expression or activity of TRIM50 is expected to become a new strategy for inhibiting the malignant progression of UC-CRC, and provides a solid theoretical basis and screening direction for the development of innovative drugs.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and medicine, specifically relating to biomarkers for ulcerative colitis and related colorectal cancer and their applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Ulcerative colitis (UC) is a chronic, relapsing, nonspecific inflammatory bowel disease (IBD) that primarily affects the mucosa and submucosa of the colon and rectum. Its pathological features include persistent inflammation and diffuse ulceration. The pathogenesis of UC is complex, involving multi-dimensional interactions of genetic susceptibility, gut microbiota dysbiosis, immune system abnormalities, and environmental factors. Notably, long-term uncontrolled inflammation can lead to genomic instability and epigenetic alterations in intestinal epithelial cells, ultimately promoting the development of colorectal cancer (CRC). UC patients have a significantly higher risk of developing colorectal cancer (CRC) than the general population, and this risk increases exponentially with disease progression. Currently, clinical prediction and screening for UC-CRC have significant limitations.

[0004] In recent years, research on S100A8 as an inflammation-related molecule in ulcerative colitis (UC) and its derivative CRC has deepened. In the association between chronic inflammation and cancer, the aberrant release of damage-related molecular patterns is a key driving factor, and S100A8, as a typical member of the calcium-binding EF-hand family of proteins, has been proven to be one of the important DAMPs in the intestinal mucosal injury microenvironment. Fecal calprotectin (CP) concentration is an important indicator for assessing intestinal inflammatory activity, but traditional detection methods mainly target the heterotetrameric structure of CP (a heterotetramer composed of S100A8 and S100A9). Studies have reported the presence of homodimers of S100A8 (or S100A9) in the feces of patients with active inflammatory bowel disease, in addition to calprotectin. Recent research shows that the homodimer of human S100A8 (or S100A9), rather than the heterotetramer calprotectin, is a driving factor for colitis. The detection of homodimers of S100A8 (or S100A9) is associated with clinical and endoscopic disease activity in IBD and can serve as a biomarker for inflammatory bowel disease.

[0005] Previous studies have reported that S100A8 not only serves as a potential biomarker for inflammatory activity in ulcerative colitis (UC), but its interaction with prostatic inflammatory markers (PRRs) may also be a target for intervening in inflammatory progression. Furthermore, in the context of colitis-associated chronic rheumatoid arthritis (CRC), the expression level of S100A8 is closely related to tumor progression and histopathological damage. Notably, S100A8 has a dual role in the tumor microenvironment: on the one hand, elevated intracellular S100A8 levels in tumor cells are directly associated with poor prognosis; on the other hand, an increase in S100A8-positive cells in the tumor stroma indirectly inhibits endometrial metastasis (EMT) and metastasis. This feedback regulatory mechanism suggests that targeting the dynamic expression of S100A8 and its interaction with tumor cells and stromal cells may provide a theoretical basis for developing more precise treatment strategies. However, existing research is mostly focused on basic experimental levels, and further in-depth clinical translational research is needed to explore S100A8 as a biomarker or therapeutic target for UC-CRC.

[0006] TRIM50, a member of the TRIM family, plays a crucial role in E3 ubiquitin ligase-mediated ubiquitination of target proteins, thus playing a vital role in protein degradation, cell signaling, and immune responses. Current technologies do not disclose the application of TRIM50 in ulcerative colitis (UC) and its derivative CRC, and its specific function remains unclear. Further research is needed to explore the regulatory targets and functional patterns of TRIM50 in the pathological characteristics of UC-CRC (such as long-term chronic inflammation, specific gene mutations, and dynamic changes in the immune microenvironment). Furthermore, whether the expression level of TRIM50 in UC-CRC is related to disease severity or prognosis, and its feasibility as a predictive or therapeutic target, all require validation and functional experiments.

[0007] In summary, existing monitoring methods for UC-CRC have significant shortcomings in terms of large-scale application and early detection capabilities. Research on potential screening indicators for this specific subtype of UC-CRC is still insufficient. Therefore, in-depth research and exploration of the application of TRIM50 in UC and related cancers has important clinical value and social significance. Summary of the Invention

[0008] To address the lack of effective early detection, prognostic assessment, and therapeutic targets for ulcerative colitis and related colorectal cancer in existing technologies, this invention provides biomarkers for ulcerative colitis and related colorectal cancer and their applications. Specifically, it discloses the application of an E3 ubiquitin ligase TRIM50 in the preparation of diagnostic and therapeutic products for ulcerative colitis and related colorectal cancer.

[0009] The significant increase in TRIM50 expression in patients suggests a high risk of disease, and understanding the interaction between S100A8 and TRIM50 is of great clinical and social value for developing a highly sensitive and specific early auxiliary detection model for UC-CRC applicable to high-risk populations.

[0010] This invention discloses the role of the E3 ubiquitin ligase TRIM50 in ulcerative colitis and its associated colorectal cancer. The key regulatory role of TRIM50 in ulcerative colitis (UC) and UC-CRC is discovered and validated for the first time, clarifying its potential as a novel auxiliary detection biomarker and therapeutic target. The gene sequence of TRIM50 is shown in Sequence: NM_178125.3, and the gene sequence of S100A8 is shown in Sequence: NM_002964.5.

[0011] Clinical sample analysis showed that the expression level of TRIM50 in UC patient tissues was significantly increased and positively correlated with disease severity. ROC curve analysis also showed that TRIM50 could serve as a validation indicator to distinguish between normal and patients. Further investigation using mouse and colon cancer cell models confirmed that TRIM50 promotes disease progression and significantly enhances malignant phenotypes such as cancer cell proliferation, colony formation, and invasion / metastasis. Mechanistic studies identified TRIM50 as a direct interacting protein of S100A8, with co-localization and a stronger binding effect after DSS induction. Treatment with S100A8 inhibitors weakened WT and... Trim50- / - The disease differences among mice indicate that TRIM50 regulates disease progression by modulating S100A8, and the experiment found a positive feedback axis of TRIM50-S100A8, which further exacerbates disease formation.

[0012] Based on the above findings, this invention provides a TRIM50-based auxiliary detection and treatment strategy: on the one hand, the expression level of TRIM50 can serve as a specific biomarker for UC and UC-CRC, and can be applied to early screening, disease assessment, or prognosis; on the other hand, targeted regulation of TRIM50 expression or activity is expected to become a new strategy to inhibit the malignant progression of UC-CRC, providing a solid theoretical basis and screening direction for the development of innovative drugs.

[0013] The technical solution of this invention provides novel molecular targets and tools for the auxiliary screening and treatment of UC and UC-CRC, which have significant clinical value and industrial transformation potential.

[0014] To achieve the above objectives, the technical solution of the present invention is as follows:

[0015] In a first aspect, the present invention provides the use of biomarkers for ulcerative colitis and its associated colorectal cancer in the preparation of products for the diagnosis, prevention, treatment and / or screening of ulcerative colitis and its associated colorectal cancer, wherein the biomarkers are TRIM50 or its encoded protein, or substances thereof that specifically bind to the molecule S100A8.

[0016] Secondly, the present invention provides the use of a substance for detecting TRIM50 or its encoded protein, or its specific binding molecule S100A8, in the preparation of any one or more of the following products:

[0017] a1) Diagnostic or auxiliary diagnostic products for ulcerative colitis and related colorectal cancer;

[0018] a2) Products for the prevention and / or treatment of ulcerative colitis and related colorectal cancer;

[0019] a3) Prognostic assessment / evaluation of ulcerative colitis and its associated colorectal cancer, or products that assist in prognostic assessment / evaluation.

[0020] The detection substance may include the detection reagent. The reagent is used to detect the expression level or protein activity of the TRIM50 gene in the sample.

[0021] The samples were selected from blood, serum, plasma, tissue biopsy samples, or fecal samples.

[0022] The detection includes detecting the mRNA expression level, protein expression level, or protein ubiquitination modification level of TRIM50.

[0023] The products include primers, probes, nucleic acid membrane strips, or kits for detecting the TRIM50 expression level, protein expression level, or protein ubiquitination modification level in the sample to be tested.

[0024] Thirdly, the present invention provides a product comprising the aforementioned substance for detecting TRIM50 or its encoded protein, or its specific binding molecule S100A8; the product having any one or more of the following uses:

[0025] a1) Diagnosis or auxiliary diagnosis of ulcerative colitis and its associated colorectal cancer;

[0026] a2) Prevention and / or treatment of ulcerative colitis and its associated colorectal cancer;

[0027] a3) Prognostic assessment / evaluation of ulcerative colitis and its associated colorectal cancer, or auxiliary prognostic assessment / evaluation.

[0028] The products include primers, probes, chips, nucleic acid membrane strips, formulations, or kits for detecting the expression level of TRIM50 in the sample to be tested.

[0029] Fourthly, the present invention provides a system for prognostic assessment or auxiliary prognostic assessment of ulcerative colitis and its associated colorectal cancer, the system comprising:

[0030] b1) An analysis unit, the analysis unit comprising: a detection substance selected from the TRIM50 expression level in the test sample of a subject;

[0031] b2) Assessment unit, the assessment unit comprising: prognostic assessment of the subject based on the TRIM50 expression level determined in b1).

[0032] Fifthly, the present invention provides a system for the diagnosis or auxiliary diagnosis of ulcerative colitis and related colorectal cancer, the system comprising:

[0033] (1) Collect samples from the subjects to be tested and collect control samples;

[0034] (2) Detect and compare the expression levels of TRIM50 in the test subject samples and control samples;

[0035] If the expression level of TRIM50 in the sample of the subject being tested is higher than the expression level of TRIM50 in the control sample, the subject being tested is diagnosed with ulcerative colitis and its associated colorectal cancer or is at risk of having ulcerative colitis and its associated colorectal cancer.

[0036] In a sixth aspect, the present invention provides a method for screening drugs for ulcerative colitis and related colorectal cancer, comprising:

[0037] c1) The system expressing and / or containing the TRIM50 was treated with the candidate substance; a parallel control was set up without the candidate substance treatment;

[0038] c2) After completing step c1), detect the expression level of TRIM50 in the system; if the expression level of TRIM50 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for ulcerative colitis and its related colorectal cancer.

[0039] In a seventh aspect, the present invention provides the application of substances that inhibit TRIM50 expression in the preparation of products;

[0040] The product has any one or more of the following functions:

[0041] d1) Inhibits the proliferation of ulcerative colitis and related colorectal cancer cells;

[0042] d2) Inhibits the malignant invasion of ulcerative colitis and its associated colorectal cancer cells;

[0043] d3) Promotes apoptosis in ulcerative colitis and related colorectal cancer cells;

[0044] d4) Inhibits the growth of ulcerative colitis and its associated colorectal cancer;

[0045] d5) Inhibits ulcerative colitis and its associated colorectal cancer metastasis;

[0046] d6) Treatment of ulcerative colitis and its associated colorectal cancer.

[0047] Substances that reduce TRIM50 expression levels include RNA interference molecules or antisense oligonucleotides targeting TRIM50, small molecule inhibitors, siRNA, and substances that induce lentiviral infection or gene knockout.

[0048] The products include pharmaceuticals.

[0049] Eighthly, the present invention provides a product whose active ingredient includes a substance for inhibiting TRIM50 expression levels;

[0050] The product has any one or more of the following functions:

[0051] d1) Inhibits the proliferation of ulcerative colitis and related colorectal cancer cells;

[0052] d2) Inhibits the malignant invasion of ulcerative colitis and its associated colorectal cancer cells;

[0053] d3) Promotes apoptosis in ulcerative colitis and related colorectal cancer cells;

[0054] d4) Inhibits the growth of ulcerative colitis and its associated colorectal cancer;

[0055] d5) Inhibits ulcerative colitis and its associated colorectal cancer metastasis;

[0056] d6) Treatment of ulcerative colitis and related colorectal cancer;

[0057] The products include pharmaceuticals.

[0058] In a ninth aspect, the present invention provides a method for diagnosis, prevention, treatment and / or prognosis assessment, by applying the above-described product to a subject.

[0059] One or more of the above technical solutions have the following advantages or beneficial effects:

[0060] 1. TRIM50 shows promise as a highly specific biomarker for UC and UC-CRC.

[0061] Experimental verification by this invention showed that TRIM50 expression levels were significantly upregulated in UC patient tissues, mouse UC models, and UC-related colorectal cancer (UC-CRC) models, and its expression intensity was positively correlated with disease severity. In analyses of normal individuals and UC patients, the area under the ROC curve was 0.708, which can serve as a validation indicator to distinguish between normal individuals and patients. More notably, the expression level of TRIM50 in the healed tissues of recovered patients was significantly lower than that in inflamed tissues. This dynamic change provides crucial evidence for monitoring disease activity and assessing prognosis. The specific expression pattern of TRIM50 fills the gaps in sensitivity and dynamic monitoring of existing UC-CRC risk-associated biomarkers, demonstrating significant clinical value.

[0062] 2. Revealing the regulatory role of the TRIM50-S100A8 positive feedback shaft.

[0063] This invention elucidates for the first time that TRIM50, as an E3 ubiquitin ligase, dually regulates the stability of the S100A8 protein through ubiquitination and oligomerization. This dual modification mechanism of TRIM50 not only directly affects the functional state of S100A8 but also forms a positive feedback loop between TRIM50 and S100A8, thus playing a core regulatory role in the inflammation-driven transformation of UC to CRC. This discovery provides novel molecular-level theoretical support for the study of the pathological mechanisms of UC-CRC.

[0064] 3. Dual potential as a risk-associated biomarker and a treatment

[0065] (1) Application of biomarkers: The expression level of TRIM50 can be used as a specific biomarker for early screening, disease assessment and prognosis of UC and UC-CRC.

[0066] (2) Therapeutic applications: Based on the core regulatory role of TRIM50 in the disease process, this invention lays the theoretical foundation for developing novel targeted therapies against TRIM50 or its downstream pathways (such as S100A8). By regulating the expression or activity of TRIM50, the malignant progression of UC-CRC can be effectively intervened.

[0067] In summary, this invention not only provides specific, dynamically monitorable risk-associated biomarkers for the malignant progression of UC and UC-CRC, but also reveals the key role of TRIM50 in disease translation at the molecular mechanism level, and provides innovative directions for the development of therapeutic targets, thus having important clinical translational prospects. Attached Figure Description

[0068] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0069] Figure 1 Figures show the results of immunoprecipitation and laser confocal microscopy analysis of the interaction between TRIM50 and S100A8. A represents the interaction between exogenous TRIM50 and S100A8 in 293T cells detected by immunoprecipitation; B represents the binding of exogenous TRIM50 and S100A8 in HEK29T cells detected by laser confocal microscopy (scale bar 5 μm); C represents the fluorescence colocalization relationship of TRIM50 and S100A8 in figure B; D represents the binding of endogenous TRIM50 and S100A8 in PM cells detected by laser confocal microscopy (scale bar 5 μm); and E represents the fluorescence colocalization relationship of TRIM50 and S100A8 in figure D.

[0070] Figure 2 The images show the spatial molecular structures of TRIM50 and S100A8, as well as the results of immunoprecipitation and in vitro protein transcription and translation systems. A shows molecular docking demonstrating the potential spatial binding of TRIM50 and S100A8; B shows immunoprecipitation demonstrating the domain binding of S100A8 and TRIM50; and C shows the direct binding of TRIM50 and S100A8 using in vitro protein transcription and translation systems.

[0071] Figure 3 TRIM50 is highly expressed in UC patients and is associated with disease activity. A shows the immunohistochemical results from colonoscopy (scale bar 20 μm for both normal and UC patients during IHC), B shows TRIM50 expression in normal and UC patients, C shows TRIM50 expression in UC patients as shown by Western blotting, D shows TRIM50 protein expression analysis, E shows TRIM50 expression in UC patients with different disease severities detected by immunohistochemical methods (scale bar 20 μm), F shows statistical analysis of TRIM50 expression in normal, mild / moderate, and severe patients, G shows the correlation between TRIM50 and UCEIS scores in UC patients, and H shows the ROC curve of univariate logistic regression analysis using TRIM50 as a validation factor for distinguishing between normal and diseased patients.

[0072] Figure 4 WT after DSS induction and Trim50- / - Data on the severity of UC in mice; where A represents the WT and WT after DSS induction. Trim50- / - The colon length phenotype diagram of mice (WT+DSS refers to WT after DSS induction) Trim50- / - +DSS refers to DSS induction after... Trim50- / -B is a bar chart showing the colon length of WT and Trim50- / - mice after DSS induction, and C is a bar chart showing the colon length of WT and Trim50- / - mice after DSS induction. Trim50- / - Weight loss in mice, D represents the statistical analysis of WT and weight loss after DSS induction. Trim50- / - Intestinal permeability in mice. E shows HE-stained sections of the colon of mice treated with the S100A8 inhibitor (scale bar 100 μm). F shows the statistical analysis of WT and WT after treatment with the S100A8 inhibitor. Trim50- / - Changes in mouse body weight, G represents statistical analysis after using S100A8 inhibitors. Trim50- / - Changes in colon length in mice;

[0073] Figure 5 This study aims to establish the positive feedback loop of the TRIM50-S100A8 axis. Specifically, A represents the expression of TRIM50 and S100A8 in bone marrow neutrophils, spleen neutrophils, and bone marrow-derived macrophages (BMDM) after DSS induction, as detected by Western blotting (WB); B represents the protein stability of TRIM50 against S100A8 as detected by the CHX assay; and C represents the mRNA and protein expression levels of TRIM50 after stimulation with recombinant S100A8 protein in peritoneal macrophages (PM) and BMDM, as detected by qPCR and WB.

[0074] Figure 6 TRIM50 ubiquitinates S100A8; where A represents the ubiquitination modification of S100A8 by TRIM50 detected by Western blotting, B represents the ubiquitination modification of S100A8 by TRIM50 and its enzyme activity-deficient mutant detected by Western blotting, C represents the direct ubiquitination modification of S100A8 by TRIM50 in vitro detected by Western blotting, and D represents the specific ubiquitination modification type of S100A8 by TRIM50 detected by Western blotting.

[0075] Figure 7 The results show that TRIM50 oligomerizes and stabilizes the S100A8 protein. Figure A shows the oligomerization effect of TRIM50 on S100A8 as detected by SDD-AGE; Figure B shows the ubiquitination effect of TRIM50 on S100A8 and its ubiquitination site deletion mutant as detected by Western blotting; Figure C shows the oligomerization effect of TRIM50 on S100A8 and its ubiquitination site deletion mutant as detected by Western blotting; Figure D shows the protein stability of S100A8 and its ubiquitination site deletion mutant as detected by CHX assay; and Figure E shows the protein quantification analysis of S100A8 at different time points in Figure D.

[0076] Figure 8The TRIM50-S100A8 axis has a prognosis assessment effect; where A represents the immunohistochemical results of TRIM50 and S100A8 in UC patients with different disease severities (scale bar 20μm), B represents the expression of TRIM50 and S100A8 in UC patients with different disease severities, C represents the colonic immunohistochemical results of TRIM50 and S100A8 in UC patients with a prognosis (scale bar 20μm), D represents the expression of TRIM50 and S100A8 in UC patients with a prognosis, E represents the expression of TRIM50, S100A8, and β-acin in normal, inflamed, and prognostic patient tissues, F represents the endoscopic images of normal, inflamed, and prognostic patients, G represents the protein expression level of TRIM50 in normal, inflamed, and prognostic patient tissues, and H represents the protein expression level of S100A8 in normal, inflamed, and prognostic patient tissues.

[0077] Figure 9 The results of constructing a mouse colon cancer model are shown in the figure below. A represents the drug treatment cycle, B shows HE-stained sections of the mouse colon after inducing the CRC model with AOM+DSS (scale bar: 100μm), C shows the expression of TRIM50 in wild-type CRC mice by immunohistochemistry (scale bar: 100μm), and D shows the corresponding data of the expression in the figure below.

[0078] Figure 10 This image shows changes in colon length in CRC model mice and the expression of TRIM50 and S100A8 in cancerous and adjacent tissues; where A represents WT and Trim50- / - The graph shows the changes in colon length in CRC mice. B represents the corresponding data for the changes in colon length in graph A. C represents the expression of TRIM50 and S100A8 in CRC cancer tissue and adjacent normal tissue microarray detected by immunohistochemistry (scale bar is 50μm). D represents the corresponding data for the quantitative statistical analysis of TRIM50 expression in graph C. E represents the corresponding data for the quantitative statistical analysis of S100A8 expression in graph C.

[0079] Figure 11 The results show the clonogenic and migration abilities of colon cancer cells. Specifically, A represents the clonogenic ability of colon cancer cells transfected with TRIM50 and its enzyme-deficient mutants as detected by a clonogenic assay; B represents the statistical analysis of the HT29 cell count corresponding to Figure A; C represents the statistical analysis of the HCT116 cell count corresponding to Figure A; D represents the migration ability of colon cancer cells transfected with TRIM50 and its enzyme-deficient mutants as detected by a Transwell assay (scale bar at 50 μm); E represents the statistical analysis of the invasive HT29 cell count corresponding to Figure C; and F represents the statistical analysis of the invasive HCT116 cell count corresponding to Figure C. Detailed Implementation

[0080] Explanation of terms involved:

[0081] Ulcerative colitis: UC. Ulcerative colitis and its associated colorectal cancer: UC-CRC. Flag-TRIM50: TRIM50 plasmid with the Flag tag (other tags and mutants are similar). HA-S100A8: S100A8 plasmid with the HA tag (other tags and mutants are similar). All other molecules are the same. UC patients: UC patients. Ubiquitin (UB): ubiquitin. UBCH5α: E2-binding enzyme. Healed: Healed tissue (patient tissue). HT29 / HCT116 cells: colon cancer cell line.

[0082] Paquinimod is a specific inhibitor of S100A8 with oral activity.

[0083] WT+DSS: WT induced by DSS. Trim50- / - +DSS: DSS-induced Trim50- / - WT+DSS+paquinimod: WT induced by DSS after using an S100A8 inhibitor. Trim50- / - +DSS+paquinimod: DSS induction after administration of S100A8 inhibitor Trim50- / - .

[0084] IP:HA: This generally refers to the immunoprecipitation of plasmids with the HA tag in the experiment. For example, if multiple plasmids are transfected into cells, and one of them is HA-S100A8, IP:HA means using an HA antibody to extract HA-S100A8 and detect any modifications or other conditions on the molecule.

[0085] IOD (Integrated Optical Density) is a display value used to quantify and describe the results of immunohistochemistry.

[0086] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0087] The terms "indicator" and "signature" are used interchangeably in this invention and refer to a sign or signal of a symptom or for monitoring a symptom. Such a "symptom" refers to the biological state of a cell, tissue, or organ, or to the health and / or disease state of an individual. An indicator can be the presence or absence of molecules, including but not limited to peptides, proteins, and nucleic acids, or a change in the expression level or pattern of such molecules in a cell, tissue, organ, or individual. An indicator can be a sign of the occurrence, development, or presence of a disease in an individual, or a sign of further progression of such a disease. An indicator can also be a sign of the risk of developing a disease in an individual.

[0088] The terms "upgraded," "increased," or "enhanced" for an indicator level mean that the level of such an indicator in the sample is lower than that in a reference or reference sample. The terms "downgraded," "decreased," or "dropped" for an indicator level mean that the level of such an indicator in the sample is lower than that in a reference or reference sample.

[0089] In one typical embodiment, the present invention provides the use of biomarkers for ulcerative colitis and its associated colorectal cancer in the preparation of products for the diagnosis, prevention, treatment and / or screening of ulcerative colitis and its associated colorectal cancer, wherein the biomarkers are TRIM50 or its encoded protein, or substances thereof that specifically bind to the molecule S100A8.

[0090] The products include pharmaceuticals.

[0091] In one typical embodiment, the present invention provides the use of a substance for detecting TRIM50 or its encoded protein, or its specific binding molecule S100A8, in the preparation of any one or more of the following products:

[0092] a1) Diagnostic or auxiliary diagnostic products for ulcerative colitis and related colorectal cancer;

[0093] a2) Products for the prevention and / or treatment of ulcerative colitis and related colorectal cancer;

[0094] a3) Prognostic assessment or adjunctive prognostic assessment products for ulcerative colitis and its associated colorectal cancer.

[0095] Furthermore, TRIM50 can be used as a biomarker as an auxiliary indicator for ulcerative colitis (UC) and / or ulcerative colitis-associated colorectal cancer (UC-CRC). The sequence of TRIM50 is shown in Sequence: NM_178125.3. This reagent is used to detect the expression level or protein activity of the TRIM50 gene in a sample.

[0096] The diagnostic or auxiliary diagnostic products include those that screen for potential indicators.

[0097] The detection substance may include the detection reagent. The reagent is used to detect the expression level or protein activity of the TRIM50 gene in the sample.

[0098] The samples were selected from blood, serum, plasma, tissue biopsy samples, or stool samples. The assays included detecting the mRNA expression level, protein expression level, or protein ubiquitination modification level of TRIM50.

[0099] The products include primers, probes, nucleic acid membrane strips, or kits for detecting the TRIM50 expression level, protein expression level, or protein ubiquitination modification level in the sample to be tested.

[0100] The detection methods include reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative PCR (qRT-PCR), immunohistochemistry (IHC), Western blotting, enzyme-linked immunosorbent assay (ELISA), or high-throughput sequencing.

[0101] In one typical embodiment, the present invention provides a product comprising the substances described above for detecting TRIM50 or its encoded protein, or its specific binding molecule S100A8; the product has any one or more of the following uses:

[0102] a1) Diagnosis or auxiliary diagnosis of ulcerative colitis and its associated colorectal cancer;

[0103] a2) Prevention and / or treatment of ulcerative colitis and its associated colorectal cancer;

[0104] a3) Prognostic assessment / evaluation of ulcerative colitis and its associated colorectal cancer, or auxiliary prognostic assessment / evaluation.

[0105] Used to assess the risk of UC patients progressing to CRC or for prognosis.

[0106] The product contains specific primers, probes, or antibodies for detecting TRIM50 gene expression levels or protein activity.

[0107] This invention reveals a novel mechanism by which TRIM50 drives disease progression by ubiquitinizing the S100A8 protein to form a positive feedback axis, and provides a therapeutic target for intervening in this pathway.

[0108] The TRIM50 was upregulated in patients with UC and CRC.

[0109] In another specific embodiment of the present invention, the substances for detecting TRIM50 include, but are not limited to, substances used for detecting the expression level of TRIM50 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

[0110] In another specific embodiment of the present invention, the product includes, but is not limited to, primers, probes, chips, nucleic acid membrane strips, formulations, or kits for detecting the TRIM50 expression level in a sample to be tested. Preferably, the formulation or kit contains a reaction reagent for detecting the TRIM50 expression level. Preferably, the formulation or kit is used in the preparation of products that serve as risk markers for ulcerative colitis and colorectal cancer.

[0111] In another specific embodiment of the present invention, the sample to be tested may be a human sample.

[0112] In one typical embodiment, the present invention provides a system for prognostic assessment or auxiliary prognostic assessment of ulcerative colitis and its associated colorectal cancer, the system comprising: b1) an analysis unit comprising: a detection substance selected from the TRIM50 expression level in a test sample of a subject; and b2) an assessment unit comprising: performing a prognostic assessment of the subject based on the TRIM50 expression level determined in b1).

[0113] In one typical embodiment, the present invention provides a system for the diagnosis or auxiliary diagnosis of ulcerative colitis and its associated colorectal cancer, the system comprising the following steps: (1) collecting samples from a subject to be tested and collecting control samples; (2) detecting and comparing the expression levels of TRIM50 in the subject to be tested and the control samples; if the expression level of TRIM50 in the subject to be tested is higher than that in the control samples, the subject to be tested is diagnosed with ulcerative colitis and its associated colorectal cancer or at risk of having ulcerative colitis and its associated colorectal cancer.

[0114] The control samples are derived from healthy individuals or healthy tissues of the subjects to be tested. The samples from the subjects to be tested are one or more of the following: serum, plasma, whole blood, pus, organs, biopsy samples, circulating tumor cells, circulating tumor DNA, or exfoliated cells from urine.

[0115] In another specific embodiment of the present invention, the ulcerative colitis and related colorectal cancer drugs are drugs for prevention and / or treatment.

[0116] In one typical embodiment, the present invention provides a method for screening drugs for ulcerative colitis and related colorectal cancer, comprising:

[0117] c1) The system expressing and / or containing the TRIM50 was treated with the candidate substance; a parallel control was set up without the candidate substance treatment;

[0118] c2) After completing step c1), detect the expression level of TRIM50 in the system; if the expression level of TRIM50 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for ulcerative colitis and its related colorectal cancer.

[0119] If the candidate substance can downregulate the expression or activity of TRIM50, it indicates that it is a potential preventive or therapeutic agent.

[0120] In another specific embodiment of the present invention, the system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0121] In another specific embodiment of the present invention, the tissue in the tissue system can be ulcerative colitis and related colorectal cancer tissue;

[0122] In another specific embodiment of the present invention, the animals in the animal system can be mammals, such as rats, mice, guinea pigs, rabbits, monkeys, humans, etc.

[0123] In one typical embodiment, the present invention provides the use of a substance that inhibits the expression of TRIM50 in the preparation of a product;

[0124] The product has any one or more of the following functions:

[0125] d1) Inhibits the proliferation of ulcerative colitis and related colorectal cancer cells;

[0126] d2) Inhibits the malignant invasion of ulcerative colitis and its associated colorectal cancer cells;

[0127] d3) Promotes apoptosis in ulcerative colitis and related colorectal cancer cells;

[0128] d4) Inhibits the growth of ulcerative colitis and its associated colorectal cancer;

[0129] d5) Inhibits ulcerative colitis and its associated colorectal cancer metastasis;

[0130] d6) Treatment of ulcerative colitis and its associated colorectal cancer.

[0131] Among them, substances that reduce TRIM50 expression levels include RNA interference molecules or antisense oligonucleotides targeting TRIM50, small molecule inhibitors, siRNA, and substances that induce lentiviral infection or gene knockout.

[0132] The product may be a drug.

[0133] In one typical embodiment, the present invention provides a product whose active ingredient includes a substance for inhibiting TRIM50 expression levels.

[0134] The product has any one or more of the following functions:

[0135] d1) Inhibits the proliferation of ulcerative colitis and related colorectal cancer cells;

[0136] d2) Inhibits the malignant invasion of ulcerative colitis and its associated colorectal cancer cells;

[0137] d3) Promotes apoptosis in ulcerative colitis and related colorectal cancer cells;

[0138] d4) Inhibits the growth of ulcerative colitis and its associated colorectal cancer;

[0139] d5) Inhibits ulcerative colitis and its associated colorectal cancer metastasis;

[0140] d6) Treatment of ulcerative colitis and its associated colorectal cancer.

[0141] Substances that reduce TRIM50 expression levels include RNA interference molecules or antisense oligonucleotides targeting TRIM50, small molecule inhibitors, siRNA, and substances used for lentiviral infection or gene knockout. These products can be pharmaceuticals.

[0142] In this invention, knocking out TRIM50 can inhibit the disease progression of ulcerative colitis (UC) and chronic myeloid fibrosis (CRC). Molecularly, TRIM50 promotes UC development by directly binding to S100A8 and ubiquitinizing it, subsequently oligomerizing and stabilizing its function. Exogenous overexpression of TRIM50 in colon cancer cell lines also promotes cancer cell formation and invasion. In summary, both animal and cell experiments demonstrate that TRIM50 can enhance the progression of UC and the development of CRC.

[0143] According to the present invention, the concept of "treatment" means any measure applicable to the treatment of ulcerative colitis and its associated colorectal cancer, or preventive treatment of such disease or its symptoms, or prevention of recurrence of such disease, such as recurrence after the end of a treatment period or treatment of symptoms of an already occurring disease, or preemptive intervention to prevent, suppress or reduce the occurrence of such disease or symptoms.

[0144] According to the present invention, the above-mentioned drug further includes at least one inactive pharmaceutical ingredient. The inactive pharmaceutical ingredient may be a carrier, excipient, or diluent commonly used in pharmaceuticals. Furthermore, it can be formulated into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays, in the form of oral, topical, suppository, and sterile injectable solutions, according to conventional methods. The inactive pharmaceutical ingredients such as carriers, excipients, and diluents that may be included are well known in the art, and those skilled in the art can determine that they meet clinical standards. The carriers, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0145] In a typical embodiment, the drug of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as target cells, biological type or tissue, the general condition of the subject to be treated, route of administration, manner of administration, etc. The drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0146] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0147] Example 1: Collection of clinical specimens from UC patients

[0148] This invention collected 101 clinical tissue specimens from Qilu Hospital of Shandong University. The clinical characteristics of the patients are shown in Table 1.

[0149] Table 1 Clinical UC cases and their clinical characteristics

[0150]

[0151] Note: The "#" in Table 1 refers to the specific number of records.

[0152] Example 2: Immunohistochemical detection of TRIM50 expression in patient tissues

[0153] All paraffin tissue sections from the patients were collected from Qilu Hospital of Shandong University.

[0154] 1) Dewaxing and hydration: The paraffin sections were placed in fresh xylene and soaked for 10 min × 3 times; after removing excess liquid, they were placed in anhydrous ethanol and soaked for 3 min × 3 times; after removing excess liquid, they were placed in 95% ethanol and soaked for 3 min × 2 times; after removing excess liquid, they were placed in 75% ethanol and soaked for 3 min × 2 times; rinsed with distilled water for 1 min and placed in PBS buffer.

[0155] 2) Antigen retrieval: Boil the prepared citrate buffer in a pressure cooker, place the slides on a slide rack, put in the buffer, and boil again for 5 minutes.

[0156] 3) Blocking endogenous peroxidase: Add an appropriate amount of endogenous peroxidase blocking agent and incubate at room temperature for 10 min; wash with PBS buffer for 3 min × 3 times.

[0157] 4) Add primary antibody: Add an appropriate amount of primary antibody (about 100 μL) according to the size of the tissue, incubate at 37°C for 60 min; rinse with PBS buffer for 3 min × 3 times.

[0158] 5) Add reaction enhancement solution: Add 100 μL or an appropriate amount of reaction enhancement solution, incubate at 37°C for 20 min; wash with PBS buffer for 3 min × 3 times.

[0159] 6) Add enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer: Add 100 μL or an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer, incubate at 37°C for 20 min; wash with PBS buffer for 3 min × 3 times.

[0160] 7) DAB color development: Add an appropriate amount of freshly prepared DAB or AEC color development solution and incubate at room temperature for 5-8 minutes.

[0161] 8) Counterstaining: Rinse with tap water, incubate with hematoxylin staining solution for 20 s; differentiate, rinse and return to blue.

[0162] 9) Dehydrated, transparent, neutral resin sealing film.

[0163] 10) Determination of film review results.

[0164] Example 3: Western blot detection of TRIM50 expression in UC tissues

[0165] 1. Extraction of tissue proteins:

[0166] 1) Cryopreserve the tissue in liquid nitrogen and prepare protein lysis buffer: the ratio of protein lysis buffer (RIPA): protease inhibitor (PMSF): phosphatase inhibitor (PI) is 100:1:1.

[0167] 2) Take 100 mg of tissue and add 200 μL of protein lysis buffer. Add an appropriate amount of tissue grinding beads and grind the tissue into a homogenate using an instrument. Place on ice for 15 min for complete lysis.

[0168] 3) Centrifuge at 12000 rpm and 4℃ for 15 min, and carefully aspirate the supernatant after centrifugation.

[0169] 4) Protein concentration determination: Follow the experimental procedures according to the BCA protein quantification kit.

[0170] 5) Measure the absorbance at 562 nm using an ELISA reader, and then convert the absorbance value into protein concentration using the formula.

[0171] 6) Add an appropriate amount of 5×SDS loading buffer to the protein solution, boil the protein for 10 min, and use it for Western blot electrophoresis or store it at -80℃ for later use.

[0172] 2. Western Blot analysis:

[0173] 1) Gel preparation: Wash the 1.5 mm glass plate with ddH2O and let it dry. After the glass plate is dry, install the gel holder and prepare 7.5%, 10% and 12.5% ​​gels according to the instructions of the new cyme kit. Gel at room temperature for about 15-40 minutes.

[0174] 2) Prepare 1× electrophoresis buffer: Weigh 18.8g of glycine, 3.02g of Tris, and 1g of SDS, add 1L of ddH2O and stir to mix well.

[0175] 3) Sample loading: Mount the gel onto the electrophoresis holder, place it in the electrophoresis tank, and pour in 1× electrophoresis buffer. Remove the comb and add 3 μL of protein molecular weight marker or an appropriate amount of protein loading solution to the sample wells in sequence.

[0176] 4) Electrophoresis: Electrophoresis is performed at a constant voltage of 130V until the protein sample bands reach the edge of the gel plate, then electrophoresis is stopped.

[0177] 5) Transfer: Prepare the semi-dry transfer buffer (semi-dry transfer refers to a transfer method; here we are referring to the transfer buffer used for this method): Dissolve 5.82g Tris and 2.93g glycine in 800 mL ddH2O, then add 200 mL methanol and mix well. Cut out 8.5cm × 5.5cm PVDF membranes and filter paper. Immerse the PVDF membrane in methanol for 1 min. Place the filter paper, PVDF membrane, and gel sequentially from bottom to top in the semi-dry transfer apparatus, taking care to avoid air bubbles. Transfer at a constant voltage of 14V for 1 h.

[0178] 6) Blocking: Prepare a 5% blocking solution: Dissolve 5g of BSA in 100mL of washing solution. Cut the PVDF membrane to an appropriate size according to the molecular weight of the target band, immerse it in the blocking solution, and incubate slowly on a shaker at room temperature for 1 hour.

[0179] 7) Primary antibody incubation: Place the sealed PVDF membrane in the antibody incubation box, add the antibody prepared in advance according to the instructions, and incubate overnight at 4°C.

[0180] 8) Membrane washing: Prepare the washing solution: Add 1 mL of Tween-20 to 1 L of PBS and mix well. Add PVDF to the washing solution and wash three times for 10 min each time.

[0181] 9) Secondary antibody incubation: Select a suitable secondary antibody based on the species of the primary antibody, prepare a secondary antibody dilution solution at a ratio of 1:5000, and place the PVDF membrane in the secondary antibody solution. Incubate slowly on a shaker at room temperature for 1 hour.

[0182] 10) Washing the membrane: Place the PVDF in the washing solution and wash three times for 10 minutes each time.

[0183] 11) Development: Prepare ECL developing solution according to the reagent instructions. Immerse the PVDF membrane evenly in the developing solution and scan it using the ECL gel imaging system.

[0184] Example 4: ROC curve analysis of TRIM50 as a validation indicator to distinguish between normal control group and UC patients

[0185] Based on tissue samples collected from normal individuals and UC patients at Qilu Hospital, the TRIM50 expression level of each sample was detected, and the samples were divided into two groups: 0 (normal group) and 1 (disease group). SPSS software was used for analysis: "State variable" represented the grouping, and "Test variable" represented the TRIM50 expression level. After selecting "ROC curve," "with diagonal reference line," "standard error and confidence interval" (usually selecting a 95% confidence interval), and "ROC curve coordinates," an ROC curve was generated. The validation of TRIM50 was determined based on the AUC value (area under the curve) and its confidence interval.

[0186] Example 5 Cell line and culture conditions

[0187] 1. Cell Culture

[0188] HT29 and HCT-116 cell lines are both human colon cancer cell lines, and 293T cells are human embryonic kidney epithelial cell lines. All were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and cultured in our laboratory for a long period after importation. Cells were cultured in DMEM + 10% FBS at 37℃ and 5% CO2 under saturated humidity conditions.

[0189] 2. Primary cell isolation

[0190] 2.1 Extraction of mouse peritoneal macrophages (PM)

[0191] 1) Six- to eight-week-old mice were injected intraperitoneally with 6% starch broth to recruit peritoneal macrophages.

[0192] 2) After 72 hours, the mice were euthanized, disinfected by immersion in alcohol, and then placed in a biosafety cabinet.

[0193] 3) Use scissors to cut open the skin of the mouse's abdomen to expose the peritoneum.

[0194] 4) Using a 20 mL syringe, draw 10 mL of serum-free DMEM culture medium incubated at 37°C. Insert the syringe needle into the mouse's peritoneal cavity, avoiding excessive force that could puncture organs or the intestines. Thoroughly aspirate and ventilate the mouse's abdomen several times until the intestinal tissue is suspended. Transfer the culture medium to a new 50 mL centrifuge tube. Repeat this step once to obtain a large number of macrophages.

[0195] 5) Centrifuge at 1000 rpm at room temperature for 5 min, and the cells will settle at the bottom of the tube.

[0196] 6) Add 10 mL of complete culture medium incubated at 37°C and remix the cells by pipetting.

[0197] 7) Cell counting, at 1×10⁻⁶ 6 Density of seed plates.

[0198] 8) After the cells adhered, wash them three times with PBS, add fresh culture medium, and continue culturing in a 37°C, 5% CO2 incubator.

[0199] 2.2 Extraction of mouse bone marrow-derived macrophages (BMDM)

[0200] 1) Sacrifice the mouse, cut off the tibia and femur from the mouse's leg and place them in a biosafety cabinet, being careful not to cut off both ends of the bones.

[0201] 2) Clean the muscles on the bone with tweezers and rinse with alcohol.

[0202] 3) Rinse the bone three times with PBS containing penicillin and streptomycin antibiotics.

[0203] 4) Use scissors to cut open the joints at both ends of the femur to expose the bone marrow.

[0204] 5) Use a 1 mL syringe to draw up the incubated DMEM medium, insert the syringe needle into the bone marrow to flush out the bone marrow, and then mix the cells in the medium by pipetting.

[0205] 6) Centrifuge at 1000 rpm for 5 min, remove the supernatant, add 5 mL of red blood cell lysis buffer and lyse on ice for 3-5 min.

[0206] 7) Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL of prepared BMDM medium (50% DMEM, 20% FBS, 30% L929 supernatant and 1% antibiotics) to resuspend the cells into a single-cell suspension.

[0207] 8) Cell counting: Cells were counted at a density of 1.5 × 10⁻⁶. 6 The cells were seeded at a high density and cultured at 37°C with 5% CO2 for 7 days. On days 3-4, the cells largely adhered to the culture medium, which was then replaced and cultured further.

[0208] Example 6 Plasmid Extraction

[0209] 1. Transformation

[0210] 1) Add 5 μL of plasmid to 30 μL of DH5α competent cells taken at -80℃ and mix gently. Incubate on ice for 30 min.

[0211] 2) Heat shock in a 42℃ water bath for 90 seconds. Immediately place on ice and let stand for 2 minutes.

[0212] 3) Add 1000 μL of antibiotic-free LB medium and incubate at 37°C with shaking for 1 h.

[0213] 4) Take 100 μL of bacterial culture, spread it evenly on an LK / LA plate, and incubate overnight at 37°C.

[0214] 5) Select a single clone and inoculate it into 15 mL of LK / LA culture medium, and culture with shaking for 12 h.

[0215] 2. Plasmid extraction

[0216] 1) Take 15 mL of overnight cultured bacterial solution, add it to a centrifuge tube (self-prepared), centrifuge at 13,000 rpm (~16,200×g) for 30 s to collect bacteria, and discard as much of the supernatant as possible.

[0217] 2) According to the Kangwei reagent plasmid extraction instructions: Add 500 μL Buffer P1 to the centrifuge tube containing bacterial precipitate, mix thoroughly, and suspend the bacterial precipitate.

[0218] 3) Add 500 μL Buffer P2 to the centrifuge tube, gently invert and mix 8-10 times to fully lyse the cells, and incubate at room temperature for 3-5 minutes.

[0219] 4) Add 500 μL of Buffer E3 to the centrifuge tube and immediately invert to mix 8-10 times. A white flocculent precipitate will appear. Let it stand at room temperature for 5 min. Centrifuge at 13,000 rpm for 5 min and collect the supernatant in a centrifuge tube (provided by yourself).

[0220] 5) Add 0.3 times the volume of the supernatant of isopropanol and mix by inverting the container.

[0221] 6) Column equilibration step: Add 200 μL of Buffer PS to the adsorption column (Spin Columns DM) that has been loaded into the collection tube, centrifuge at 13,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0222] 7) Transfer the mixture of filtrate and isopropanol from step 5 to the adsorption column (already loaded into the collection tube).

[0223] 8) Centrifuge at 13,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0224] Add 750 μL of Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 1 min, and discard the waste liquid in the collection tube. Place the adsorption column back into the collection tube and centrifuge at 13,000 rpm for 1 min.

[0225] 9) Place the adsorption column in a new collection tube, add 50-100 μL of BufferEB to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 13,000 rpm for 2 min, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20℃.

[0226] Example 7 Cell Transfection

[0227] 1) Select cells in good growth condition, digest and resuspend the cells, and count them. HEK293T cells were counted at a rate of 4 × 10⁻⁶. 6 Cells / mL were seeded into 6-well plates or cell culture dishes and cultured in a 37°C, 5% CO2 cell culture incubator.

[0228] 2) When the cell density reaches 70%-80% after 16-20 hours, replace the culture medium with Opti-MEM medium.

[0229] 3) For plasmid transfection, dissolve 2-6 μg of plasmid in 200 μL of opti-MEM medium, and add 2-6 μL of lipo2000 dissolved in 200 μL of opti-MEM medium. Mix the diluted plasmid and liposome thoroughly and incubate at room temperature for 15 min. For small interfering RNA transfection, dilute 5 μL of siRNA in 200 μL of opti-MEM medium, add 10 μL of INTERFERin to 200 μL of opti-MEM medium, add the diluted small interferon to the diluted INTERFERin, and incubate at room temperature for 15 min.

[0230] 4) Add the mixed plasmid or small interference drop by drop into the culture plate and gently shake to mix.

[0231] 5) After culturing the cells in an incubator at 37°C and 5% CO2 for 6-8 hours, replace the culture medium with complete culture medium and continue culturing.

[0232] Example 8: Co-immunoprecipitation (Co-IP) experiment

[0233] 1) Transfect FLAG-TRIM50 and HA-S100A8 plasmids into 293T cells, culture for 24 h, discard the cell culture medium, and wash the cells three times with 2 mL of pre-cooled PBS.

[0234] 2) After completely removing PBS, add an appropriate amount of IP buffer (with protease inhibitor), lyse the cells on ice, scrape off the cells at the bottom of the culture plate with a cell scraper, transfer the lysis buffer to a 1.5 mL EP tube, vortex for 1 min, and then continue to lyse the cells on ice for 20 min.

[0235] 3) Centrifuge at 14000 rpm and 4℃ for 20 min.

[0236] 4) Take 40 μL of cell lysis supernatant as the input group and add 10 μL of 5×SDS protein loading buffer. Mix well, heat at 100℃ for 10 min, centrifuge at 12000 rpm for 2 min to allow the sample to settle to the bottom, and store at -20℃.

[0237] 5) Add the required antibody to the remaining supernatant, mix by inversion in a 4°C chromatography freezer for 2 h, then add 40 μL of Protein A / G agarose beads, mix by inversion in a 4°C chromatography freezer overnight.

[0238] 6) Centrifuge the IP sample at 1000 rpm for 5 min at 4℃, gently aspirate the supernatant (avoiding the beads), add 400 μL of IP buffer, mix by inversion, and centrifuge at 1000 rpm for 5 min at 4℃. Repeat 5 times. Aspirate the supernatant, add 40 μL of 1×SDS protein loading buffer, heat at 100℃ for 5 min, and then centrifuge to allow the sample to settle at the bottom of the tube. Store at -20℃ for Western blotting.

[0239] Example 9 Immunofluorescence (IF) Experiment

[0240] 1) Seed an appropriate number of cells onto a 24-well cell slide, culture the cells overnight, and then perform appropriate treatment.

[0241] 2) After the treatment, the cells were removed from the smear and washed three times with PBS for 5 minutes each time.

[0242] 3) Add 1 mL of methanol to fix the cells for 15 min, and wash the cell slides with PBS three times for 5 min each time.

[0243] 4) Add 500 μL of 5% BSA and block at room temperature for 1 h.

[0244] 5) Remove the BSA and add the primary antibody diluted according to the instructions. Incubate overnight at 4°C.

[0245] 6) Remove the primary antibody, add PBST to wash the cell slide three times, 5 min each time.

[0246] 7) Add the immunofluorescent secondary antibody corresponding to the species of the primary antibody according to the instructions, and incubate at 37°C in the dark for 1 hour.

[0247] 8) Remove the secondary antibody and wash the cells three times with PBST for 5 minutes each time (avoid light).

[0248] 9) Add DAPI and incubate in the dark for 5 min to stain cell nuclei. Wash with PBST 3 times, 5 min each time.

[0249] 10) Aspirate the liquid from the stem cell slide and place the slide onto a glass slide with an anti-fluorescence quenching mounting medium. Use a laser confocal microscope to acquire cell fluorescence images.

[0250] Example 10 In vitro experiment

[0251] 10.1 In vitro protein synthesis

[0252] 1) Take the reagents out of the -80℃ freezer, quickly melt the TnT in a 37℃ water bath, quickly mix the stock solution and place it on ice, and thaw the other reagents at room temperature and place them on ice.

[0253] 2) Prepare the reaction mixture on ice according to the following ingredients.

[0254] Table 2 In vitro translation system

[0255]

[0256] 3) Incubate at 30℃ for 60-90 min.

[0257] 4) Take 1 μL of the reaction product and use Western blot to detect the translation product. Use the remaining part for subsequent experiments.

[0258] 10.2 In vitro ubiquitination detection

[0259] 1. Preparation of reaction system: Prepare ubiquitination reaction buffer: 2 µL UBE1 (5 μM), 2 µL UBcH5α (50 μM), 10 µL Flag-TRIM50, 10 µL HA-S100A8, 5 µL Ubiquitin (1 mM), 5 µL Mg-ATP, 5 µL E3 ligase buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 1 mM TCEP), and add enzyme-free sterile water to a final volume of 50 µL.

[0260] 2. Incubate the reaction system at 37°C for 1 h, add SDS-PAGE loading buffer to terminate the reaction, and heat at 100°C for 10 min.

[0261] 3. SDS-PAGE electrophoresis: The reaction products were separated into ubiquitinated proteins of different molecular weights by electrophoresis. After transfer to a membrane, the S100A8 ubiquitinated band was detected using an anti-HA antibody.

[0262] Example 11 Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE)

[0263] 1) Preparation of SDD gel: Weigh 0.6g of agar powder and add it to 40mL of 0.5×TBE solution. Dissolve the agar powder completely in a microwave oven. Allow the solution to cool slowly to about 60℃ at room temperature. Add 0.04g of SDS and gently shake to dissolve the SDS completely. Pour the solution into a pre-placed glass plate interlayer and insert a 10-well comb. Allow the gel to solidify completely at room temperature.

[0264] 2) Install the glass plate after the gel has solidified into the electrophoresis tank, add 0.5×TBE (containing 0.1% SDS) buffer, remove the comb and add the protein marker and protein sample in sequence.

[0265] 3) Electrophoresis: Place the electrophoresis tank on ice and electrophoresis at a constant voltage of 100V for 35 minutes.

[0266] 4) Continue with the relevant operations for Western blot.

[0267] Example 12 Cycloheximide (CHX) Experiment

[0268] 1) When the cell density reaches 80%, transfect the cells with HA-S100A8 and Flag-TRIM50 plasmids and the control group Flag-vec plasmid.

[0269] 2) 24 h after transfection, replace with full culture medium containing CHX, with a CHX concentration of 1 uM and 1 mL per well.

[0270] 3) Collect cells treated with CHX for 0h, 2h, and 4h respectively, extract proteins, and detect changes in S100A8 protein levels using Western blot.

[0271] Example 13: Detection of cell proliferation viability using the CCK-8 assay

[0272] Colon cancer cells at 1×10 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and cultured overnight.

[0273] Transfect the expression plasmids Si-S100A8 or TRIM50, and use the Si-NC or empty vector transfection group as a control. Change the medium 6 h after transfection and set it as 0 h. Add CCK-8 reagent at 0 h, 24 h, 48 h and 72 h after transfection, respectively, and incubate at 37℃ for 1 h. Detect the OD value at 450 nm with a microplate reader.

[0274] Example 14 Transwell migration experiment

[0275] 1) Colon cancer cells at 3×10 5 Cells were seeded at a density of 1 / mL in 6-well plates, with 2 mL of cell suspension added to each well, and cultured overnight. When the cell density reached 60%-80%, the cells were transfected with the Si-S100A8 or TRIM50 expression plasmid, with the Si-NC or empty vector transfection group serving as a control, and a blank control group also included. After culturing for another 24 h, the cells were trypsinized and prepared into a single-cell suspension using serum-free medium. Cells were counted, and the cell density of each group was adjusted to 2 × 10⁶ cells / well. 5 / mL.

[0276] 2) Take 100 μL of cell suspension, add it to a Transwell chamber, add 600 μL of complete culture medium containing 10% FBS to the lower chamber, and continue culturing for 24 h.

[0277] 3) Remove the chamber, rinse three times with PBS, add 100 μL of methanol, and fix for 15 min.

[0278] 4) Remove the methanol, rinse three times with PBS, and then add 100 μL of crystal violet for staining for 15 min.

[0279] 5) Aspirate the crystal violet, wipe the crystal violet dye inside the chamber clean with a cotton swab, rinse three times with PBS to remove unmigrated cells inside the chamber.

[0280] 6) Observe the cell migration of each group under an electron microscope, randomly select 5 different fields of view to take pictures, count them respectively, and calculate the cell migration rate.

[0281] Example 15 Cloning Experiment

[0282] Colon cancer cells at 3×10 5 Cells were seeded at a density of 1 / mL in 6-well plates, with 2 mL of cell suspension added to each well, and cultured overnight. When the cell density reached 60%-80%, colon cancer cells were transfected with the Si-S100A8 or TRIM50 expression plasmid. The Si-NC or empty vector transfection group served as a control, and a blank control group was also included. After culturing for another 24 h, the cells were trypsinized to prepare a single-cell suspension. Cells were counted, and the cell density of each group was adjusted to 500 cells / mL. 2 mL of cell suspension was added to each 6-well plate, and the plates were incubated at 37°C in a 5% CO2 incubator for 7-10 days. Cell clones were stained with crystal violet, photographed, counted, and analyzed. Clones containing more than 50 cells were analyzed.

[0283] Example 16 Enzyme-linked immunosorbent assay (ELISA)

[0284] 1) According to the instructions, before the experiment, equilibrate the ELISA pre-coated plate, washing buffer (50×), Dilution buffer R, and TMB at room temperature for 30 min.

[0285] 2) Sample addition: Dilute the supernatant or serum sample with Dilution buffer R, add 100 μL to the sample well, and set up a sub-well for the sample and a blank control well at the same time.

[0286] 3) Add antibody: Dilute the primary antibody with Dilution buffer R at a ratio of 1:100. Add 50 μL of the diluted antibody to each well, cover with the sealing film, and incubate at 37°C for 90 min. During incubation, you can manually shake it every 15 min to make the reaction more uniform.

[0287] 4) Washing the plate: Remove the liquid in the wells, add 300 μL of washing solution to each well, dilute the washing solution with double-distilled water, let it stand in the plate for 1 min, remove the liquid, and repeat the washing 4 times.

[0288] 5) Add enzyme: Dilute Streptavidin-HRP with Dilution buffer R at a ratio of 1:100, add 100 μL to each well, cover with sealing film, and incubate at 37°C for 30 min.

[0289] 6) Wash the plate: Wash the plate 4 times as per step 4.

[0290] 7) Color development: Add 100 μL TMB to each well and develop the color at 37℃ in the dark. The development time varies from 5 to 30 minutes. Stop the color development based on the color result.

[0291] 8) Termination: Add 100 μL of termination solution to the well to terminate the reaction.

[0292] 9) Plate reading: Within 10 minutes after the reaction is terminated, the absorbance value at 450 nm is measured, and it can be corrected using 630 nm.

[0293] Example 17 Flow Cytometry

[0294] 1) Cell collection: Centrifuge to collect cells, discard the supernatant, and wash the cells three times with pre-cooled PBS.

[0295] 2) Cell staining: Discard the supernatant, add 100 μL PBS to resuspend the cells, add the corresponding flow cytometry antibody to the resuspended solution, mix gently, and incubate in the dark for 15-30 min.

[0296] 3) Flow cytometry analysis: Wash cells twice with PBS, discard the supernatant, and resuspend the cells in 300 μL PBS. Perform flow cytometry detection according to the standard procedure provided by the instrument company, generally counting 20,000 cells. Statistical analysis of the obtained flow cytometry results is performed using GraphPad software.

[0297] Example 18: Construction of a mouse UC model

[0298] 1. Select male C57BL / 6 mice aged 6-8 weeks, weighing approximately 23g. Randomly divide the mice into experimental group and normal control group, and weigh and mark them.

[0299] 2. Prepare a 3% DSS aqueous solution using sterile drinking water. After preparation, filter it through a 0.22μm filter membrane for sterilization.

[0300] 3. Starting from day 1 of the experiment, mice were given free access to a freshly prepared 3% DSS aqueous solution for 7 days. The DSS solution was replaced with fresh solution every 2-3 days to ensure a stable concentration. During the same period, mice were also given normal sterile drinking water.

[0301] 4. Seven days after DSS treatment (i.e., day 8 of the experiment), the experimental group mice were given normal sterile drinking water and continued to be fed normally.

[0302] 5. During the modeling period, the weight of the mice was monitored daily. After the experiment, the mice were sacrificed, their colon length was isolated and measured, and the colon tissue was taken for HE staining and microscopic observation.

[0303] Example 19 Construction of a mouse CRC model

[0304] 1. Azomethane (AOM) pretreatment: First, each mouse is given an intraperitoneal injection of AOM, usually at a dose of 10-20 mg / kg body weight.

[0305] 2. After injection of AOM, mice were allowed to drink normal water for one week to allow them to adapt to the environment and recover from the injection stress.

[0306] 3. Periodic treatment phase: Days 1-7: Dissolve DSS in drinking water to a concentration of 2%-3% and replace all normal drinking water. Days 8-21: Discontinue DSS and revert to normal drinking water for 14 days.

[0307] 4. Repeat the cycle: Repeat the above process of "DSS treatment for 7 days + normal drinking water for 14 days" twice, for a total of 3 complete cycles.

[0308] 5. Regularly weigh the mice and observe their activity, detect blood in their stool, observe the colon tissue, perform HE staining and histopathological analysis, etc.

[0309] Example 20 Statistical Analysis

[0310] Statistical analysis was performed using SPSS 16.0 and GraphPad Prism 5.0 software. Data are expressed as mean plus standard deviation (mean + SD). The difference between the means of the two groups was analyzed by unpaired t-test and two-way ANOVA. P < 0.05 was considered statistically significant.

[0311] Figure 1 and Figure 2The figure shows the verification results of TRIM50's ability to interact with S100A8. Figure 1 In the figure, A represents the interaction between exogenous TRIM50 and S100A8 in HEK293T cells as detected by the immunoprecipitation forward and reverse interaction IP experiment. The results showed that TRIM50 and S100A8 can bind to each other under exogenous conditions. Figure 1 B, C, D, and E in the figure represent the binding of exogenous TRIM50 and S100A8 in HEK293T cells and endogenous TRIM50 and S100A8 in macrophages detected by laser confocal microscopy. The results show that TRIM50 and S100A8 have an interaction. Figure 2 In the figure, A represents the possibility of two molecules binding in molecular docking simulation. The results show that TRIM50 and S100A8 may bind spatially. Figure 2 In the figure, B represents the immunoprecipitation assay used to detect the binding of S100A8 and TRIM50 domains. The results showed that S100A8 and TRIM50 bind to the C-C2 domains. Figure 2 In the figure, C represents the translation of TRIM50 and S100A8 proteins using an in vitro protein transcription and translation system. Immunoprecipitation experiments showed that TRIM50 and S100A8 bind directly to each other.

[0312] Figure 3 This figure shows the results of high TRIM50 expression in UC patients and its correlation with disease activity. Figure 3 In the figures A and B, the expression of TRIM50 in UC patients was detected by immunohistochemistry and colonoscopy. The results showed that TRIM50 expression was significantly upregulated in UC patients. Figure 3 C and D in the figure represent the expression of TRIM50 in UC patients detected by Western blot (WB) assay. The WB bands also showed that TRIM50 expression was significantly upregulated in UC patients. Figure 3 E in the figure represents the expression of TRIM50 in UC patients with different disease severity detected by immunohistochemical experiments. The results show that TRIM50 increases with the increase of disease severity. Figure 3 F in the figure represents the expression of TRIM50 in the above patients in a statistical analysis. The results also show that TRIM50 has a positive correlation with the disease. Figure 3 G in the figure represents the UCEIS scores collected from clinical patients. The correlation between TRIM50 and UC patients was analyzed, and the results showed that TRIM50 and UCEIS scores were positively correlated, indicating a positive correlation with inflammation. Figure 3 H in the figure represents the ROC curve analysis of TRIM50 in clinical patients. The results show that the area under the ROC curve is 0.708, which indicates that it has clinical significance.

[0313] Figure 4 For DSS induction Trim50- / -The severity of UC in mice was lower. Figure 4 In the figure, A and B are the statistical analyses of WT and WT after DSS induction. Trim50- / - The length of the mouse colon, the results showed Trim50- / - The mice had longer colons and were in better condition. Figure 4 In the figure, C represents the statistical analysis of WT and WT after DSS induction. Trim50- / - The results showed that the mice lost weight. Trim50- / - The mice lost less weight than the WT mice. Figure 4 In the figure, D represents the statistical analysis of WT and WT after DSS induction. Trim50- / - The results showed intestinal permeability in mice. Trim50- / - The mice had lower intestinal permeability, suggesting that their intestinal tissue function was better preserved. Figure 4 In the diagram, E represents two groups of mice treated with an S100A8 inhibitor after DSS induction. The results are HE-stained sections of the colon of mice treated with the S100A8 inhibitor. Trim50- / - The mouse intestinal tissue showed more intact morphology and less damage. Figure 4 In the figure, F represents the WT and WT values ​​after using the S100A8 inhibitor in the statistical analysis. Trim50- / - The results of the study on changes in mouse body weight showed no significant difference between the two groups of mice. Figure 4 In the figure, G represents the WT and WT values ​​after using the S100A8 inhibitor in the statistical analysis. Trim50- / - The study examined changes in colon length in mice, and the results showed no significant difference between the two groups. These results indicate that S100A8 inhibitors can reduce the degree of inflammation, but this effect is only achieved in the presence of TRIM50.

[0314] Figure 5 The diagram shows the effect of the TRIM50-S100A8 axis positive feedback. Figure 5 In the figure, A represents the expression of TRIM50 and S100A8 in neutrophils and bone marrow-derived macrophages after DSS induction, as detected by Western blot (WB) assay. The results showed that both increased under inflammatory conditions. Figure 5 B in the figure represents the effect of the CHX experiment on the protein stability of S100A8 by TRIM50. The results show that TRIM50 can significantly enhance the protein stability of S100A8. Figure 5 C in the figure represents the expression of TRIM50 in peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) after stimulation with S100A8 recombinant protein by qPCR and WB experiments. The results showed that S100A8 could inversely stimulate and promote the increase of TRIM50 protein level.

[0315] Figure 6 TRIM50 will ubiquitinate S100A8. Figure 6In the figure, A represents the Western blot analysis of the ubiquitination modification of S100A8 by the E3 ubiquitin ligase TRIM50. The results show that TRIM50 can increase the ubiquitination modification of S100A8. Figure 6 B in the figure represents the Western blot (WB) assay used to detect the ubiquitination modification of S100A8 by TRIM50 and its enzyme activity-deficient mutant (TRIM50-C53A). The results showed that TRIM50-C53A significantly rescued the ubiquitination modification of S100A8, indicating that TRIM50 acts on S100A8 through its enzyme activity. Figure 6 In the figure, C represents the direct ubiquitination modification of S100A8 by TRIM50 in vitro as detected by Western blot (WB) experiment. The results show that the ubiquitination modification of S100A8 by TRIM50 is a direct effect. Figure 6 In the figure, D represents the type of ubiquitination modification of S100A8 by TRIM50 as detected by WB experiment. The results confirm that TRIM50 significantly promotes the ubiquitination modification of S100A8 at position K63.

[0316] Figure 7 TRIM50 oligomerizes and modifies S100A8, thereby stabilizing the S100A8 protein. Figure 7 In the figure, A represents the oligomerization modification of S100A8 by TRIM50 detected by the SDD-AGE experiment. The results show that TRIM50 significantly promotes the oligomerization modification of S100A8. Figure 7 B in the figure represents the Western blot (WB) experiment used to detect the ubiquitination modification of S100A8 and its ubiquitination site deletion mutant by TRIM50. The results showed that the modification of S100A8 by TRIM50 was weakened after mutations at K36 and K56, and the weakening effect was stronger after two-site mutations. Figure 7 In the figure, C represents the Western blot (WB) experiment used to detect the oligomerization modification of S100A8 and its ubiquitination site deletion mutant by TRIM50. The results showed that only after the K36 mutation of S100A8 was the modification of TRIM50 significantly weakened, suggesting that K36 is the site of action of S100A8. Figure 7 D and E in the figure represent the effects of the CHX experiment on the protein stability of S100A8 and its ubiquitination site deletion mutants by TRIM50. The results showed that the effect of TRIM50 on enhancing protein stability was weakened after the K36 mutation of S100A8.

[0317] Figure 8 The TRIM50-S100A8 axis has a post-treatment assessment effect. Figure 8 In the figures, A and B represent the expression of TRIM50 and S100A8 in UC patients with different disease severity as detected by immunohistochemistry and colonoscopy. The results show that the expression of both TRIM50 and S100A8 increases with the increase of disease severity. Figure 8C and D in the figure represent the expression of TRIM50 and S100A8 in UC patients with a prognostic condition as detected by immunohistochemistry and colonoscopy. The results showed that the expression of both TRIM50 and S100A8 increased with the degree of inflammation, but the expression decreased in patients with a prognostic condition. Figure 8 E, F, G, and H in the figure represent the expression of TRIM50 and S100A8 in UC patients with a prognostic outcome, as detected by Western blotting. The results showed that the expression of both TRIM50 and S100A8 increased with the degree of inflammation, but decreased in patients with a prognostic outcome. This suggests that they are predictive of the severity of the disease.

[0318] Figure 9 , Figure 10 and Figure 11 The generation of UC-related CRCs facilitated by TRIM50. Figure 9 A and B in the figure represent mouse CRC models induced by AOM+DSS. The results are HE-stained sections of the colon of the two CRC mouse models, indicating the successful construction of the models. Figure 9 C and D in the figure represent the expression of TRIM50 in wild-type CRC mice as detected by immunohistochemistry. The results showed that TRIM50 was significantly increased in the CRC model. Figure 10 A and B in the figure represent statistical analysis WT and Trim50- / - The colon length of CRC mice was measured, and the results showed... Trim50- / - CRC mice have a longer colon. Figure 10 C, D, and E in the figure represent the expression of TRIM50 and S100A8 in 80 pairs of CRC patient tissue microarrays detected by immunohistochemistry. The results showed that the levels of TRIM50 and S100A8 in CRC patient microarrays were increased. Figure 11 A, B, and C in the figure represent the effects of exogenous TRIM50 and its enzyme-deficient mutant on the clonogenic ability of colon cancer cells in a clonogenic assay. The results showed that TRIM50 promoted the clonogenic ability of colon cancer cells, and this was achieved through its enzyme activity effect. Figure 11 D, E, and F in the figure represent the effects of transgenic TRIM50 and its enzyme-deficient mutant on the migration ability of colon cancer cells as determined by Transwell assays. The results showed that TRIM50 promoted the migration ability of colon cancer cells, and this was achieved through its enzyme activity effect.

[0319] To verify the effects of TRIM50 on ulcerative colitis (UC) and colorectal cancer (CRC) in mice, this invention first collected clinical patient tissues and compared TRIM50 expression at different disease severities. Then, TRIM50 knockout mice, DSS-induced UC, and AOM+DSS-induced CRC models were constructed. The results showed that TRIM50 knockout could inhibit the disease progression of UC and CRC. Molecularly, TRIM50 promotes UC by directly binding to S100A8 and ubiquitinizing it, followed by oligomerization to stabilize its function. Exogenous overexpression of TRIM50 in colon cancer cell lines also promotes cancer cell formation and invasion. In conclusion, both animal and cell experiments confirm that TRIM50 can enhance the progression of UC and the development of CRC.

[0320] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of substances for detecting TRIM50 expression levels in the preparation of diagnostic or auxiliary diagnostic products for ulcerative colitis and related colorectal cancer.

2. A method for screening drugs for ulcerative colitis and related colorectal cancer, characterized in that, include: c1) The system expressing and / or containing TRIM50 was treated with the candidate substance; a parallel control was set up without the candidate substance treatment; c2) After completing step c1), detect the expression level of TRIM50 in the system; if the expression level of TRIM50 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate drug for ulcerative colitis and its related colorectal cancer.

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