Use of trim21-related biologicals in the manufacture of ymel1-related products

Abstract

This invention discloses the application of TRIM21-related biopharmaceuticals in the preparation of YME1L1-related products. It reveals the interaction between TRIM21 and YME1L1 and its key regulatory mechanism in the development and progression of bladder cancer. Studies show that TRIM21 specifically binds to the YME1L1 protein through its SPRY domain, mediating K63-linked ubiquitination modification of YME1L1 and thus promoting its protein degradation. Functional experiments confirm that TRIM21 inhibits the proliferation, migration, invasion, and mitochondrial function of bladder cancer cells and induces apoptosis by downregulating YME1L1 protein levels. This invention elucidates for the first time the tumor-suppressive mechanism of the TRIM21-YME1L1 signaling axis in bladder cancer, providing a new molecular marker for the diagnosis and prognostic assessment of bladder cancer, and offering a theoretical basis and intervention strategy for developing anti-tumor drugs targeting the TRIM21 / YME1L1 pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of TRIM21-related biological products in the preparation of products related to YME1L1. Background Technology

[0002] Bladder cancer (BLCA) is a leading cause of cancer-related death due to its high tumor heterogeneity, which leads to complex biological behavior and varied treatment responses. Targeted therapies exert their effects by acting on specific signaling pathways or molecular targets in cancer cells. Immune checkpoint inhibitors, FGFR3-targeting drugs, and antibody-drug conjugates have made significant progress in clinical applications. However, drug resistance due to tumor heterogeneity remains a major clinical challenge. Identifying novel signaling pathways or molecular targets is crucial for exploring innovative targeted therapies for bladder cancer. Therefore, investigating treatments for bladder cancer is a highly valuable area of ​​research. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides the application of TRIM21-related biological products in the preparation of YME1L1-related products. This invention reveals the interaction between TRIM21 and YME1L1 and its key regulatory mechanism in the occurrence and development of bladder cancer, providing new molecular markers for the diagnosis and prognostic assessment of bladder cancer, and providing a theoretical basis and intervention strategy for the development of anti-tumor drugs targeting the TRIM21 / YME1L1 pathway.

[0004] The technical solution provided by this invention is as follows:

[0005] This invention provides the use of TRIM21-related biological products in the preparation of reagents that promote the degradation of YME1L1, wherein the TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing a nucleic acid molecule encoding TRIM21; A nucleic acid molecule containing the encoded TRIM21 or a vector containing the expression cassette.

[0006] Furthermore, TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the K63-linked polyubiquitination modification of YME1L1, thereby promoting the degradation of YME1L1.

[0007] This invention also provides the use of TRIM21-related biological products in the preparation of reagents that shorten the half-life of YME1L1 in cells, wherein the TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing a nucleic acid molecule encoding TRIM21; A nucleic acid molecule containing the encoded TRIM21 or a vector containing the expression cassette.

[0008] Furthermore, TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the K63-linked polyubiquitination modification of YME1L1, thereby promoting the degradation of YME1L1 and shortening its half-life in cells.

[0009] This invention also provides the use of TRIM21-related biological products in the preparation of medicaments for treating bladder cancer, wherein the TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing the aforementioned nucleic acid molecule encoding TRIM21; A vector containing the above-described nucleic acid molecule encoding TRIM21 or containing the above-described expression cassette; The TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the polyubiquitination modification of YME1L1 linked to K63, thereby promoting the degradation of YME1L1 to treat bladder cancer.

[0010] Furthermore, the drug is used for: Inhibits the proliferation of bladder cancer cells; And / or, inhibit the migration of bladder cancer cells; And / or, inhibit the invasion of bladder cancer cells; And / or, induce apoptosis in bladder cancer cells; And / or, impairs mitochondrial function in bladder cancer cells.

[0011] Furthermore, the damage to mitochondrial function in bladder cancer cells includes reducing mitochondrial membrane potential, decreasing ATP production in bladder cancer cells, and increasing reactive oxygen species levels in bladder cancer cells.

[0012] This invention also provides the use of biological products related to the inhibition of TRIM21 expression or activity in the preparation of reagents for inhibiting YME1L1 degradation, wherein the biological products related to the inhibition of TRIM21 expression or activity include any of the following: Monoclonal antibody against TRIM21; Antisense nucleic acid molecules that inhibit TRIM21 expression; Gene editing tools that inhibit TRIM21; Among them, biopharmaceuticals that inhibit TRIM21 expression or activity inhibit YME1L1 degradation by inhibiting the interaction between TRIM21 and YME1L1.

[0013] Furthermore, the antisense nucleic acid molecule that inhibits TRIM21 expression is an shRNA molecule that specifically targets the TRIM21 coding sequence, and the shRNA contains a sense strand and an antisense strand with complementary base pairing; the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO:3~SEQ ID NO:4 or SEQ ID NO:5~SEQ ID NO:6.

[0014] Furthermore, the gene editing tool for inhibiting TRIM21 is selected from homologous recombination, TALEN, ZFN, and CRISPR / Cas9.

[0015] Beneficial effects

[0016] This invention reveals the interaction between TRIM21 and YME1L1 and its key regulatory mechanism in the development and progression of bladder cancer. TRIM21 overexpression can inhibit bladder cancer cell proliferation and motility; TRIM21 can reduce the protein abundance of YME1L1 through ubiquitination and degradation; the SPRY domain of TRIM21 mediates the interaction and ubiquitination and degradation of YME1L1; and TRIM21 inhibits bladder cancer development by degrading YME1L1.

[0017] The results showed that TRIM21 specifically binds to the YME1L1 protein through its SPRY domain, mediating K63-linked ubiquitination modification of YME1L1 and thus promoting its protein degradation. Functional experiments confirmed that TRIM21 significantly inhibited the proliferation, migration, invasion, and mitochondrial function of bladder cancer cells and induced apoptosis by downregulating YME1L1 protein levels; while knockdown of TRIM21 reversed the above effects and restored the pro-cancer phenotype of YME1L1. Clinical data analysis showed that high expression of TRIM21 was positively correlated with a good prognosis in bladder cancer patients, and that TRIM21 and YME1L1 exhibited spatial co-localization in tissue sections.

[0018] This invention elucidates for the first time the tumor-suppressing mechanism of the TRIM21-YME1L1 signaling axis in bladder cancer, providing a new molecular marker for the diagnosis and prognostic assessment of bladder cancer, and providing a theoretical basis and intervention strategy for the development of anti-tumor drugs targeting the TRIM21 / YME1L1 pathway. Attached Figure Description

[0019] Figure 1YME1L1 expression is associated with poor survival outcomes in bladder cancer. A shows the overall survival and progression-free interval analysis of bladder cancer patients (TCGA) based on YME1L1 expression levels (low vs. high). B shows the mRNA expression analysis of YME1L1 in bladder cancer (T=408) and normal tissue samples (N=19) from the TCGA database (top image), and the mRNA expression analysis of YME1L1 in low-grade (L=20) and high-grade bladder cancer samples (H=388) from the TCGA database (bottom image). C shows the immunofluorescence localization of DAPI (blue, cell nucleus) and YME1L1 (red) in normal bladder tissue (top image) and bladder cancer tissue (bottom image). D shows the Western blot analysis of protein extracts from 6 pairs of paired tumor tissue and adjacent normal tissue samples. E shows the YME1L1 protein expression in bladder cancer (n=55) tissue detected by immunohistochemistry. F shows the analysis of E, with YME1L1 expression levels stratified by pathological stage, grade, and invasiveness.

[0020] Figure 2 This is a single-cell sequencing identification of the main cell subpopulations and their characteristic biomarkers in the sample; where A is a single-cell atlas and cell type annotation, UMAP projection of bladder cancer sample cells, colored according to the annotated cell type; B is a dot plot showing the expression level (Z-score) and expression percentage of each cell subpopulation-specific biomarker gene.

[0021] Figure 3 This section presents the single-cell expression characteristics and cell type distribution of YME1L1 in normal and tumor tissues. A shows the spatial expression distribution of YME1L1 in normal / tumor specimens, visualized using UMAP dimensionality reduction to display the single-cell expression level of the YME1L1 gene in both normal and tumor tissue specimens. B shows the expression distribution and detection rate of YME1L1 in various cell types. The left figure (YME1L1 expression distribution): a combination of violin plot and scatter plot, showing the expression level distribution of YME1L1 in eight major cell types (T cells, myeloid cells, epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, B cells, and plasma cells). Blue represents normal tissue, and red represents tumor tissue. The right figure (YME1L1 detection rate): a bar chart showing the percentage of positive expression of YME1L1 in each cell type (blue = normal tissue, red = tumor tissue), providing a direct comparison of the differences in the positive expression rate of YME1L1 in different cell populations between normal and tumor tissues.

[0022] Figure 4 This is a differential expression analysis of tumor groups with high and low expression of YME1L1 in different cell types.

[0023] Figure 5It is an enrichment of GO biological processes in tumor groups with high and low expression of YME1L1 in different cell types.

[0024] Figure 6 The study investigated the effect of YME1L1 knockdown on the proliferation, migration, and invasion of bladder cancer cells. Specifically, A represents Western blotting verification of the knockdown efficiency of YME1L1 in T24 and 5637 cells, with cell invasion using lentiviruses encoding either a non-target control shRNA (shCtrl) or two different shRNAs targeting YME1L1 (sh-YM-1 and sh-YM-2); B represents cell proliferation as assessed by a CCK-8 assay after YME1L1 knockdown; C represents the EdU assay to assess cell proliferation levels after YME1L1 knockdown; D represents a Transwell assay to evaluate the effect of YME1L1 knockdown on cell invasion; and E represents a scratch healing assay to assess changes in cell migration after YME1L1 knockdown.

[0025] Figure 7 The study showed that knocking down YME1L1 induced apoptosis in bladder cancer cells and disrupted mitochondrial function. A represents the level of apoptosis detected by Annexin V-FITC / PI double staining flow cytometry after YME1L1 knockdown; B is a quantitative analysis of the apoptosis level (A); C is the effect of YME1L1 knockdown on mitochondrial membrane potential levels assessed by JC-1 staining; D shows the intracellular reactive oxygen species (ROS) levels observed by fluorescence microscopy after YME1L1 knockdown (green: ROS; blue: DAPI); and E shows the cellular ATP production detected after YME1L1 knockdown.

[0026] Figure 8 The study found that overexpression of YME1L1 promotes the proliferation, migration, and invasion of bladder cancer cells. Specifically, A is the Western blotting assay verifying the overexpression efficiency of YME1L1 in T24 and 5637 cells; B is the CCK-8 assay analyzing cell proliferation after YME1L1 overexpression; C is the EdU assay confirming increased cell proliferation after YME1L1 overexpression; D is the Transwell assay assessing the effect of YME1L1 overexpression on cell invasion ability; and E is the scratch healing assay assessing cell migration ability after YME1L1 overexpression.

[0027] Figure 9 Overexpression of YME1L1 enhances mitochondrial function and inhibits apoptosis in bladder cancer cells. A shows the intracellular reactive oxygen species (ROS) level observed under fluorescence microscopy after YME1L1 overexpression (green: ROS; blue: DAPI); B shows the mitochondrial membrane potential level assessed by JC-1 staining after YME1L1 overexpression; C shows the ATP production of cells after YME1L1 overexpression; and D shows apoptosis analysis by Annexin V-FITC / PI double staining flow cytometry after YME1L1 overexpression.

[0028] Figure 10 This is the interaction between TRIM21 and YME1L1; where A is a schematic diagram of the LC-MS / MS experimental procedure for screening YME1L1 interacting proteins; B is three candidate E3 ubiquitin ligases identified by mass spectrometry; C is the verification of the interaction between YME1L1 and TRIM21 by endogenous immunoprecipitation; D is the structural model of the YME1L1-TRIM21 complex predicted by molecular simulation docking; E is the determination of the TRIM21 domain required for YME1L1 binding: (left) schematic diagram of wild-type and truncated mutant TRIM21, (right) immunoprecipitation experiment in HEK293T cells expressing Flag-YME1L1 and HA-tag TRIM21 mutants.

[0029] Figure 11 TRIM21 promotes the ubiquitination and degradation of YME1L1 at position K63: A shows the quantitative RT-PCR analysis of YME1L1 mRNA levels after overexpression of TRIM21 in designated bladder cancer cell lines (T24, 5637, UMUC3, RT4) and normal ureteral epithelial cell line (SV-HUC-1); B shows the Western blotting analysis of YME1L1 protein levels after overexpression of TRIM21 in the same cell line series, with GAPDH as a loading control; C shows the ubiquitination experiment performed in HEK293T cells by co-expression of Flag-YME1L1, HA-ubiquitin, and TRIM21, with cell lysates immunoprecipitated using anti-Flag magnetic beads and Western blotting performed using a designated antibody; D shows the ubiquitination experiment using HA-tagged ubiquitin mutants K48R and K63R.

[0030] Figure 12 TRIM21 regulates the stability of YME1L1 protein: A shows the YME1L1 protein level after gradient overexpression of TRIM21 in HEK293T cells, as analyzed by Western blotting; B shows the actinomycete tracing experiment in HEK293T cells overexpressing TRIM21.

[0031] Figure 13High TRIM21 expression is associated with a favorable prognosis in bladder cancer tissues and exhibits spatial co-localization with YME1L1. A represents the prognostic value of TRIM21 expression in the TCGA bladder cancer cohort, based on Kaplan-Meier survival analysis of 408 bladder cancer patients (n=408) from the TCGA database stratified by TRIM21 mRNA expression levels. B shows the correlation between TRIM21 and YME1L1 at the mRNA level. C represents the spatial co-localization of the transcriptomes of TRIM21 and YME1L1. D shows representative double-labeled immunofluorescence staining of bladder cancer patient tissue sections (green: TRIM21; red: YME1L1; blue: DAPI). The lower image shows the cancerous region, YME1L1 is mainly expressed in proliferating epithelial cells, and the upper image shows paired adjacent normal tissues. E shows the TRIM21 protein expression level analyzed by Western blotting in 6 pairs of fresh bladder tumor tissues (T) and paired adjacent normal tissues (N), with GAPDH as a loading control.

[0032] Figure 14 Knocking down TRIM21 promotes the proliferation, migration, and invasion of bladder cancer cells. A shows the efficiency of TRIM21 knockdown in T24 and 5637 cells infected with either a control (shCtrl) or TRIM21-targeting shRNA (sh-T-1, sh-T-2) viruses, verified by Western blotting. B shows cell proliferation as detected by the CCK-8 assay after TRIM21 knockdown. C shows cell proliferation as detected by the EdU assay after TRIM21 knockdown. D shows cell migration ability as assessed by the scratch healing assay after TRIM21 knockdown. E shows cell invasion ability as assessed by the Transwell assay after TRIM21 knockdown.

[0033] Figure 15 The study showed that TRIM21 knockdown inhibited apoptosis in bladder cancer cells and improved mitochondrial function. Figure A shows the apoptosis level analyzed by Annexin V-FITC / PI double staining flow cytometry after TRIM21 knockdown; Figure B is a quantitative statistical graph of Figure A; Figure C shows the mitochondrial membrane potential assessed by JC-1 staining after TRIM21 knockdown; Figure D shows the intracellular reactive oxygen species (ROS) level observed by fluorescence microscopy after TRIM21 knockdown (green: ROS; blue: DAPI); and Figure E shows the cellular ATP production detected after TRIM21 knockdown.

[0034] Figure 16The study investigated the effects of TRIM21 overexpression on the proliferation, migration, and invasion of bladder cancer cells. Specifically, A was a Western blotting assay to verify the effect of stable overexpression of YME1L1 in T24 and 5637 cells; B was a CCK8 assay to detect the effect of TRIM21 overexpression on bladder cancer cell proliferation; C was an EdU assay to detect the effect of TRIM21 overexpression on bladder cancer cell proliferation; D was a scratch healing assay to assess the effect of TRIM21 overexpression on the migration ability of bladder cancer cells; and E was a Transwell assay to assess the effect of TRIM21 overexpression on the invasive ability of bladder cancer cells.

[0035] Figure 17 Overexpression of TRIM21 promotes apoptosis in bladder cancer cells and impairs mitochondrial function. A shows the effect of TRIM21 overexpression on apoptosis levels in bladder cancer cells detected by Annexin V-FITC / PI double staining flow cytometry (left image) and the corresponding quantitative analysis (right image); B shows the intracellular reactive oxygen species (ROS) level observed by fluorescence microscopy after TRIM21 overexpression (green: ROS; blue: DAPI); C shows the mitochondrial membrane potential level assessed by JC-1 staining after TRIM21 overexpression; and D shows the ATP production of cells after TRIM21 overexpression.

[0036] Figure 18 Knocking down TRIM21 can partially reverse the inhibitory effect of YME1L1 deletion on the malignant phenotype of bladder cancer cells; where A is a schematic diagram of experimental design for four genetic backgrounds: control (shCtrl), YME1L1 knockdown (sh-Y), TRIM21 knockdown (sh-T), and double knockdown of YME1L1 and TRIM21 (sh-Y+T). B is the CCK-8 assay used to detect the dynamic changes in cell proliferation in each group over 72 hours; C is the EdU assay used to detect the dynamic changes in cell proliferation in each group; D is the Transwell assay used to assess the invasive ability of each group of cells; E is the scratch healing assay used to assess the migration ability of each group of cells.

[0037] Figure 19 Knockdown of TRIM21 partially reversed YME1L1 deficiency-induced oxidative stress, mitochondrial dysfunction, apoptosis, and in vivo tumorigenesis inhibition in bladder cancer cells. A shows intracellular reactive oxygen species (ROS) levels observed under fluorescence microscopy (green: ROS; blue: DAPI); B shows ATP production in each group; C shows JC-1 staining to assess mitochondrial membrane potential; D shows Annexin V-FITC / PI double staining flow cytometry analysis of apoptosis levels; EG shows a nude mouse subcutaneous tumorigenesis experiment (in vivo rescue validation) to verify the effect of YME1L1 / TRIM21 regulation on the in vivo tumorigenesis ability of bladder cancer cells.

[0038] *p <0.05,** p <0.01,*** p <0.001,**** p <0.0001. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0040] The experimental materials used in the embodiments of this invention are as follows:

[0041] 1. Cell lines

[0042] T24, 5637, and 293T cells were all purchased from Shanghai Fuheng Biotechnology Co., Ltd. T24 and 5637 cells were cultured in DMEM high-glucose basal medium supplemented with 10% fetal bovine serum (FBS) as the complete medium. 5637 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. The culture environment was maintained at a constant temperature of 37°C, 5% CO2 concentration, and 95% relative humidity.

[0043] 2. Antibodies and reagents

[0044] Anti-YME1L1 (11510-1-AP; Wuhan Sanying), anti-Myc (16286-1-AP, Wuhan Sanying), anti-ubiquitin (10201-2-AP, Wuhan Sanying), anti-GAPDH (GB15004-100, Wuhan Saiweier), anti-TRIM21 (12108-1-AP, Wuhan Sanying), anti-Flag (F1804, Sigma) antibody, goat anti-rabbit secondary antibody (GAR007, Wuhan Sanying), goat anti-mouse secondary antibody (GAM007, Wuhan Sanying). EdU Cell Proliferation Detection Kit (C0075L, Shanghai Beyotime), CCK-8 Kit (C0037, Beyotime), DMEM High Glucose Medium (PM150210, Wuhan Pronosai), RPMI-1640 Medium (PM150110, Wuhan Pronosai), Apoptosis Detection Kit (AP101C, Linko Biotechnology), Lipofectamine 3000 Transfection Reagent (L3000015, Thermo Fisher Scientific), Reverse Transcription Kit (E047-01B, Nearshore Protein), Premium Fetal Bovine Serum (164210, Wuhan Pronosai).

[0045] 3. Plasmid construction and lentivirus packaging

[0046] YME1L1 and TRIM21 overexpression plasmids, YME1L1 and TRIM21 knockdown shRNA plasmids, lentiviral packaging helper plasmids psPAX2 and pMD2.G, ubiquitin mutant plasmids, and truncated mutant plasmids (pcDNA3.1-Myc-TRIM21 ΔRING (Δ15–58); pcDNA3.1-Myc-TRIM21 ΔB-box (Δ91–128); pcDNA3.1-Myc-TRIM21 ΔPRY (Δ286–334); pcDNA3.1-Myc-TRIM21 ΔSPRY (Δ333–463)) were all purchased from UBO Biotechnology (Changsha, China).

[0047] Virus packaging: 293T cells were plated the day before transfection, with a density of 70%-90% at transfection. The packaging plasmids psPAX2 and pMD2.G were co-transfected with the target plasmid using liposomes, with a plasmid ratio of transfer plasmid:psPAX2:pMD2.G = 4:2:1 (mass ratio). The supernatant was collected at 48h and 72h post-transfection and filtered through a 0.45μm filter membrane.

[0048] 4. Construction of stable cell lines

[0049] One day before infection, target cells in the logarithmic growth phase are plated to achieve a confluence of 70%-80% at infection. On the day of infection, the cell supernatant is discarded, and cell supernatant containing the virus is added. 8-12 hours later, the medium is replaced with fresh complete medium. 48-72 hours after infection, puromycin (final concentration 2 μg / ml) is added to the culture system to select stable cells. Selection typically continues for at least 14 days, with the medium replaced with fresh antibiotic-containing medium every 2-3 days. At this point, polyclonal stable transfectants are obtained, and infection efficiency is assessed using Western blot (WB).

[0050] 5. Bioinformatics Analysis

[0051] 5.1 Association analysis of pan-cancer level YME1L1 or TRIM21 expression with overall survival (OS)

[0052] YME1L1 mRNA expression profiles and corresponding patient clinical follow-up data for 33 cancer types, including adrenocortical carcinoma (ACC) and bladder urothelial carcinoma (BLCA), were downloaded from the TCGA (The Cancer Genome Atlas) database. Cox proportional hazards regression analysis was performed using RStudio software (R language) to assess the relationship between YME1L1 expression levels (as a continuous variable or divided into high / low expression groups based on median expression) and overall survival (OS). The results are presented as forest plots showing the hazard ratio (HR) and its 95% confidence interval (CI) for each cancer type. Red dots indicate HR>1 (risk factor), blue dots indicate HR<1 (protective factor), and dashed lines indicate HR=1. A difference was considered statistically significant (p<0.05) if the confidence interval did not intersect the dashed line.

[0053] 5.2 Effects of YME1L1 or TRIM21 expression on overall survival (OS) and progression-free survival (PFI) in bladder cancer patients

[0054] Based on RNA-seq data and clinical information of bladder urothelial carcinoma (BLCA) patients in the TCGA database, patients were divided into a high-expression group (High) and a low-expression group (Low) according to YME1L1 expression levels (the grouping threshold was usually the median or the optimal cutoff value). Kaplan-Meier survival curves for overall survival (OS) and progression-free survival interval (PFI) were plotted for both groups, and the survival differences between the two groups were compared using the log-rank test. p < 0.05 was considered statistically significant.

[0055] 5.3 Single-cell transcriptome data analysis

[0056] Single-cell RNA-seq data of bladder cancer samples (3 tumor tissues and their paired normal tissues) were obtained from the GEO database (GSE222315). Single-cell objects were constructed using the Seurat package, and the following quality control (QC) criteria were applied: each gene was expressed in at least 5 cells, and at least 200 genes were detected per cell. Mitochondrial genes (MT-) and hemoglobin genes (HBA / HBB) were used as QC indicators. A dynamic filtering threshold based on median absolute deviation (MAD) was used for rigorous quality control and batch effect correction. The filtering criteria included: retaining cells with 200 to 10,000 genes, 500 to 50,000 UMI counts, and less than 25% mitochondrial gene percentage. Batch effect correction was performed using the Harmony algorithm, followed by dimensionality reduction using PCA. Multi-resolution clustering was performed using the Louvain algorithm (resolution range: 0.01–1.0). The optimal resolution (0.1) was determined by the clustering tree, and a UMAP / t-SNE visualization was generated. Cells were annotated using classical marker genes, and high / low expression subgroups of tumor cells were defined based on the detection rate of YME1L1. Differential gene analysis was performed (|logFC|>0.5, adj.p<0.05). GO biological process enrichment analysis was performed on differentially expressed genes using the clusterProfiler package, and the top 3 pathways with significantly enriched pathways (adj.p<0.05) were visualized.

[0057] 5.4 Spatial Transcriptome Data Analysis

[0058] Spatial transcriptome data (sample B1) was downloaded from GSE246011, and the gene expression matrix was integrated with spatial coordinates. Quality control criteria included: UMI count > 200, number of detected genes > 200, and mitochondrial gene percentage < 10%. Blobs meeting these criteria were retained for subsequent analysis. Spatial data were normalized using SCTransform, and the top 3,000 hypervariable genes were selected. Dimensionality reduction was performed using PCA, and spatial domains were identified using Louvain clustering (resolution = 0.2). The spatial coordinates were integrated with the transcriptome data to generate a high-resolution spatial expression map of YME1L1 / TRIM21.

[0059] 6. Survival rate determination

[0060] The viability of 786-O and 769-P cells was determined using the CCK-8 colorimetric method. After cell treatment, the culture medium in the 96-well plates was removed, and 100 µL of fresh culture medium containing 10 µL of CCK-8 was added to each well. After incubation at 37°C for 1–2 h, the absorbance at 450 nm was measured using a microplate reader. For EDU assay: Cells were seeded in 24-well plates, and after adhesion, incubation and staining were performed according to the instructions of the EDU proliferation kit (Beyotime, USA). The absorbance at 450 nm was then measured using a microplate reader.

[0061] 7. Immunoblotting

[0062] Select a suitable lysis buffer according to experimental requirements, and add protease inhibitors (such as PMSF) and phosphatase inhibitors a few minutes before use. Lyse cells on ice for 15 min using the lysis buffer, then centrifuge and collect the supernatant. Quantify protein using BCA and separate an equal volume of protein (30 μg) by 10% SDS-PAGE electrophoresis, transferring to a PVDF membrane (300 mA, 90 min). Block with 5% BSA at room temperature for 1 h, add YME1L1 primary antibody (1:1000) and incubate overnight at 4°C. Wash three times with TBST, add HRP-goat anti-rabbit secondary antibody (1:5000) and incubate at room temperature for 1 h. Expose to ECL substrate for development.

[0063] 8. Immunofluorescence

[0064] Cell slides were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. Primary antibodies (anti-YME1L1, 1:200; TRIM21, 1:200) were incubated overnight at 4°C, followed by incubation with fluorescent secondary antibody (Alexa Fluor 568, 1:500) at room temperature in the dark for 1 h. Nuclei were stained with DAPI for 5 min, and the slides were mounted and observed and photographed under a fluorescence microscope.

[0065] 9. Immunohistochemistry

[0066] Tissue sections were dewaxed with xylene and hydrated with graded ethanol; microwave antigen retrieval was performed for 15 min using sodium citrate buffer (pH 6.0); endogenous enzymes were inactivated by incubation with 3% H2O2 for 10 min; and the sections were blocked with 5% goat serum at room temperature for 30 min. Primary antibodies (anti-YME1L1, 1:200; TRIM21, 1:200) were added and incubated overnight at 4°C. The next day, HRP-labeled secondary antibody (1:500) was added and incubated at room temperature for 1 h, followed by DAB staining for 1–3 min, and counterstaining with hematoxylin for 30 s. After dehydration and clearing, the sections were mounted with neutral resin and images were acquired using an optical microscope (×200).

[0067] 10. CCK8 Experiment

[0068] Cell viability was determined using the CCK-8 colorimetric assay. After cell treatment, the culture medium in the 96-well plate was removed, and 100 µL of fresh culture medium containing 10 µL of CCK-8 was added to each well. After incubation at 37°C for 1–2 h, the absorbance at 450 nm was measured using a microplate reader.

[0069] 11. EDU Experiment

[0070] Cells at 5 × 10 3 / well seeding, labeled with 10 μmol / L EdU for 2 h, fixed with 4% PFA, and permeabilized with 0.5% Triton X-100. Incubate with EdU reaction solution in the dark for 30 min, then stain nuclei with DAPI. Image taken using a confocal microscope, counted using ImageJ, and the proliferation rate was calculated as EdU / Ed ... + / DAPI + ×100%. Statistical analysis was performed using t-tests or ANOVA.

[0071] 12. Transwell test

[0072] A layer of Matrigel (e.g., Matrigel) is placed on the upper surface of the Transwell chamber and solidified at 37°C. Cells are digested, resuspended in serum-free medium, and the density is adjusted to 1 × 10⁶ cells / mL. 5 / mL. Add 200μL of cell suspension to the upper chamber and 500μL of culture medium containing chemokines to the lower chamber. Incubate at 37℃ for 24 hours, remove the chamber, wash with PBS, wipe away uninvaded cells from the upper chamber with a cotton swab, fix with 4% paraformaldehyde, and stain with crystal violet. Randomly select at least 5 fields of view under a microscope and count the cells that have invaded the lower chamber surface.

[0073] 13. JC-1 staining Treat cells according to experimental requirements, discard the culture medium, and gently wash once with PBS. Add JC-1 working solution to a final concentration of 2 μg / mL and incubate at 37°C in the dark for 30 minutes. Discard the staining solution and wash 1-2 times with JC-1 buffer or PBS. Immediately detect cells using a fluorescence microscope (red excitation / green emission) or flow cytometry. Normal cells have a high mitochondrial membrane potential, forming red J-aggregates; apoptotic cells have a decreased membrane potential, and JC-1 exists as green monomers.

[0074] 14. Measurement of mitochondrial reactive oxygen species (ROS)

[0075] Intracellular ROS levels were detected using a reactive oxygen species (ROS) detection kit (S0033S, Beyotime Biotechnology, Shanghai). Cells cultured in confocal culture dishes were loaded with 10 μM DCFH-DA probe (1:1000 dilution) diluted in extracellular fluid (C0216) and incubated at 37°C for 20 min. After incubation, cells were washed three times with extracellular fluid to remove excess probe. Where applicable, cells were treated with Rosup for 20–30 min as a positive control. Confocal imaging was performed using a FITC equivalent setting with 488 nm excitation and 525 nm emission detection. Fluorescence intensity was quantified using ImageJ software, and data were normalized to the control group.

[0076] 15. Apoptosis experiment

[0077] Apoptosis rate was detected using the Annexin V / PI kit (Linken Biotech, China). After treatment, cells were washed with pre-chilled PBS and then resuspended in 500 μL of binding buffer. PI and Annexin V-FITC staining was performed at room temperature for 5 min. Apoptosis was analyzed using flow cytometry (Beckman, Germany).

[0078] 16. ATP Measurement

[0079] Intracellular ATP levels were determined using an ATP assay kit (S0026, Beyotime Biotechnology Co., Ltd., Shanghai). For adherent cells, after aspirating the culture medium, 200 µL of lysis buffer was added to each well (6-well plate) on ice for lysis. After complete lysis, the lysis buffer was centrifuged at 4°C, 12,000 × g for 5 minutes, and the supernatant was collected for analysis. For assay, 100 µL of working solution was added to each well of the luminescent plate and incubated at room temperature for 3–5 minutes, followed by 20 µL of sample. The luminescence intensity (RLU) was measured after a 2-second delay. The ATP concentration for each sample was calculated based on the standard curve and corrected for by the total protein concentration determined by the BCA method.

[0080] The amino acid sequences of TRIM21 and YME1L1 in this embodiment are shown below:

[0081] The amino acid sequence of TRIM21 (SEQ ID NO:1): MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY

[0082] Amino acid sequence of YME1L1 (SEQ ID NO:2): MFSLSSTVQPQVTVPLSHLINAFHTPKNTSVSLSGVSVSQNQHRDVVPEHEAPSSECMFSDFLTKLNIVSIGKGKIFEGYRSMFMEPAKRMKKSLDTTDNWHIRPEPFSLSIPPSLNLRDLGLSELKIGQIDQLVENLLPGFCKGKNISSHWHTSHVSAQSFFENKYGNLDIFSTLRSSCLYRHHSRALQSICSDLQYWPVFIQSRGFKTLKSRTRRLQSTSERLAETQNIAPSFVKGFLLRDRGSDVESLDKLMKTKNIPEAHQDAFKTGFAEGFLKAQALTQKTNDSLRRTRLILFVLLLFGIYGLLKNPFLSVRFRTTTGLDSAVDPVQMKNVTFEHVKGVEEAKQELQEVVEFLKNPQKFTILGGKLPKGILLVGPPGTGKTLLARAVAGEADVPFYYASGSEFDEMFVGVGASRIRNLFREAKANAPCVIFIDELDSVGGKRIESPMHPYSRQTINQLLAEMDGFKPNEGVIIIGATNFPEALDNALIRPGRFDMQVTVPRPDVKGRTEILKWYLNKIKFDQSVDPEIIARGTVGFSGAELENLVNQAALKAAVDGKEMVTMKELEFSKDKILMGPERRSVEIDNKNKTITAYHESGHAIIAYYTKDAMPINKATIMPRGPTLGHVSLLPENDRWNETRAQLLAQMDVSMGGRVAEELIFGTDHITTGASSDFDNATKIAKRMVTKFGMSEKLGVMTYSDTGKLSPETQSAIEQEIRILLRDSYERAKHILKTHAKEHKNLAEALLTYETLDAKEIQIVLEGKKLEVR

[0083] The shRNAs used in this example are shown in Table 1:

[0084] Table 1 shRNA Sequence List

[0085]

[0086] Example 1 YME1L1 Expression Is Associated with Poor Survival Outcomes in Bladder Cancer

[0087] We analyzed the association between YME1L1 mRNA expression and prognosis in bladder cancer patients from the TCGA database. The results showed that in the bladder cancer samples (n=408), YME1L1 expression was significantly associated with shorter overall survival and progression-free survival. Figure 1 (A). Furthermore, we found no significant difference in YME1L1 expression between normal bladder tissue and cancerous tissue, but it was significantly upregulated in high-grade bladder cancer compared to low-grade bladder cancer. Figure 1 Based on the analysis results, we hypothesize that YME1L1 expression is associated with bladder cancer progression. Immunofluorescence analysis showed that YME1L1 is widely localized in normal bladder tissue; however, notably, its expression is highly concentrated in the epithelial region in cancerous tissue. This significant change in expression distribution suggests that YME1L1 may be involved in regulating cancer cell proliferation ( ). Figure 1 (C). To verify this hypothesis, we performed Western blot analysis on six pairs of paired bladder cancer and adjacent normal tissue samples. The results showed that, compared with paired adjacent normal tissues, most cancer tissues had higher protein expression levels of YME1L1 (C). Figure 1 (D). Subsequently, we compared the protein expression of YME1L1 by immunohistochemical staining of bladder cancer tissue sections (n=55). Negative expression of YME1L1 appeared to be associated with better clinicopathological features, including lower T stage, lower tumor grade, and non-invasive characteristics (D). Figure 1 (E and F in the middle).

[0088] Example 2: Single-cell transcriptome analysis reveals the expression pattern of YME1L1 in bladder cancer and its association with immune regulation.

[0089] We first analyzed the overall expression distribution of YME1L1 using single-cell transcriptome data based on UMAP clustering. Figure 2 (A). The detected cells were divided into multiple subpopulations and manually annotated using established cell type markers to identify the major cell types, such as T cells, epithelial cells, and fibroblasts. Figure 2 (B). Our analysis showed a significant difference in the expression levels of YME1L1 between normal and tumor samples. Figure 3 (A). Furthermore, the detection rate of YME1L1 (defined as the proportion of cells expressing this gene) also showed significant differences within the tumor group. Figure 3 (B). Further research showed that YME1L1 exhibited heterogeneous distribution in tumor subpopulations with different detection rates. When comparing cells with high and low YME1L1 detection rates, volcano plots revealed a large number of differentially expressed genes ( Figure 4This suggests that YME1L1 may influence specific functional states of tumor cells. We then performed differential expression analysis on cells with high and low YME1L1 expression in the tumor group, and conducted gene ontology enrichment analysis of biological processes. This analysis revealed a significant enrichment of immune-related pathways associated with differential YME1L1 expression. Figure 5 The enrichment patterns were cell type specific: B cells were involved in T cell and lymphocyte differentiation pathways; endothelial cells and fibroblasts were associated with chemotaxis and leukocyte migration; myeloid cells and plasma cells were significantly enriched in T cell activation and immune cell activation pathways; and T cells were associated with positive regulation of leukocyte proliferation and the JAK-STAT signaling pathway. In summary, these results suggest that YME1L1 may participate in regulating tumor immune responses by modulating the differentiation, chemotaxis, and activation of immune cells in the tumor microenvironment.

[0090] Example 3: YME1L1 enhances the proliferation, invasion, and mitochondrial function of bladder cancer cells.

[0091] To investigate the biological role of YME1L1 in bladder cancer cells, we used lentiviruses carrying either a non-targeted control shRNA (shCtrl) or short hairpin RNAs targeting YME1L1 (sh-YM-1, sh-YM-2) to construct T24 and 5637 cell lines that stably knocked down YME1L1. Figure 6 (A). CCK8 and EdU experiments confirmed that knocking down YME1L1 significantly weakened the proliferative capacity of bladder cancer cells. Figure 6 (B and C). Transwell and scratch assay results showed that the knockdown group had lower invasive and migration abilities than the control group ( Figure 6 (D and E). Flow cytometry analysis showed that downregulation of YME1L1 significantly increased the apoptosis rate of bladder cancer cells (D and E). Figure 7 (A and B in the original text). To investigate the role of YME1L1 in mitochondrial function, we conducted JC-1, reactive oxygen species, and ATP experiments. The results showed that knockdown of YME1L1 impaired mitochondrial function (…). Figure 7 (C to E). Subsequently, we constructed T24 and 5637 cell lines that stably overexpress YME1L1 (C to E). Figure 8 (A) to further investigate its pro-cancer effect. In contrast to the knockdown effect, overexpression of YME1L1 significantly promoted the proliferation of bladder cancer cells ( ). Figure 8 (B and C), invasion ( Figure 8 D), migration ( Figure 8 (E) and mitochondrial function ( Figure 9 (From A to C), while a reduction in cell apoptosis was detected ( Figure 9 (Middle D). These results collectively indicate that YME1L1 enhances the malignant phenotype of bladder cancer cells in vitro.

[0092] Example 4: TRIM21 interacts with YME1L1 and targets it for ubiquitin-mediated degradation.

[0093] We used liquid chromatography-tandem mass spectrometry (LC-MS / MS) to screen for potential regulators of YME1L1 in intracellular signaling pathways. Figure 10 (A). In short, Flag-tagged YME1L1 was transiently overexpressed in HEK293T cells, and its binding complex was isolated for LC-MS / MS analysis. Based on the mass spectrometry results, we identified three E3 ubiquitin ligases in the protein co-precipitated with YME1L1. Two of them are involved in the ubiquitination pathway, and one is involved in the SUMOylation pathway (…). Figure 10 (B). We then performed endogenous immunoprecipitation experiments in T24 and 5637 cells. The results showed that only the interaction between TRIM21 and YME1L1 produced a positive signal in the Western blot analysis ( ). Figure 10 TRIM21 contains five major domains: an N-terminal RING domain (amino acids 15-58), a B-Box domain (amino acids 98-128), a coil-and-coil domain, and C-terminal PRY (amino acids 286-334) and SPRY (amino acids 335-462) domains. We used PyMOL to predict the binding interface between YME1L1 and TRIM21, and found that most predicted interaction sites were located within the PRY or SPRY domains. Figure 10 Based on known functional domains, we constructed a series of single or combined deletion mutants (D). Figure 10 (E). Protein-protein interaction experiments showed that the loss of the SPRY domain alone disrupts the binding of TRIM21 to YME1L1 ( Figure 10 (E). In summary, our results indicate that TRIM21 primarily interacts with YME1L1 through its SPRY domain. To investigate the regulatory role of TRIM21 on YME1L1 at the RNA or protein level, we overexpressed TRIM21 in four bladder cancer cell lines and one immortalized normal ureteral epithelial cell line. No significant changes in YME1L1 RNA levels were observed in these cells (E). Figure 11 (A). However, Western blot analysis showed that YME1L1 protein expression was significantly reduced in T24 and 5637 cells, but not significantly changed in UMUC3 cells. Figure 11 In RT4 and SV-HUC-1 cells, the basal expression level of YME1L1 was too low to detect a precise change. Figure 11(Middle B). In summary, these findings indicate that TRIM21 regulates YME1L1 at the protein level. TRIM21 is a well-characterized E3 ubiquitin ligase known to mediate its own ubiquitination and the ubiquitination of various substrates, thereby regulating their degradation or activation. This prompted us to investigate whether its interaction with YME1L1 induces YME1L1 ubiquitination. Interestingly, we observed significant YME1L1 ubiquitination in cells overexpressing TRIM21, while basal ubiquitination levels were low under control conditions (Middle B). Figure 11 The results (C) indicate that TRIM21 binding promotes the ubiquitination of YME1L1. Since TRIM21 has been reported to mediate K63- and K48-linked ubiquitination, this study investigated the effects of TRIM21 on the ubiquitination level and chain type of YME1L1 in 293T cells to clarify the molecular mechanism by which TRIM21 regulates YME1L1. The results showed that TRIM21 overexpression significantly promoted YME1L1 ubiquitination. Even with co-transfection with the ubiquitin mutant ub-K48R, TRIM21 still significantly enhanced YME1L1 ubiquitination; however, with co-transfection with ub-K63R, TRIM21-mediated YME1L1 ubiquitination essentially disappeared. This suggests that TRIM21 primarily mediates K63-linked ubiquitination of YME1L1, rather than K48-linked ubiquitination. Figure 11 (D). Our studies on the stability of the YME1L1 protein show that TRIM21 promotes its degradation. Specifically, gradient expression of TRIM21 leads to a corresponding decrease in the abundance of YME1L1 (D). Figure 12 (A). Consistently, overexpression of TRIM21 in the presence of actinomycete ketone directly reduced the stability of YME1L1 and shortened its half-life. Figure 12 (B)

[0094] Example 5: High expression of TRIM21 is associated with a favorable prognosis in bladder cancer tissues and exhibits spatial co-localization with YME1L1.

[0095] Based on the analysis of transcriptome data from 408 bladder cancer patients in the TCGA cohort, we found that patients with high TRIM21 expression had better clinical outcomes, suggesting that TRIM21 may act as a protective factor in bladder cancer. Figure 13 (A). This observation is consistent with our previous finding that TRIM21 promotes the ubiquitination and degradation of the oncoprotein YME1L1. Subsequent analysis of TCGA data showed that TRIM21 and YME1L1 expression were not correlated at the mRNA level. Figure 13(B) Interestingly, spatial transcriptomic analysis revealed significant spatial co-localization of TRIM21 and YME1L1 within tissue sections—both genes exhibited synergistic high expression in different cell subpopulations, suggesting potential functional synergy or interaction within the local microenvironment. Figure 13 (C). We further performed double-label immunofluorescence staining (green: TRIM21, red: YME1L1, blue: DAPI) on bladder cancer tissue sections from one patient to differentiate cancerous areas based on nuclear morphology and structural arrangement. Figure 13 (Middle D, top image) and adjacent normal tissue ( Figure 13 (See figure below). Results showed that TRIM21 was mainly expressed in epithelial cells of adjacent normal tissue, while YME1L1 was mainly expressed in proliferating epithelial cells within the cancerous region. Notably, both proteins are located in epithelial cells and have similar spatial distributions, providing topological evidence for their potential interaction. Furthermore, Western blot analysis of six pairs of paired fresh tumor tissue and adjacent normal tissue samples showed that in five of the pairs, TRIM21 expression in adjacent normal tissue was higher than that in tumor tissue. Figure 13 These findings collectively indicate that TRIM21 is primarily expressed in normal tissues and inhibits tumorigenesis.

[0096] Example 6: Knocking down TRIM21 promotes the proliferation and migration of bladder cancer cells and improves mitochondrial function.

[0097] To investigate the biological role of TRIM21 in bladder cancer cells, we constructed stable TRIM21 knockdown T24 and 5637 cell lines using lentiviruses carrying either a non-targeted control shRNA (shCtrl) or a TRIM21-targeting shRNA (sh-T-1, sh-T-2). Figure 14 (A). CCK8 and EdU experiments showed that knockdown of TRIM21 significantly increased the proliferative capacity of bladder cancer cells. Figure 14 (B and C). Furthermore, scratch and Transwell invasion assays showed that knocking down TRIM21 significantly promoted the migration and invasion of bladder cancer cells (B and C). Figure 14 (C and D). Flow cytometry analysis showed that downregulation of TRIM21 significantly reduced the apoptosis rate of bladder cancer cells (C and D). Figure 15 (A and B). We used JC-1, reactive oxygen species, and ATP assays to assess the function of TRIM21 in mitochondria. The results showed that knocking down TRIM21 improved mitochondrial function (A and B). Figure 15 (C to E). To investigate the tumor-suppressive function of TRIM21 in more detail, we constructed T24 and 5637 cell clones stably overexpressing exogenous TRIM21 and used Western blotting to detect the knockdown efficiency (C to E). Figure 16(A). In contrast to the TRIM21 knockdown phenotype, overexpression of TRIM21 significantly inhibited the proliferation of bladder cancer cells ( ). Figure 16 (B and C), migration invasion ( Figure 16 D and E) and mitochondrial function ( Figure 17 (B and C). Furthermore, overexpression of TRIM21 leads to increased apoptosis in bladder cancer cells ( Figure 17 (A). All these results indicate that TRIM21 inhibits the proliferation, migration, invasion, and mitochondrial activity of bladder cancer in vitro.

[0098] Example 7: Knockdown of TRIM21 reverses the inhibitory effect of YME1L1 deficiency on bladder cancer cells.

[0099] To determine whether the oncogenic phenotype induced by TRIM21 inhibition is functionally dependent on YME1L1, we constructed four stable bladder cancer cell lines with different genetic backgrounds (control, TRIM21 knockdown, YME1L1 knockdown, and double knockdown) and performed rescue experiments: control (shCtrl), YME1L1 knockdown (sh-Y), TRIM21 knockdown (sh-T), and combined double knockdown of YME1L1 and TRIM21 (sh-Y+T). Figure 18 (A). The results showed that knockdown of TRIM21 largely reversed the tumor suppressive effect induced by YME1L1 deficiency. Specifically, knockdown of YME1L1 alone significantly inhibited the proliferation of bladder cancer cells ( Figure 18 (B and C), invasion ( Figure 18 D), transferability ( Figure 18 E) also leads to the downregulation of key parameters of mitochondrial function ( Figure 19 The upregulation of apoptosis levels (from A to C) and (from A to C) Figure 19 In the group with the combined knockdown of TRIM21 and YME1L1, significant recovery was observed. Furthermore, to simulate the in vivo environment, we conducted xenograft tumor experiments in nude mice using four cell lines derived from 5637. Figure 19 (Middle E). Consistent with our previous findings, knockdown of TRIM21 reverses the tumor suppressive effect induced by YME1L1 knockdown. Figure 19 (F and G). This functional rescue experiment shows that the tumorigenic function of TRIM21 is mainly mediated by the inhibition of YME1L1, thus positioning TRIM21 as a key upstream effector in this regulatory pathway.

[0100] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of TRIM21-related biological products in the preparation of reagents that promote the degradation of YME1L1, characterized in that, The TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing a nucleic acid molecule encoding TRIM21; A nucleic acid molecule containing the encoded TRIM21 or a vector containing the expression cassette.

2. The application according to claim 1, characterized in that, The TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the K63-linked polyubiquitination modification of YME1L1, thereby promoting the degradation of YME1L1.

3. The application of TRIM21-related biological products in the preparation of reagents that shorten the half-life of YME1L1 in cells, characterized in that, The TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing a nucleic acid molecule encoding TRIM21; A nucleic acid molecule containing the encoded TRIM21 or a vector containing the expression cassette.

4. The application according to claim 3, characterized in that, The TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the K63-linked polyubiquitination modification of YME1L1, thereby promoting the degradation of YME1L1 and shortening its half-life in cells.

5. The application of TRIM21-related biological products in the preparation of drugs for treating bladder cancer, characterized in that, The TRIM21-related biological products include any of the following: Nucleic acid molecules encoding TRIM21; TRIM21 protein molecule; An expression cassette containing the aforementioned nucleic acid molecule encoding TRIM21; A vector containing the above-described nucleic acid molecule encoding TRIM21 or containing the above-described expression cassette; The TRIM21 interacts directly with YME1L1 through its SPRY domain and mediates the polyubiquitination modification of YME1L1 linked to K63, thereby promoting the degradation of YME1L1 to treat bladder cancer.

6. The application according to claim 5, characterized in that, The drug is used for: Inhibits the proliferation of bladder cancer cells; And / or, inhibit the migration of bladder cancer cells; And / or, inhibit the invasion of bladder cancer cells; And / or, induce apoptosis in bladder cancer cells; And / or, impairs mitochondrial function in bladder cancer cells.

7. The application according to claim 6, characterized in that, The damage to mitochondrial function in bladder cancer cells includes reducing mitochondrial membrane potential, decreasing ATP production, and increasing reactive oxygen species levels.

8. The application of biological products related to the inhibition of TRIM21 expression or activity in the preparation of reagents for inhibiting the degradation of YME1L1, characterized in that, The biological products associated with inhibition of TRIM21 expression or activity include any of the following: Monoclonal antibody against TRIM21; Antisense nucleic acid molecules that inhibit TRIM21 expression; Gene editing tools that inhibit TRIM21; Among them, biopharmaceuticals that inhibit TRIM21 expression or activity inhibit YME1L1 degradation by inhibiting the interaction between TRIM21 and YME1L1.

9. The application according to claim 8, characterized in that, The antisense nucleic acid molecule that inhibits TRIM21 expression is an shRNA molecule that specifically targets the TRIM21 coding sequence. The shRNA contains a sense strand and an antisense strand with complementary base pairing. The nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID NO:3~SEQ ID NO:4 or SEQ ID NO:5~SEQ ID NO:

6.

10. The application according to claim 8, characterized in that, The gene editing tool for inhibiting TRIM21 is selected from homologous recombination, TALEN, ZFN, and CRISPR / Cas9.

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