Application of USP15 in preparation of bladder cancer radiotherapy sensitizer

Abstract

The invention discloses application of USP15 in preparation of a bladder cancer radiotherapy sensitizer, and belongs to the technical field of biological medicine. The objective of the invention is to improve the radiotherapy sensitivity of bladder cancer. The invention relates to an application of USP15 in preparation of a bladder cancer radiotherapy sensitizer. The USP15 is used for preparing the bladder cancer radiotherapy sensitizer. The radiotherapy sensitivity of the bladder cancer is improved by overexpressing the USP15. The USP15 can regulate and control the stability of FIS1 protein through ubiquitination modification so as to inhibit the mitochondrial function, and the activation of the regulation and control pathway is the core mechanism of the USP15 for exerting the radiotherapy sensitization effect. In conclusion, the invention not only provides a new thought for molecular mechanism research of bladder cancer radiotherapy sensitization, but also provides a potential strategy for treatment of clinical radiotherapy resistant bladder cancer patients, and has important drug development value and clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of USP15 in the preparation of radiosensitizers for bladder cancer. Background Technology

[0002] Bladder cancer is the most common malignant tumor of the urinary system, posing a serious threat to human health. Chemotherapy combined with radiotherapy is one of the core strategies in current clinical treatment. However, chemotherapy alone has limited efficacy in advanced invasive bladder cancer, resulting in low patient survival rates. Therefore, the clinical need for highly effective combination therapy regimens is increasingly urgent. More critically, the current lack of biomarkers that can accurately guide radiotherapy decisions leads to uncertainty in treatment selection. Therefore, developing drugs that enhance radiosensitivity has become a crucial issue that urgently needs to be addressed to overcome the bottlenecks in radiotherapy for advanced invasive bladder cancer and improve patient prognosis.

[0003] DNA double-strand breaks (DSBs) are among the most lethal types of genomic damage, and defects in repair mechanisms are associated with a wide range of human diseases. Deubiquitinating enzymes (DUBs) play a crucial role in maintaining genomic stability and regulating tumorigenesis and development, and their functional mechanisms at the chromatin level have received widespread attention in recent years. Ubiquitin-specific protease 1 (USP1) is one of the earliest identified and most thoroughly studied members of the DUB family. It promotes homologous recombination (HR) repair by deubiquitinizing Fanconi anemia complement D2 (FANCD2) and proliferating cell nuclear antigen (PCNA). Ubiquitin-specific protease 39 (USP39) regulates HR repair through a unique mechanism independent of poly(ADP-ribose) polymerase (PARP) by binding to the spliceosome. In esophageal cancer, ubiquitin-specific protease 49 (USP49) can deubiquitinate replication protein A70 (RPA70), promoting its co-recruitment with RAD51 to DNA damage sites, thereby enhancing HR repair efficiency. The aforementioned research findings highlight the central role of DUBs in regulating the radiosensitivity of tumor cells. Recent studies have confirmed that ubiquitin-specific protease 15 (USP15) is closely related to the regulation of HR repair and PARP inhibitor resistance in breast cancer; however, its radiosensitizing effect in bladder cancer still requires further investigation. Summary of the Invention

[0004] The problem to be solved by this invention is to improve the radiosensitivity of bladder cancer, and proposes the application of USP15 in the preparation of radiosensitizers for bladder cancer.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] An application of USP15 in the preparation of a radiosensitizer for bladder cancer, wherein the USP15 is used to prepare a radiosensitizer for bladder cancer.

[0007] The radiosensitivity of bladder cancer was improved by overexpressing USP15. The target sequence for USP15 overexpression is shown in SEQ ID NO:1.

[0008] Furthermore, USP15 enhances the sensitivity of tumor cells to radiotherapy by inhibiting mitochondrial function through deubiquitination to stabilize FIS1.

[0009] A radiosensitizer for bladder cancer, including USP15.

[0010] The beneficial effects of this invention are:

[0011] This invention describes the application of USP15 in the preparation of a radiosensitizer for bladder cancer. Cellular and animal experiments revealed that USP15 significantly enhances the radiosensitivity of bladder cancer at both cellular and animal levels, exhibiting high safety profile, thus making it suitable for development and application as a radiosensitizer. This invention, through its mechanism of action, discovered that USP15 overexpression can inhibit tumor growth and enhance radiosensitivity. Simultaneously, USP15 enhances the radiosensitivity of tumor cells by deubiquitinizing and stabilizing FIS1, thereby inhibiting mitochondrial function.

[0012] The application of USP15 in the preparation of radiosensitizers for bladder cancer, as described in this invention, can provide new research ideas and directions for radiosensitization, and at the same time, it can provide new treatment options for patients with radiotherapy-resistant bladder cancer, showing good potential for drug development.

[0013] This invention discloses the application of USP15 in the preparation of a radiosensitizer for bladder cancer. Through in vitro cell experiments and in vivo animal models, it was found that USP15 significantly enhances the radiosensitivity of bladder cancer cells at both the cellular and animal levels, effectively strengthening the killing effect of radiotherapy on tumor cells. Further investigation into the mechanism of action elucidates that USP15 can regulate the stability of the FIS1 protein through ubiquitination modification, thereby inhibiting mitochondrial function. Activation of this regulatory pathway is the core mechanism by which USP15 exerts its radiosensitizing effect. In summary, this invention not only provides new insights into the molecular mechanism of radiosensitization in bladder cancer but also offers a potential strategy for the treatment of radioresistant bladder cancer patients, possessing significant drug development value and clinical application prospects. Attached Figure Description

[0014] Figure 1 The results of the effect of USP15 on radiosensitivity in vitro are as follows: (a) shows the colony formation of cells in the empty control group and the USP15 overexpression group at radiation doses of 0 GY, 4 GY and 8 GY, respectively; (b) shows the count of the colonies in (a) and presents the number of colonies in each group in the form of a bar chart.

[0015] Figure 2 This invention involves Western blot analysis of protein expression in T24 cells overexpressing USP15.

[0016] Figure 3 The following is a graph showing the Annexin V / PI staining results of this invention, where (a) is a flow cytometry scatter plot of Annexin V / PI staining, and (b) is a bar chart calculated based on the flow cytometry scatter plot in (a) to show the total percentage of "early apoptosis + late apoptosis" cells in each sample.

[0017] Figure 4 This is a graph showing the half-life detection results of the FIS1 protein in this invention;

[0018] Figure 5 The results of the adjacent connection assay of the present invention are shown in the figure. (a) is the experimental group image. The empty control group and the USP15 overexpression group of T24 cells were detected by FIS1 and USP15 antibodies at the same time, and the PLA signal (red fluorescent signal) in the image was observed. (b) is the count and statistics of the PLA signal of the two groups of samples.

[0019] Figure 6 This is an experimental diagram of ubiquitination in this invention;

[0020] Figure 7 The present invention describes the effects of USP15 knockdown on BIU87 cells in a mouse subcutaneous xenograft model on tumor weight and volume through radiotherapy and control experiments. (a) shows the average tumor volume of mice in each group, measured every three days, and (b) shows the tumor weight of mice in each group on day 18.

[0021] Figure 8 In this invention, T24-pUSP15 cells were fluorescently labeled and then used in a mouse orthotopic xenograft model to observe the fluorescence imaging intensity in each group of animals through radiotherapy and control experiments and a small animal imaging system. (a) shows the average bioluminescence signal of each group of mice on day 21, and (b) shows the growth curve of the average bioluminescence intensity of each group of mice over time.

[0022] Figure 9 To verify the overexpression and knockdown efficiency of USP15 in this invention, (a) shows the overexpression efficiency of USP15 in T24 cells and (b) shows the knockdown efficiency of USP15 in BIU87 cells. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described specific embodiments are merely a part of the embodiments of the invention, and not all of them. The components of the specific embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations, and the invention may also have other embodiments.

[0024] Therefore, the following detailed description of specific embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected specific embodiments of the invention. All other specific embodiments obtained by those skilled in the art based on these specific embodiments without inventive effort are within the scope of protection of this invention.

[0025] To further understand the invention's content, features, and effects, the following specific embodiments are provided, along with accompanying drawings. Figure 1 -Appendix Figure 9 Detailed explanation is as follows:

[0026] Example 1:

[0027] In this embodiment, clonogenic experiments were performed on T24 cells overexpressing the USP15 gene and T24 cells transfected with the empty pENTER vector, as follows:

[0028] I. Experimental Methods:

[0029] 1. Tumor cell culture: Human bladder cell lines (BIU87, T24) were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 (Gibco, USA) or DMEM (Gibco, USA) + 10% fetal bovine serum (FBS, Gibco, USA). Cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0030] 2. Construction and culture of T24 cells stably overexpressing the USP15 gene: To establish USP15 overexpressing cells, T24 cells were seeded in 24-well plates and infected with the USP15 overexpression plasmid provided by WZ Biosciences (Shandong, China). Forty-eight hours after infection, cells were selected with puromycin for two weeks. Overexpression efficiency was confirmed by Western blot analysis. The target USP15 overexpression sequence is shown in SEQ ID NO:1.

[0031] 3. Colony Formation Assay: T24 cells overexpressing the USP15 gene and T24 cells transfected with the empty pENTER vector (empty vector control group) were divided into irradiated and non-irradiated groups for colony formation assays. The irradiated groups received 4 Gy and 8 Gy of radiation therapy, respectively. In the colony formation assay, cells were cultured in 6-well plates at 37°C for 2 weeks. Cells were fixed with methanol, stained with 0.5% crystal violet (Sorabio, Beijing, China), and colonies were quantified.

[0032] II. Experimental Results:

[0033] Western blot analysis confirmed the USP15 overexpression efficiency in T24 cells, as shown in the figure. Figure 9 (a) In the non-irradiated group, colony formation in the USP15 overexpression group was lower than that in the empty vector control group, and in the irradiated group, the reduction in colony formation in the USP15 overexpression group was more pronounced. Figure 1 The results indicate that increased USP15 levels inhibited cell proliferation, with the inhibitory effect being more pronounced after irradiation. This suggests that USP15 can enhance the radiosensitivity of bladder cancer cells.

[0034] Example 2:

[0035] The purpose of this embodiment is to investigate the mechanism of action of USP15 in enhancing radiosensitivity in bladder cancer and the relationship between USP15 and FIS1.

[0036] I. Experimental Methods:

[0037] 1. Western Blot detection of protein expression:

[0038] Construction and culture of T24 cells stably overexpressing the USP15 gene: To establish USP15 overexpressing cells, T24 cells were seeded in 24-well plates and infected with the USP15 overexpression plasmid provided by WZ Biosciences (Shandong, China). Forty-eight hours after infection, cells were selected with puromycin for two weeks. Overexpression efficiency was confirmed by Western blot analysis. The target USP15 overexpression sequence is shown in SEQ ID NO:1.

[0039] After culturing USP15-overexpressing T24 cells in good growth condition for 48 h, the cells were collected and lysed with RIPA lysis buffer for 30 min. After centrifugation at 4 °C, the supernatant was transferred to a new EP tube, and the protein concentration was determined by the BCA method. After sample denaturation, SDS-PAGE electrophoresis was performed. The desired target protein was excised according to the molecular weight of the pre-stained marker and electroporated at a constant current of 300 mA for 1.5 h to transfer the target protein onto a PVDF membrane. The transferred bands were placed in rapid blocking buffer and blocked at room temperature for 1 h. The bands were then incubated overnight on a shaker at 4 °C with primary antibodies (USP15, mtCO2, OXPHOS, Actin). The bands were washed with PBST and incubated at room temperature for 2 h with horseradish peroxidase (HRP)-labeled secondary antibody. After thorough washing with PBST, ECL chemiluminescence buffer was added for development.

[0040] 2. Annexin V / PI staining:

[0041] Construction and culture of BIU87 bladder cancer cells with USP15 gene knockdown: BIU87 cells provided by the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were seeded in 24-well plates and infected with the USP15 shRNA plasmid provided by WZ Biosciences (Shandong, China) according to their instructions. Forty-eight hours after infection, cells were screened with puromycin for two weeks. The knockdown efficiency was confirmed by Western blot analysis. After verification, the cells were expanded for further experiments. The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2.

[0042] BIU87 cells transfected with USP15-shRNA1 knocked down from USP15 and BIU87 bladder cancer cells transfected with negative control shRNA (non-targeting USP15 sequence) were stained with Mito Tracker Green (100 nm) for 30 min at 37°C for mitochondrial labeling. Annexin V-FITC / PI staining was used to detect apoptosis rate. After 48 h of treatment, BIU87 cells were washed with PBS and collected for Annexin V-FITC / PI staining. Each cell particle was resuspended in 500 μL binding buffer, supplemented with 5 μL of FITC and 5 μL of PI, and incubated for 15 min. Flow cytometry was used to detect apoptosis rate. The mean fluorescence intensity of labeled mitochondria was analyzed by gating individual cells. Samples were analyzed on a flow cytometer (BD Biosciences) and processed using FlowJo software (Tree Star). Among them, the mitochondrial-targeting antioxidant Mito-Tempo (20 μmol) was added to the cells 24 hours before Annexin V-FITC / PI staining to detect cell apoptosis rate, with a radiation dose of 8 Gy.

[0043] 3. FIS 1 protein half-life assay:

[0044] Construction and culture of BIU87 bladder cancer cells with USP15 gene knockdown: BIU87 cells provided by the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were seeded in 24-well plates and infected with the USP15 shRNA plasmid provided by WZ Biosciences (Shandong, China) according to their instructions. Forty-eight hours after infection, cells were screened with puromycin for two weeks. Western blot analysis was used to confirm the knockdown efficiency. After verification, the cells were expanded for further experiments. The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2.

[0045] BIU87 cells and BIU87 cells with USP15 knocked down using USP15-shRNA1 were cultured in 2 ml of DMEM medium in 6-well plates to reach 50% confluence. At the specified time, cells were treated with 10 μM actinomycin (CHX, MedChemExpress, #HY-12320). After treatment, cells were harvested and lysed in EBC lysis buffer. The cell lysates were then subjected to Western blotting using rabbit anti-FIS 1 antibody.

[0046] 4. Proximity Adjacency Test (PLA):

[0047] Construction and culture of T24 cells stably overexpressing the USP15 gene: To establish USP15 overexpressing cells, T24 cells were seeded in 24-well plates and infected with the USP15 overexpression plasmid provided by WZ Biosciences (Shandong, China). Forty-eight hours after infection, cells were selected with puromycin for two weeks. Overexpression efficiency was confirmed by Western blot analysis. The target USP15 overexpression sequence is shown in SEQ ID NO:1.

[0048] T24 cells (PEnter: T24 bladder cancer cells transfected with the empty pENTER vector and pUSP15) were seeded on glass slides. Cells were fixed with 4% paraformaldehyde and infiltrated with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. After incubation overnight at 4°C with USP15 and FIS1 antibodies, polylactic acid (PLA) was detected using Duolink in situ polylactic acid probes against rabbits (+) and mice (-), and Duolink in situ detection reagent Green (Sigma-Aldrich) according to the manufacturer's instructions.

[0049] 5. Ubiquitination experiment:

[0050] Construction and culture of BIU87 bladder cancer cells with USP15 gene knockdown: BIU87 cells provided by the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were seeded in 24-well plates and infected with the USP15 shRNA plasmid provided by WZ Biosciences (Shandong, China) according to their instructions. Forty-eight hours after infection, cells were screened with puromycin for two weeks. Western blot analysis was used to confirm the knockdown efficiency. After verification, the cells were expanded for further experiments. The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2, and the nucleotide sequence of USP15-shRNA2 is shown in SEQ ID NO:3.

[0051] Ubiquitin from USP15 and FIS1 is expressed in BIU87 cells. BIU87 cells and BIU87 cells with USP15 knocked down using USP15-shRNA1 and USP15-shRNA2 were each added to 10 µL of MG132 (MedChemExpress, #HY-13259), and immunoprecipitated with an anti-ubiquitin antibody. These complexes were then incubated overnight at 4°C with Protein A / G agarose beads using rotation. FIS1 ubiquitination was analyzed by Western blotting.

[0052] II. Experimental Results:

[0053] 1. Western blot confirmed the negative regulatory role of USP15 in mitochondrial function, showing that USP15 overexpression downregulates oxidative phosphorylation. Figure 2 ).

[0054] 2. Western blot analysis confirmed the USP15 knockdown efficiency in BIU87 cells, as shown in Figure (). Figure 9 (b) Annexin V / PI staining results showed that, compared with the empty vector control group, the USP15 knockdown group had reduced apoptosis, which could be reversed by the mitochondrial-targeting antioxidant Mito-Tempo, indicating that USP15 sensitizes cells to radiation by impairing mitochondrial function. Figure 3 ).

[0055] 3. Compared with the control group, the half-life of FIS1 was shortened in the USP15 knockdown group in the presence of actinomycete ketone (CHX). Figure 4 ).

[0056] 4. Western blot analysis confirmed the USP15 overexpression efficiency in T24 cells, as shown in Figure (). Figure 9 (a) Significant PLA signaling was observed in T24 cells overexpressing USP15, indicating a strong interaction between endogenous FIS1 and USP15. Figure 5 ).

[0057] 5. The ubiquitination experiment results show that USP15 stabilizes FIS1 through deubiquitination ( Figure 6 ).

[0058] In summary, this study first confirmed that USP15 plays a negative regulatory role in mitochondrial function, and its overexpression significantly inhibits cellular oxidative phosphorylation. Secondly, functional experiments showed that USP15 knockdown reduces radiation-induced apoptosis, while the mitochondrial-targeting antioxidant Mito-Tempo reverses this protective effect, suggesting that USP15 mediates cellular radiosensitivity by impairing mitochondrial function. Mechanistically, it was found that USP15 can stabilize the mitochondrial fission-related protein FIS1 by deubiquitination, prolonging its half-life; proximity connectivity (PLA) experiments further verified the direct interaction between the two. In conclusion, this study elucidates the crucial role of the USP15-FIS1 signaling axis in regulating mitochondrial homeostasis and cellular radiosensitivity.

[0059] Example 3:

[0060] The mouse comparison experiment in this embodiment is as follows:

[0061] I. Experimental Methods:

[0062] 1. Construction and culture of BIU87 bladder cancer cells with USP15 gene knockdown: BIU87 cells provided by the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were seeded in 24-well plates and infected with the USP15 shRNA plasmid provided by WZ Biosciences (Shandong, China) according to their instructions. Forty-eight hours after infection, cells were screened with puromycin for two weeks. The knockdown efficiency was confirmed by Western blot analysis. After verification, the cells were expanded for further experiments. The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2.

[0063] Establishment of a subcutaneous xenograft model in BALB / c nude mice (20-22g) (purchased from Liaoning Changsheng Biotechnology Co., Ltd.): Mice were randomly divided into four groups (shNC, shUSP15, shNC+IR, shUSP15+IR).

[0064] ① shNC group: BIU87 bladder cancer cells were transfected with negative control shRNA (non-targeting USP15 sequence). After verifying that USP15 gene expression remained normal, each mouse was subcutaneously injected with approximately [missing information - likely a specific type of injection] along the midline of its back. The experimental group that was successfully transfected with BIU87 bladder cancer cells and did not receive ionizing radiation (radiotherapy) treatment.

[0065] ②shUSP15 group: BIU87 bladder cancer cells were transfected with USP15-targeting shRNA. After verification of USP15 gene knockdown, approximately [amount missing] was subcutaneously injected into each mouse along the midline of the back. The experimental group consisting of successfully transfected BIU87 bladder cancer cells and untreated ionizing radiation (radiotherapy). The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2.

[0066] ③ shNC+IR group: BIU87 bladder cancer cells were transfected with negative control shRNA (non-targeting USP15 sequence). After verifying that USP15 gene expression remained normal, each mouse was subcutaneously injected with approximately [missing information - likely a specific type of injection] along the midline of its back. The successfully transfected BIU87 bladder cancer cells underwent radiation therapy every seven days for the next 18 days, with a radiation dose of 8 Gy.

[0067] ④ shUSP15+IR group: BIU87 bladder cancer cells were transfected with USP15-targeting shRNA. After verification of USP15 gene knockdown, approximately [amount missing] was subcutaneously injected into each mouse along the midline of the back. Successfully transfected BIU87 bladder cancer cells underwent radiation therapy every seven days for the next 18 days, with a radiation dose of 8 Gy. The nucleotide sequence of USP15-shRNA1 carried by the USP15-targeting shRNA vector is shown in SEQ ID NO:2.

[0068] Four groups of mice were housed in individually ventilated cages under controlled temperature and humidity conditions, with free access to food and water. Tumor volume was measured every 3 days, and tumors were collected for analysis on day 18.

[0069] 2. Construction and culture of T24-luc cells stably overexpressing the USP15 gene: To establish USP15 overexpressing cells, T24 cells were seeded in 24-well plates and infected with the USP15 overexpression plasmid provided by WZ Biosciences (Shandong, China). Forty-eight hours after infection, cells were selected with puromycin for two weeks. Overexpression efficiency was confirmed by Western blot analysis. The target USP15 overexpression sequence is shown in SEQ ID NO:1. To establish T24-pUSP15-luc cells, T24-pUSP15 cells were seeded in 24-well plates and infected with the luciferase plasmid provided by WZ Biosciences (Shandong, China). Forty-eight hours after infection, cells were selected with blastocysteine ​​for two weeks. Successful plasmid infection was confirmed by small animal in vivo imaging analysis.

[0070] Establishment of BALB / c nude mouse (20~22g) orthotopic xenograft model (purchased from Liaoning Changsheng Biotechnology Co., Ltd.): Mice were randomly divided into four groups (pENTER, pUSP15, pENTER+IR, pUSP15+IR).

[0071] ① pENTER group: T24-luc bladder cancer cells transfected with the empty pENTER vector (empty vector control group) were injected into each mouse via the bladder after verification that USP15 gene expression remained normal. T24-luc bladder cancer cells that were successfully transfected, in the experimental group that did not receive ionizing radiation (radiotherapy).

[0072] ②pUSP15 group: T24-luc bladder cancer cells were transfected with a plasmid overexpressing USP15. After verification of USP15 gene overexpression, approximately [amount missing] were injected into each mouse along the bladder. T24-pUSP15-luc bladder cancer cells successfully transfected, in an experimental group that was not treated with ionizing radiation (radiotherapy). The target nucleotide sequence for USP15 overexpression is shown in SEQ ID NO:1.

[0073] ③ pENTER+IR group: T24-luc bladder cancer cells transfected with the empty pENTER vector (empty vector control group) were injected into each mouse via the bladder after verification that USP15 gene expression remained normal. The successfully transfected T24-luc bladder cancer cells underwent radiation therapy on days 7, 12, 17, and 22, with a radiation dose of 8 Gy.

[0074] ④ pUSP15+IR group: T24-luc bladder cancer cells were transfected with a USP15 overexpression plasmid. After verification of USP15 gene overexpression, approximately [amount missing] was injected into each mouse along the bladder. Successfully transfected T24-pUSP15-luc bladder cancer cells underwent radiation therapy on days 7, 12, 17, and 22, with a radiation dose of 8 Gy. The target sequence for USP15 overexpression is shown in the nucleotide sequence of SEQ ID NO:1.

[0075] Four groups of mice were housed in individually ventilated cages under controlled temperature and humidity conditions, with free access to food and water. Bioluminescence imaging was performed on mice carrying in situ tumors in different groups on days 7, 14, and 21. Tumor capture and survival were assessed on day 27.

[0076] II. Experimental Results:

[0077] The results showed that, in the USP15 knockdown group, the subcutaneous tumor volume was significantly larger than that in the control group compared to the non-irradiated group. Furthermore, after radiotherapy, the tumor suppression rate in the control group was significantly higher than that in the USP15 knockdown group. Figure 7 )

[0078] In the USP15 overexpression group, the mean bioluminescence signal was significantly lower in the unirradiated group than in the control group. Furthermore, after radiotherapy, the bioluminescence signal in the control group was significantly higher than that in the USP15 overexpression group. Figure 8 (a) Curve of average biofluorescence intensity increasing over time ( Figure 8 (b) also supports this result. This further demonstrates that USP15 enhances the radiosensitivity of bladder cancer cells.

[0079] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0080] Although this application has been described above with reference to specific embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of this application. In particular, as long as there is no structural conflict, the features in the specific embodiments disclosed in this application can be combined with each other in any way. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, this application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The application of USP15 in the preparation of a radiosensitizer for bladder cancer, characterized in that, The USP15 is used to prepare a radiosensitizer for bladder cancer. The radiosensitivity of bladder cancer was improved by overexpressing USP15. The target sequence for USP15 overexpression is shown in SEQ ID NO:

1.

2. The application of USP15 according to claim 1 in the preparation of a radiosensitizer for bladder cancer, characterized in that, USP15 enhances the sensitivity of tumor cells to radiotherapy by inhibiting mitochondrial function through deubiquitination and stabilization of FIS1.

3. A radiosensitizer for bladder cancer, characterized in that, Including USP15.

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