Application of UBR5 in treatment and prognosis evaluation of bladder cancer

Abstract

The invention relates to application of UBR5 in treatment and prognosis evaluation of bladder cancer. According to the invention, the key carcinogenic regulation effect of the UBR5 in NMIBC is determined, the high expression of the UBR5 can predict early recurrence and poor prognosis, and the value of the UBR5 as a biomarker for guiding a future precise treatment strategy is highlighted. Targeted UBR5 can effectively inhibit tumor cell proliferation and movement ability, and manifests the potential of the targeted UBR5 as a therapeutic target. By inhibiting the UBR5 or reducing the biological activity of the UBR5, the development of the NMIBC disease can be obviously delayed, the prognosis is improved, the survival rate of a patient is prolonged, the prognosis is improved, and the potential of developing a personalized treatment strategy for a high-risk NMIBC patient by targeting the UBR5 is manifested; the invention provides sufficient scientific basis and theoretical basis for exploring new bladder cancer diagnosis, prognosis judgment and treatment molecular targets and developing new targeted drugs, is helpful for better realizing accurate treatment, and has important social value and scientific significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of UBR5 in the treatment and prognostic assessment of bladder cancer. Background Technology

[0002] Bladder cancer ranks ninth in incidence worldwide, and it is more common in men—accounting for approximately three-quarters of all confirmed cases. Non-muscle-invasive bladder cancer (NMIBC), including Ta, T1, and carcinoma in situ (CIS), accounts for approximately 75% of all bladder cancer cases. A typical clinical feature of NMIBC is its significant tendency to recur. According to the European Association of Urology (EAU) risk stratification system, the recurrence rate varies significantly across different risk strata, ranging from approximately 30% to 70%. Furthermore, NMIBC exhibits great heterogeneity in disease progression: low-grade tumors rarely progress to muscle-invasive disease, while the five-year progression rate for high-grade lesions (especially CIS) can be as high as 50%. Following transurethral resection of bladder tumor (TURBT), intravesical instillation with BCG or chemotherapy is the standard treatment for NMIBC, aiming to reduce the risk of recurrence and delay disease progression. However, despite adjuvant intravesical instillation therapy, approximately 30%–50% of patients experience disease recurrence.

[0003] In recent years, the search for reliable prognostic biomarkers for non-malignant jaundice (NMIBC) has received increasing attention. Multiple molecules, including p53, Ki-67, and HER2, have been reported to be associated with recurrence and progression, and some non-invasive urinary mRNA-based detection methods have shown potential in predicting clinical outcomes. However, due to the molecular heterogeneity of NMIBC and the limitations of existing biomarkers, reliably predicting postoperative recurrence and long-term prognosis remains challenging. Therefore, novel and robust molecular biomarkers are urgently needed to better predict the recurrence and progression of NMIBC.

[0004] Ubiquitin protein ligase E3 component n-recognition protein 5 (UBR5) is a highly conserved HECT domain E3 ubiquitin ligase located on chromosome 8q22.3. It encodes a protein of approximately 300 kDa, composed of 2799 amino acids. As a key enzyme in the ubiquitin-proteasome system, E3 ligases confer substrate specificity and mediate ubiquitin transfer, thereby regulating protein degradation and functional modification. UBR5 is involved in various tumor-related biological processes, including protein turnover, DNA damage repair, transcriptional regulation, and cell cycle control. Numerous studies have shown that aberrant expression or mutations of UBR5 promote the development and progression of various malignancies, including breast cancer, colorectal cancer, and ovarian cancer, and are associated with poor patient prognosis. Furthermore, a genomics study reported that UBR5 mutations may have prognostic value for overall survival in patients with small cell bladder cancer. However, research exploring the biological role and clinical relevance of UBR5 in non-MIBC remains scarce, and its mechanistic function in this context remains to be elucidated. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the treatment of lethal bladder cancer in the prior art, and to conduct an in-depth study on its glycolytic regulation mechanism. It has been clarified that UBR5 is a key regulatory gene in the glycolytic pathway of lethal bladder cancer, and it has been confirmed that UBR5 can serve as a potential therapeutic target for intervening in glycolysis-driven lethal bladder cancer, providing a new strategy for the treatment and prognostic assessment of bladder cancer.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides the use of UBR5 inhibitors in the preparation of medicaments for the prevention and / or treatment of non-muscle-invasive bladder cancer.

[0008] Preferably, the UBR5 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the UBR5 gene.

[0009] Preferably, the UBR5 inhibitor is selected from siRNA designed based on the UBR5 gene; the siRNA is selected from one or more of si-1, si-2, and si-3; wherein the si-1 sequence is as shown in SEQ ID NO: 1 (5'-GAGAGTTTGCGGCAACTAA-3'), the si-2 sequence is as shown in SEQ ID NO: 2 (5'-GGCTAGTACTAGCATAGAT-3'), and the si-3 sequence is as shown in SEQ ID NO: 3 (5'-GGATTGTAGGTTACTTAGA-3').

[0010] A second aspect of the present invention provides the use of a reagent for detecting UBR5 expression levels in the preparation of products for the auxiliary diagnosis and / or prognostic assessment of non-muscle-invasive bladder cancer.

[0011] Preferably, the reagent for detecting UBR5 expression level includes primers for detecting UBR5 gene expression level and / or reagents for detecting UBR5 protein content.

[0012] Preferably, the primers for detecting the UBR5 gene expression level are selected from the following primer pairs:

[0013] The upstream sequence of the primer pair is shown in SEQ ID NO: 4 (5'-TGGGACCAAATCATGCTGCC-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-GAGAGTCGCTTGTCCTACCAG-3').

[0014] Preferably, the reagent for detecting UBR5 protein content is selected from anti-UBR5 Antibody; for example, it can be selected from YT4809 (Immunoway), etc.

[0015] A third aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of non-muscle-invasive bladder cancer, comprising a UBR5 inhibitor and a pharmaceutically acceptable carrier.

[0016] Preferably, the UBR5 inhibitor is selected from one or more of siRNA, shRNA, and sgRNA designed based on the UBR5 gene.

[0017] Preferably, the UBR5 inhibitor is selected from siRNA designed based on the UBR5 gene; the siRNA is selected from one or more of si-1, sh-2, and sh-3; wherein the si-1 sequence is shown in SEQ ID NO: 1, the sh-2 sequence is shown in SEQ ID NO: 2, and the sh-3 sequence is shown in SEQ ID NO: 3.

[0018] Preferably, the pharmaceutically acceptable pharmaceutical excipient is selected from one or more of fillers, disintegrants, binders, lubricants, flavoring agents, preservatives, antioxidants, and colorants.

[0019] A fourth aspect of the present invention provides a kit for the auxiliary diagnosis and / or prognostic assessment of non-muscle-invasive bladder cancer, including reagents for detecting UBR5 expression levels.

[0020] Preferably, the reagent for detecting UBR5 expression level includes primers for detecting UBR5 gene expression level and / or reagents for detecting UBR5 protein content.

[0021] Preferably, the primers for detecting the UBR5 gene expression level are selected from the following primer pairs:

[0022] The upstream sequence of the primer pair is shown in SEQ ID NO: 4, and the downstream sequence is shown in SEQ ID NO: 5.

[0023] Preferably, the reagent used to detect the UBR5 protein content is selected from anti-UBR5 Antibody.

[0024] Preferably, the kit further includes one or more of PCR enzyme, PCR buffer, dNTPs, and fluorescent substrate.

[0025] Preferably, the fluorescent substrate is selected from Syber Green or fluorescently labeled probes.

[0026] It should be understood that, unless otherwise specified, in the context of this invention, UBR5 includes UBR5 nucleotides and the UBR5 protein encoded by those nucleotides. The UBR5 inhibitor refers to a substance capable of specifically downregulating UBR5 expression levels and / or the transcriptional level of its mature mRNA and / or the expression level or activity of the UBR5 protein. For example, methods such as antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA erasers, target masking, and / or multi-target methods can be used to downregulate UBR5 expression levels and / or activity; any method that can reduce UBR5 levels and / or activity is acceptable. The primers and / or primer pairs refer to PCR primers used to synthesize the UBR5 gene cDNA strand in PCR, thereby detecting the expression level of the UBR5 gene mRNA. In addition to the primers and / or primers listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the UBR5 gene sequence using conventional methods and techniques in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the UBR5 expression level; alternatively, conventional reagents and methods in the art can be used to detect the UBR5 protein expression level; the same applies to other genes / proteins.

[0027] Non-muscle-invasive bladder cancer (NMIBC) poses a significant challenge to clinical diagnosis and treatment due to its high recurrence rate. Recurrence necessitates continuous cystoscopic monitoring and repeated local interventions, significantly impairing patients' quality of life and imposing a heavy socioeconomic burden. Although standard treatment regimens combining TURBT with postoperative intravesical chemotherapy or BCG are widely used, recurrence still occurs in 30-50% of cases. For patients who fail BCG treatment, treatment options are mostly limited to radical cystectomy. For patients unsuitable for or refusing surgery, intravesical penrubicin or systemic pembrolizumab may be considered as alternative treatment strategies. Therefore, there is an urgent need to discover novel molecular biomarkers to improve risk stratification and optimize the long-term management of NMIBC.

[0028] UBR5, a member of the E6-AP C-terminal (HECT) family, was initially identified in breast cancer cells as an E3 ubiquitin ligase mediating the ubiquitination and degradation of specific substrates. Ubiquitination is a key post-translational modification that regulates oncogenic and tumor-suppressive pathways in cancer by modulating protein stability and signal transduction. Against this backdrop, increasing evidence suggests that UBR5 is involved in the development and progression of various cancers. Studies by Wang et al. have shown that UBR5 is upregulated by SUB1 and activates the NF-κB signaling pathway by promoting the Lys11-type polyubiquitination and degradation of UBXN1, thereby driving colorectal cancer progression. In ovarian cancer, tumor-derived UBR5 drives tumor growth and metastasis by promoting the recruitment and activation of immunosuppressive macrophages and maintaining β-catenin signaling. Notably, UBR5 is often downregulated in clear cell renal cell carcinoma, and its low expression is associated with poor prognosis. Although UBR5 plays diverse roles in other cancers, its function in NMIBC remains unexplored; this knowledge gap is the key issue this invention aims to address.

[0029] This invention, through integrated genomics and multicenter NMIBC cohort survival analysis, identified UBR5 as a carcinogenic driver and key gene for poor prognosis in NMIBC. Internal cohort validation confirmed a strong association between UBR5 expression and recurrence-free survival, reinforcing the robustness of this biomarker and highlighting its potential for clinical translation. Furthermore, in vitro experiments demonstrated that knockdown of UBR5 significantly inhibited NMIBC progression, indicating its potential as a novel therapeutic target. Functional enrichment analysis revealed that UBR5 primarily promotes cell cycle and genetic regulatory pathway activation while inhibiting immune-related signaling. This molecular characteristic may partially explain the limited response of NMIBC to BCG immunotherapy.

[0030] The development and progression of bladder cancer are driven by a series of well-defined functional gene alterations, such as FGFR3 and TP53. FGFR3 alterations are one of the most common molecular events in bladder cancer, present in approximately 60% of non-invasive bladder cancer (NMIBC) cases. Notably, UBR5 has been shown to affect genomic integrity and promote somatic mutation accumulation by regulating histone ubiquitination at DNA double-strand breaks. Our analysis shows that high UBR5 expression is associated with TP53 and STAG2 mutations. As a classic tumor suppressor gene, the p53 protein encoded by TP53 plays a central role in preventing tumorigenesis. Aberrant TP53 expression is closely associated with NMIBC progression, and p53 overexpression in T1 stage tumors is considered an important indicator of transformation to muscle-invasive lesions. Previous studies have shown that UBR5 can indirectly inhibit the p53 signaling pathway by ubiquitinizing and degrading CDC73, thus promoting breast cancer metastasis. STAG2, a key cell cycle regulator involved in DNA damage repair, is recurring in NMIBC due to its inactivation, leading to genomic instability and abnormal cell cycle regulation, thereby driving tumor progression. Although there are currently no reports of direct interaction between UBR5 and STAG2, this invention hypothesizes that UBR5 may participate in the NMIBC process by regulating the STAG2-mediated DNA damage response. These findings reveal a previously unknown aspect of genomic regulation in bladder cancer.

[0031] In summary, this invention clarifies the key oncogenic regulatory role of UBR5 in non-invasive bladder cancer (NMIBC), and its high expression can predict early recurrence and poor prognosis, highlighting its value as a biomarker to guide future precision treatment strategies. Targeting UBR5 can effectively inhibit tumor cell proliferation and motility, demonstrating its potential as a therapeutic target. Inhibiting UBR5 or reducing its biological activity can significantly delay the progression of NMIBC, improve prognosis, and prolong patient survival, demonstrating the potential of targeting UBR5 to develop personalized treatment strategies for high-risk NMIBC patients. This provides sufficient scientific evidence and theoretical basis for exploring new molecular targets for bladder cancer diagnosis, prognosis, and treatment, and for developing new targeted drugs, contributing to better precision treatment and possessing significant social and scientific value. Attached Figure Description

[0032] Figure 1 A Venn diagram is used to show the overlap of prognostic genes in the four NMIBC cohorts.

[0033] Figure 2 This is a schematic diagram showing the results of the differential expression level analysis of UBR5 in NMIBC tissue and adjacent normal tissue.

[0034] Figure 3 This is a schematic diagram illustrating the association between UBR5 expression and patient clinical prognosis in a univariate Cox proportional hazards regression analysis.

[0035] Figure 4 This is a schematic diagram illustrating the association between UBR5 expression and patient clinical prognosis in a multivariate Cox proportional hazards regression analysis.

[0036] Figure 5 This is a schematic diagram showing the results of PFS analysis on queues UROMOL2021 and GSE154261.

[0037] Figure 6 This is a schematic diagram showing the OS analysis results for queues GSE48075 and GSE13507.

[0038] Figure 7 This is a schematic diagram of the PFI analysis results for TCGA-BLCA.

[0039] Figure 8 This is a schematic diagram of the immunohistochemical analysis results of clinical samples from NMIBC patients.

[0040] Figure 9 This is a schematic diagram showing the comparative analysis results of UBR5 expression levels in clinical samples of NMIBC patients grouped by T stage and recurrence status.

[0041] Figure 10 This is a schematic diagram of the results of univariate Cox regression analysis of relapse-free survival based on UBR5 expression.

[0042] Figure 11 This is a schematic diagram of the KM curve results for relapse-free survival grouped according to UBR5 expression levels.

[0043] Figure 12 This is a schematic diagram illustrating the ROC curve evaluation of UBR5's predictive performance on NMIBC recurrence.

[0044] Figure 13 This is a schematic diagram showing the inhibitory activity of siRNA on intracellular UBR5 expression levels.

[0045] Figure 14 A schematic diagram illustrating the effect of inhibiting UBR5 expression levels on cell proliferation.

[0046] Figure 15 A schematic diagram illustrating the effect of inhibiting UBR5 expression levels on cell proliferation.

[0047] Figure 16 A schematic diagram illustrating the quantitative analysis results of the effect of inhibiting UBR5 expression levels on cell proliferation.

[0048] Figure 17 A schematic diagram illustrating the effect of inhibiting UBR5 expression levels on cell invasion.

[0049] Figure 18 The bubble plot shows the top 10 significantly enriched KEGG pathways associated with UBR5.

[0050] Figure 19 This is a schematic diagram showing the mutation overview of 18 high-frequency NMIBC mutant genes in the UROMOL2021 cohort.

[0051] Figure 20 A schematic diagram showing the comparison of mutation frequencies of selected genes between the UBR5 high / low expression groups.

[0052] Figure 21 This is a schematic diagram of the genomic variation map results for key driver gene copy number variation events.

[0053] Figure 22 This is a schematic diagram showing the comparison of copy number variation frequencies of key driver genes between the UBR5 high / low expression groups. Detailed Implementation

[0054] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0055] Unless otherwise specified, the cell lines listed in this invention, including RT-4 and SW1710, were all purchased from Pronosei Biotechnology (RT-4: CL-0431, SW1710: CL-0823B) and cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available.

[0056] In this invention, informed consent was obtained from all patients using clinical samples, and the relevant procedures and methods were approved by the ethics committee, complying with medical ethics requirements and the Good Clinical Practice (GCP) guidelines for drug clinical trials. All experimental procedures adhered to the Declaration of Helsinki. The experimental methods used in this invention, such as bioinformatics analysis, molecular biology experiments, cell biology experiments, and immunohistochemical analysis, are all conventional methods and techniques in the field. Bioinformatics analysis was performed using R software version 4.4.0, with the "limma" package used for differential analysis and the "survival" and "survminer" packages used for KM survival curve plotting. Representative results from biological experiment replicates are presented in the contextual figures, and data are displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments were repeated twice. Data were analyzed using GraphPad Prism 10.0 software. Conventional medical statistical methods such as t-tests, chi-square tests, and analysis of variance were used to compare the differences in means between two or more groups. *p < 0.05 was considered a significant difference.

[0057] Example 1

[0058] To identify biomarkers associated with NMIBC, transcriptomic data and corresponding survival information for GSE13507, GSE154261, and GSE48075, as well as UROMOL2021 cohort information (including transcriptomic, mutation, and clinical data), were obtained from the GEO database. The TCGA and GTEx datasets covering 32 solid tumor types, as well as the TCGA-BLCA cohort, were obtained using the UCSC Xena platform; and UBR5 protein expression data in cell lines were obtained from the Human Protein Atlas (HPA) database.

[0059] First, four independent cohorts were analyzed: UROMOL2021, GSE154261, GSE13507, and GSE48075. Univariate Cox regression analysis of progression-free survival (UROMOL2021, GSE154261) and overall survival (GSE13507, GSE48075) screened genes with hazard ratios greater than 1 and p-values ​​less than 0.05. The intersection of these genes showed that only UBR5 consistently identified high risk across all datasets (see [link to relevant documentation]). Figure 1 UBR5 expression was significantly increased in NMIBC tissue compared to paired normal bladder tissue (see [link to original text]). Figure 2 This suggests its potential role in tumor development and progression.

[0060] Subsequently, univariate and multivariate Cox proportional hazards regression analyses were performed using the UROMOL2021, GSE13507, GSE48075, and TCGA-BLCA cohorts to assess the prognostic value of candidate genes, and clinical variables were adjusted to confirm that UBR5 is an independent prognostic risk factor (see [link to relevant documentation]). Figure 3-4 Furthermore, patients in each cohort were divided into UBR5 high-expression and low-expression groups. Kaplan-Meier survival analysis was performed using the survival (version 3.7.0) and survminer (version 0.5.0) software packages. The log-rank test was used to compare survival differences between groups to investigate the influencing factors on progression-free survival (PFS) and overall survival (OS) in NMIBC. KM survival curve results showed that high UBR5 expression was associated with significantly poor prognosis (see...). Figure 5-7 Notably, a similar trend was observed in the TCGA-BLCA cohort for muscle-invasive bladder cancer, providing further evidence. These multi-cohort analyses collectively demonstrate that UBR5 is a key molecule closely associated with poor prognosis in NMIBC.

[0061] Considering the potential for population differences and systemic bias in public datasets, we collected tissue specimens from 19 patients with non-invasive bladder cancer (NMIBC) who underwent transurethral resection of bladder tumor (TURBT) for immunohistochemical analysis to verify the prognostic robustness of UBR5. The inclusion criteria for patients were as follows: (1) NMIBC diagnosed by pathology and cystoscopy as originating from urothelial carcinoma; (2) newly diagnosed and not having received anti-tumor therapy; (3) no history of previous bladder cancer-related treatment; and (4) having received at least 6 cycles of adjuvant bladder instillation therapy postoperatively. Patients with other malignant tumors, autoimmune diseases, or incomplete follow-up data were excluded. Recurrence-free survival (RFS) was defined as the time from TURBT surgery to tumor recurrence, progression to muscle-invasive bladder cancer, or the last follow-up. The specific steps of immunohistochemistry are as follows:

[0062] (1) The prostate cancer tissue was sliced ​​to a thickness of 4μm using a pathological tissue slicer, spread on a non-slip glass slide, and dried at 65℃ for 2 hours for later use.

[0063] (2) Dewaxing: Immerse the glass slide in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → ddH2O I for 3 min → ddH2O II for 3 min.

[0064] (3) Blocking peroxidase: Soak in 3% hydrogen peroxide for 10 min, then wash with PBS for 5 min.

[0065] (4) High-pressure antigen retrieval: Prepare EDTA retrieval solution and add it to the pressure cooker. Place the glass slide in the cooker, heat at 800W for 20 minutes, and then let it cool naturally.

[0066] (5) Wash the slide twice with PBS for 3 minutes each time. Use a small piece of paper to absorb the moisture around the tissue. Use a biochemical pen to draw circles 0.5 cm away from the tissue boundary.

[0067] (6) Oily tissue surface: Immerse the glass slide in 0.1% PBST, lift it up and down and soak for 3 minutes.

[0068] (7) Primary antibody incubation: Dilute the antibody (anti-UBR5, Immunoway, YT4809) with antibody diluent according to the instructions, incubate at 50uL at 4℃ overnight, wash with PBS for 3min, and then wash with PBST for 3min.

[0069] (8) Secondary antibody incubation: 1 drop of DAKO secondary antibody per tissue, covering the tissue surface, incubated at 37°C for 1 hour, washed with PBS for 3 minutes, and then washed with PBST for 3 minutes.

[0070] (9) Prepare DAB chromogenic solution: Prepare the chromogenic solution according to the ratio. After shaking off the liquid on the slide, add freshly prepared DAB chromogenic solution to the circle. Control the chromogenic time under the microscope. The positive result is brownish-yellow. Soak the slide in PBS to stop the chromogenic process.

[0071] (10) Counterstaining cell nuclei: After terminating DAB staining, the slides were counterstained with hematoxylin for about 3 minutes, rinsed with running water, mounted with 20 μL mounting medium, and observed under a microscope. UBR5 immunoreactivity was assessed based on the staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positive cells (0: <5%, 1: 5-25%, 2: 25-50%, 3: 50-75%, 4: >75%). The final IHC score was the product of the two.

[0072] Experimental results are as follows Figure 8-9 As shown in the figure. Results showed that UBR5 was mainly located in the cytoplasm, exhibiting diffuse cytoplasmic staining. Stratification by T stage and relapse status showed that UBR5 expression was significantly higher in patients in the T1 stage and relapse group than in the control group. Subsequent univariate Cox regression analysis with relapse-free survival as the endpoint showed that high UBR5 expression was a significant risk factor for relapse in NMIBC (see [link to relevant data]). Figure 10 KM survival analysis further confirmed that patients with high UBR5 expression had significantly shorter relapse-free survival (see...). Figure 11 ROC analysis showed that UBR5 had good discriminative power in predicting NMIBC recurrence, with an area under the curve of 0.85 (see ROC analysis). Figure 12The above results collectively indicate that UBR5 can serve as a reliable biomarker for the clinical diagnosis, treatment, and prognostic assessment of NMIBC.

[0073] Example 2

[0074] To more precisely elucidate the role of UBR5 in the biological behavior of NMIBCs, a series of in vitro functional experiments were conducted. First, three specific siRNAs targeting UBR5 were designed and transfected into the NMIBC cell lines SW1710 and RT4. The knockdown efficiency was then verified by qPCR. The specific steps are as follows:

[0075] (1) Using siRNA-mate (Qingke Biotechnology, Beijing), small interfering RNAs (siRNAs) targeting UBR5 (Qingke Biotechnology, Beijing) were transfected into RT4 and SW1710 cells respectively (si-1 sequence as shown in SEQ ID NO: 1, 5'-GAGAGTTTGCGGCAACTAA-3'; si-2 sequence as shown in SEQ ID NO: 2, 5'-GGCTAGTACTAGCATAGAT-3'; si-3 sequence as shown in SEQ ID NO: 3, 5'-GGATTGTAGGTTACTTAGA-3', and cells transfected with blank vector si-NC were set as controls, and cultured under normal culture conditions.

[0076] (2) After digestion, cells in the logarithmic growth phase were collected into 1.5 mL EP tubes. NucleoZol (740404.200, biofive) was added to each sample for cell lysis, followed by the addition of ddH2O at a ratio of 1 mL:200 µL (NucleoZol:ddH2O). The lysis buffer was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was transferred to a fresh tube. An equal volume of isopropanol was then added, followed by another centrifugation at 12,000 rpm for 10 minutes. The supernatant was discarded, and the RNA was washed twice with 500 µL of 75% ethanol, centrifuged at 8000 rpm for 3 minutes each time. The RNA pellet was resuspended in 50–100 µL of ddH2O.

[0077] (3) RNA concentration was detected using NanoDrop 2000. Reverse transcription was performed using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit from Beijing TransGen. qPCR was performed using the PerfectStart Green qPCR SuperMix kit from Beijing TransGen. After the reaction, the UBR5 mRNA expression level was calculated based on the CT value of each reaction well, with the internal reference gene GAPDH as the internal reference. The primer pair sequences for detecting the UBR5 expression level are as follows: the upstream sequence is shown in SEQ ID NO: 4 (5'-TGGGACCAAATCATGCTGCC-3'), and the downstream sequence is shown in SEQ ID NO: 5 (5'-GAGAGTCGCTTGTCCTACCAG-3').

[0078] Test results as follows Figure 13 As shown in the figure. The results showed that, compared with the blank vector si-NC group, the expression of UBR5 in cells transfected with si-1, si-2, or si-3 was significantly inhibited (p < 0.0001), and two of them (si-1 and si-2) were selected for subsequent cell proliferation, colony formation, and Transwell invasion experiments. The specific steps of the cell proliferation experiment are as follows:

[0079] (1) When RT4 cells and SW1710 cells (si-NC as control) transfected with si-1 or si-2 grow to the logarithmic phase, trypsin digestion and counting are performed. The appropriate cell density is selected according to the doubling time of each cell type and seeded into 96-well plates (3 replicates).

[0080] (2) The cells were cultured in an incubator at 37°C and collected after 48 hours of culture. 10 μL of CCK-8 was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured to assess the cell proliferation status.

[0081] Experimental results are as follows Figure 14 As shown in the figure. The results showed that, compared with the control group (si-NC), inhibiting the expression of UBR5 could effectively inhibit the proliferation of bladder cancer cells, and the difference was statistically significant (***p<0.001).

[0082] The specific experimental steps for cell clone formation are as follows:

[0083] (1) When RT4 cells and SW1710 cells (si-NC as control) transfected with si-1 or si-2 grow to the logarithmic phase, trypsin digest and count them. Select an appropriate cell density (about 1000 cells / well) according to the doubling time of each cell type, seed them into 6-well plates containing 2 mL of 37℃ pre-warmed culture medium, and gently rotate them to disperse the cells evenly. Incubate them in a cell culture incubator at 37℃ with 5% CO2 and saturated humidity.

[0084] (2) When visible clones appear in the culture dish, stop the culture, discard the supernatant, carefully wash twice with PBS, add 1 mL of methanol containing 0.5% crystal violet to each well, and stain for 30 min; discard the methanol and wash the residual methanol with water; cell clones can then be observed; under a microscope, a number of cells > 50 is considered a valid clone.

[0085] Test results as follows Figure 15-16 As shown in the figure. The results showed that, compared with the blank vector si-NC group, the ability of bladder cancer cells to form colonies was significantly reduced after silencing the UBR5 gene with siRNA, and the colony formation of bladder cancer cells was significantly inhibited. The difference was statistically significant (*p<0.05, **p<0.01, ***p<0.001).

[0086] The specific steps of the Transwell invasion experiment are as follows:

[0087] (1) One day before the experiment, put a tube of Matrigel matrix gel that had been dispensed from -20°C into a 4°C refrigerator overnight to melt it from a solid state to a liquid state.

[0088] (2) Prepare 10% Matrigel matrix gel on ice, take 50 μL to coat the upper chamber surface of the bottom membrane of the Transwell chamber, place it at 37°C, and wait for the matrix gel to solidify for 30 min.

[0089] (3) After trypsin digestion of SW1710 cells in the logarithmic growth phase, the cells were resuspended in basal culture medium to form a cell suspension, counted, and the cell density was adjusted to 1×10⁻⁶. 5 per mL.

[0090] (4) Remove and discard the basal culture medium from the small chamber and the 24-well plate. Add 200 μL of cell suspension to the upper chamber of the Transwell chamber and 600 μL of complete culture medium (basal culture medium + 10% fetal bovine serum) to the lower chamber of the 24-well plate.

[0091] (5) The culture plate was placed in a CO2 incubator at 37°C and cultured for another 48 hours.

[0092] (6) Remove the chamber, rinse twice with PBS, fix with 4% paraformaldehyde in a 24-well plate for 20 min, and stain with crystal violet solution for 15 min.

[0093] (7) Carefully wipe away the cells and matrix gel in the upper layer of the microporous membrane of the chamber with a cotton swab, and take a picture under an inverted microscope.

[0094] Experimental results are as follows Figure 17 As shown in the figure. The results showed that the number of cells migrating across the basement membrane was significantly reduced in the Transwell assay. These experiments collectively demonstrate that inhibiting UBR5 expression can effectively suppress the progression of NMIBC.

[0095] Example 3

[0096] To elucidate the biological function of UBR5 in NMIBC, Spearman rank correlation analysis was performed between UBR5 and all other mRNAs using the UROMOL2021 dataset. Genes were ranked in descending order based on their correlation coefficient with UBR5 expression. Gene set enrichment analysis (GSEA) was performed using the KEGG pathway database from ClusterProfiler R package (version 4.15.2), with pathways showing a false discovery rate (FDR) less than 0.05 considered significantly enriched. After ranking pathways by normalized enrichment scores, the top ten upregulated and downregulated pathways most significantly correlated with UBR5 were visualized (see [link to relevant documentation]). Figure 18 The results indicate that UBR5 may activate pathways related to cellular processes and genetic information processing, including the cell cycle and homologous recombination. Conversely, pathways inhibited by UBR5 are primarily involved in infectious diseases and immune system function, such as the IgA gut immune network, asthma, and autoimmune thyroid diseases.

[0097] Given the ubiquitin ligase activity of UBR5 and its crucial role in maintaining genome integrity, somatic mutation and copy number variation (CNV) data of NMIBC were obtained from the UROMOL2021 cohort. The mutation spectrum was analyzed using maftools (version 2.22.0), and visualization was performed using ComplexHeatmap (version 2.22.0). Subsequently, the Wilcoxon rank-sum test was used to compare the mutation frequency differences between high and low UBR5 expression groups to explore its function at the genome level. Based on RNA-Seq mutation call and copy number variation data from the UROMOL2021 cohort, the association between 18 frequently mutated bladder cancer-related genes and 7 common driver genes and UBR5 expression was visualized. The results showed that FGFR3 had the highest mutation frequency (34%), followed by PIK3CA (25%) (see [link to relevant documentation]). Figure 19 Analysis of samples grouped by UBR5 expression levels revealed that high UBR5 expression indicated a significantly higher STAG2 mutation frequency (see [link to relevant documentation]). Figure 20 At the copy number level, CDKN2A deletion (42%) and TP53 deletion (12%) were the most common variants (see [link to original text]). Figure 21 However, these CNVs were not significantly correlated with UBR5 expression (see [link to CNV documentation]). Figure 22 As a classic tumor suppressor gene, TP53 encodes the p53 protein, which plays a central role in preventing tumorigenesis. Abnormal TP53 expression is closely associated with the progression of non-invasive bladder cancer (NMIBC), and p53 overexpression in T1 stage tumors is considered an important indicator of transformation into muscle-invasive lesions. Previous studies have shown that UBR5 can indirectly inhibit the p53 signaling pathway by ubiquitinizing and degrading CDC73, thus promoting breast cancer metastasis. STAG2, a key cell cycle regulator involved in DNA damage repair, exhibits recurring genetic alterations in NMIBC due to its inactivation, leading to genomic instability and abnormal cell cycle regulation, thereby driving tumor progression. Although there are currently no reports of direct interaction between UBR5 and STAG2, based on the aforementioned studies, this invention hypothesizes that UBR5 may participate in the NMIBC process by regulating STAG2-mediated DNA damage responses. These findings collectively reveal a previously unknown aspect of genomic regulation in bladder cancer.

[0098] Due to the significant clinical heterogeneity of non-muscle-invasive bladder cancer (NMIBC), the search for reliable prognostic biomarkers remains an unmet clinical need. Ubiquitin-E3 ligase component n-recognition protein 5 (UBR5), as a ubiquitin-E3 ligase involved in multiple oncogenic processes, has been shown to play a role in various cancers. However, its prognostic and therapeutic relevance in NMIBC remains unclear. This invention integrates multiple NMIBC cohorts and assesses the prognostic value of UBR5 using Kaplan-Meier curves and Cox regression analysis. The results were further validated by immunohistochemical staining and in vitro experiments. Its biological function was explored through functional enrichment analysis and genomic analysis, and potential UBR5-targeting compounds were identified by combining drug sensitivity analysis and molecular docking. This invention identifies UBR5 as a key prognostic molecule for NMIBC, with high expression consistently predicting poor prognosis. Immunohistochemical staining of internal cohorts further validated UBR5 expression and its robustness in prognostic assessment. In vitro experiments demonstrated that UBR5 enhances the proliferation and invasion of NMIBC cells. Functional enrichment analysis revealed that UBR5 promotes cell cycle progression and genetic regulatory pathways while inhibiting immune-related signaling. Mutation profile analysis revealed the association between UBR5 expression and alterations in key genes such as TP53 and STAG2.

[0099] In summary, this invention clarifies the key oncogenic regulatory role of UBR5 in non-invasive bladder cancer (NMIBC), and its high expression can predict early recurrence and poor prognosis, highlighting its value as a biomarker to guide future precision treatment strategies. Targeting UBR5 can effectively inhibit tumor cell proliferation and motility, demonstrating its potential as a therapeutic target. Inhibiting UBR5 or reducing its biological activity can significantly delay the progression of NMIBC, improve prognosis, and prolong patient survival, demonstrating the potential of targeting UBR5 to develop personalized treatment strategies for high-risk NMIBC patients. This provides sufficient scientific evidence and theoretical basis for exploring new molecular targets for bladder cancer diagnosis, prognosis, and treatment, and for developing new targeted drugs, contributing to better precision treatment and possessing significant social and scientific value.

[0100] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. Use of a UBR5 inhibitor in the preparation of a medicament for preventing and / or treating non-muscle invasive bladder cancer.

2. Use according to claim 1, characterized in that, The UBR5 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on UBR5 gene.

3. Use according to claim 1, characterized in that, The UBR5 inhibitor is selected from siRNA designed based on UBR5 gene; the siRNA is selected from one or more of si-1, sh-2, sh-3; wherein the sequence of si-1 is shown as SEQ ID NO: 1, the sequence of sh-2 is shown as SEQ ID NO: 2, and the sequence of sh-3 is shown as SEQ ID NO:

3.

4. Use of a reagent for detecting the expression level of UBR5 in the preparation of a product for the auxiliary diagnosis and / or prognosis evaluation of non-muscle invasive bladder cancer.

5. Use according to claim 4, characterized in that, The reagent for detecting the expression level of UBR5 comprises primers for detecting the expression level of UBR5 gene and / or reagents for detecting the content of UBR5 protein.

6. Use according to claim 5, characterized in that, The primers for detecting the expression level of UBR5 gene are selected from the following primer pairs: The upstream sequence of the primer pair is shown as SEQ ID NO: 4, and the downstream sequence is shown as SEQ ID NO:

5.

7. A pharmaceutical composition for preventing and / or treating non-muscular invasive bladder cancer, characterized by, The UBR5 inhibitor and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein, The UBR5 inhibitor is selected from one or more of siRNA, shRNA, sgRNA designed based on UBR5 gene.

9. The pharmaceutical composition of claim 7, wherein, The UBR5 inhibitor is selected from siRNA designed based on UBR5 gene; the siRNA is selected from one or more of si-1, sh-2, sh-3; wherein the sequence of si-1 is shown as SEQ ID NO: 1, the sequence of sh-2 is shown as SEQ ID NO: 2, and the sequence of sh-3 is shown as SEQ ID NO:

3.

10. A kit for the aided diagnosis and / or prognosis evaluation of non-muscular invasive bladder cancer, characterized in that, The reagent for detecting the expression level of UBR5.

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