Application of ufsp2 as a target in preparation of prostate cancer treatment product

Abstract

This invention discloses the application of UFSP2 as a target in the preparation of prostate cancer therapeutic products. This invention relates to the biomedical field, and research has found that UFM1-specific protease 2 (UFSP2) is highly expressed in prostate cancer tissues, with protein levels higher than in adjacent normal tissues, and is positively correlated with the expression of proliferation-related molecules c-Myc and Cyclin D1. Functional experiments further show that inhibiting UFSP2 expression significantly inhibits the proliferation, colony formation, and migration of prostate cancer cells, and inhibits in vivo tumor growth and metastasis; conversely, overexpression of UFSP2 promotes prostate cancer cell proliferation, indicating that UFSP2 can serve as a therapeutic target for prostate cancer and can be used to screen anti-prostate cancer drugs. This invention provides the application of UFSP2 expression inhibitors and / or functional inhibitors in the preparation of prostate cancer therapeutic products, offering a new direction for drug development and targeted intervention strategies for prostate cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of UFSP2 as a target in the preparation of prostate cancer treatment products. Background Technology

[0002] Prostate cancer (PCa) is one of the most common malignant tumors in men and a major cause of cancer-related death. Because prostate cancer cells are highly dependent on the androgen receptor signaling pathway, androgen deprivation therapy is currently an important treatment for prostate cancer. However, with continued treatment, tumor cells gradually adapt to the inhibition of androgen receptor signaling, eventually progressing to castration-resistant prostate cancer (CRPC), severely impacting patient survival. For early-stage prostate cancer, treatments such as surgery and radiotherapy can improve patient prognosis to some extent, but most patients ultimately die from tumor metastasis and drug resistance.

[0003] Current clinical treatments mainly include androgen deprivation therapy (ADT), androgen receptor pathway inhibitors, taxane chemotherapy, radionuclide therapy, and targeted therapy or combination therapy after molecular stratification. Although these regimens can prolong the survival of some patients, they generally have limitations such as decreased treatment sensitivity, acquired drug resistance, cumulative toxic side effects, and inadequate control of metastasis. Therefore, there is still a lack of effective drugs for treating prostate cancer.

[0004] In recent years, abnormal tumor metabolic reprogramming and post-translational modifications have been considered important mechanisms driving tumor progression. UFMylation is a ubiquitin-like modification system whose core components include UFM1, the activator UBA5, the conjugator UFC1, the ligase UFL1, and the UFSP family of demodification-related proteins. It plays a crucial role in various biological processes, with its demodification process mediated by UFSP1 and UFSP2. Previous studies have suggested that UFMylation-related molecules are involved in endoplasmic reticulum homeostasis, protein quality control, stress response, and the regulation of tumor biological behavior. However, the role of UFSP2 in prostate cancer remains unclear, especially its expression characteristics in clinical prostate cancer tissues and its relationship with tumor proliferation, migration, in vivo tumor growth, and metastasis. Therefore, there is an urgent need to screen key therapeutic targets for prostate cancer, particularly advanced prostate cancer and castration-resistant prostate cancer, and to develop effective treatment strategies. This invention application is thus proposed. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the limited efficacy and lack of effective molecular targets in existing treatments for prostate cancer, especially castration-resistant prostate cancer. This invention uses UFSP2 as a new therapeutic target for prostate cancer, and provides application schemes for UFSP2 expression inhibitors and / or function inhibitors in the preparation of prostate cancer therapeutic products. Through systematic validation of cell, animal and clinical samples, it is demonstrated that this target has the potential to inhibit tumor proliferation, migration, in vivo tumorigenesis and metastasis.

[0006] The first objective of this invention is to provide the application of UFSP2 as a drug target in screening drugs for prostate cancer.

[0007] A second objective of this invention is to provide the use of UFSP2 expression inhibitors and / or functional inhibitors in the preparation of drugs for treating prostate cancer.

[0008] A third objective of this invention is to provide a drug for treating prostate cancer.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention discovers that UFSP2 (UFM1-specific protease 2) can serve as a novel therapeutic target for prostate cancer. In vitro experiments show that knocking down UFSP2 significantly inhibits the proliferation, colony formation, and migration of DU145, PC3, and 22Rv1 prostate cancer cells, while overexpression of UFSP2 promotes the proliferation of these cells. Animal experiments further demonstrate that targeting UFSP2 can significantly inhibit in vivo tumor growth and metastasis of prostate cancer. Clinical sample analysis results show that the protein expression level of UFSP2 in prostate cancer tissues is higher than that in adjacent normal tissues, and it is positively correlated with the expression of proliferation-related molecules c-Myc and Cyclin D1, suggesting that UFSP2 is closely related to the proliferative activity and malignant phenotype of prostate cancer. Based on this, this invention provides a novel application of UFSP2 as a drug target in screening anti-prostate cancer drugs, and uses UFSP2 expression inhibitors and / or functional inhibitors to prepare prostate cancer prevention and treatment drugs, providing a new targeted therapy for prostate cancer, especially advanced prostate cancer, metastatic prostate cancer, and castration-resistant prostate cancer. Using UFSP2 as a therapeutic target for prostate cancer has significant clinical implications.

[0011] The UFSP2 provided by this invention is a human UFM1-specific peptidase 2 (UFSP2). The NCBI Gene ID of this protease is 55325, the protein RefSeq accession number is NP_060829.2, and the UniProtKB / Swiss-Prot accession number is Q9NUQ7.

[0012] This invention provides the application of UFSP2 as a drug target in screening drugs for prostate cancer.

[0013] Preferably, the drug exerts its effect by inhibiting the expression or function of UFSP2.

[0014] Therefore, this invention provides the use of UFSP2 expression inhibitors and / or functional inhibitors in the preparation of drugs for treating prostate cancer.

[0015] This invention provides the use of UFSP2 expression inhibitors and / or functional inhibitors in the preparation of drugs for the prevention of prostate cancer.

[0016] Preferably, the drug can inhibit the proliferation, colony formation, and migration of prostate cancer cells.

[0017] Preferably, the drug can inhibit tumor growth, reduce tumor size, and slow down tumor formation.

[0018] Preferably, the expression inhibitor is a small interfering RNA, short hairpin RNA, antisense oligonucleotide, nucleic acid aptamer, CRISPR intervention tool, or other molecules that can reduce the transcription or translation level of UFSP2.

[0019] Preferably, the functional inhibitor is a polypeptide, small molecule compound, protein degrader, or other agent that can reduce the biological function of UFSP2 and inhibit the demodification activity of UFSP2.

[0020] More preferably, the expression inhibitor is a small interfering RNA, specifically one of si-UFSP2-1: 5'-GAACAAGGATGCATACTAT-3', si-UFSP2-2: 5'-GACGGGAACTGGCTAATCA-3', and si-UFSP2-3: 5'-GTCTAATGCTTATCACTTT-3'.

[0021] Preferably, the prostate cancer is advanced prostate cancer, metastatic prostate cancer, or castration-resistant prostate cancer.

[0022] This invention provides a prostate cancer treatment drug containing a UFSP2 expression inhibitor and / or function inhibitor, preferably one of si-UFSP2-1: 5'-GAACAAGGATGCATACTAT-3', si-UFSP2-2: 5'-GACGGGAACTGGCTAATCA-3', and si-UFSP2-3: 5'-GTCTAATGCTTATCACTTT-3'.

[0023] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0024] More preferably, the drug may be a nucleic acid drug formulation, a viral vector formulation, a liposome or nanoparticle delivery formulation, a sustained-release formulation, or a formulation formed in combination with pharmaceutically acceptable excipients; the administration route may be intravenous administration, local injection, tumor-targeted delivery, or other administration routes acceptable in the art.

[0025] In terms of treatment strategies, UFSP2 targeted agents can be used alone or in combination with existing prostate cancer treatment regimens, including but not limited to androgen receptor pathway inhibitors, taxane chemotherapy drugs, radiotherapy, PARP inhibitors or other targeted drugs.

[0026] The present invention has the following beneficial effects:

[0027] This invention provides a novel application of UFSP2 as a target in the preparation of prostate cancer therapeutic products. Studies have found that UFSP2 is highly expressed in prostate cancer tissues, with protein levels higher than in adjacent normal tissues, and is positively correlated with the expression of proliferation-related molecules c-Myc and Cyclin D1. Functional experiments further demonstrate that inhibiting UFSP2 expression or function significantly inhibits the proliferation, colony formation, and migration of prostate cancer cells, and suppresses in vivo tumor growth and metastasis. Conversely, overexpression of UFSP2 promotes prostate cancer cell proliferation, indicating that UFSP2 can serve as a therapeutic target for prostate cancer and can be used to screen anti-prostate cancer drugs. This invention provides the application of UFSP2 expression inhibitors and / or functional inhibitors in the preparation of prostate cancer therapeutic products, offering a new direction for drug development and targeted intervention strategies for prostate cancer treatment. Attached Figure Description

[0028] Figure 1 Figure 1 shows the results of UFSP2 regulating the proliferation and colony formation of prostate cancer cells (A shows the immunoblotting results of si-UFSP2 in DU145, PC3 and 22Rv1 cells; B shows the cell proliferation experiment results after UFSP2 knockdown; C shows the cell proliferation experiment results after UFSP2 overexpression; D shows the immunoblotting results of UFSP2 protein expression after stable knockdown; E shows a representative figure of the colony formation experiment; F shows the statistical analysis of the colony formation experiment results).

[0029] Figure 2 The results of UFSP2 as a target inhibiting the migration of prostate cancer cells are shown in the figure (A in the figure is a representative figure of the Transwell migration experiment; B is a statistical analysis of the Transwell migration experiment results; C is a representative figure of the cell scratch experiment; D is a statistical analysis of the cell scratch healing experiment results).

[0030] Figure 3The figure shows the results of UFSP2 as a target to inhibit the growth of prostate cancer in vivo (A in the figure is a grouped arrangement of 22Rv1 subcutaneous xenografts; B is the tumor growth curve recorded periodically after the mice were tumor-bearing; C is the statistical results of the body weight of the tumor-bearing mice; D is the statistical analysis of the final tumor weight after removal).

[0031] Figure 4 The results of using UFSP2 as a target to inhibit in vivo metastasis of prostate cancer (A in the figure is a representative image of animal in vivo imaging; B is a representative image of ex vivo organ imaging; CE is a statistical analysis of the number of metastatic lesions in different organs).

[0032] Figure 5 Figure 5 shows the results of UFSP2 expression in clinical prostate cancer tissues and its relationship with proliferation-related molecules in Example 5 (A in the figure is the immunoblotting results of UFSP2 protein expression in 8 pairs of prostate cancer tissues and paired adjacent normal tissues; B is the quantitative analysis of UFSP2 protein expression in A; C is the representative immunohistochemical staining map of UFSP2, c-Myc and CyclinD1 in adjacent normal tissues and cancer tissues; D is the paired statistical results of the immunohistochemical staining intensity of UFSP2, c-Myc and CyclinD1 after analysis by ImageJ software; E is the correlation analysis of UFSP2 expression with c-Myc and CyclinD1 expression in clinical tissue samples; F is the correlation analysis of UFSP2 with c-Myc and CyclinD1 expression based on the GEPIA 3.0 database). Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: UFSP2 as a target to inhibit prostate cancer proliferation

[0036] 1. Cell Culture

[0037] Prostate cancer cell lines 22Rv1, DU145, and PC3 were purchased from the American Type Culture Collection (ATCC, Rockefeller, MD, USA). 22Rv1 cells were cultured in RPMI-1640 medium (Gibco, Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum; DU145 and PC3 cells were cultured in DMEM / F12 medium (Gibco, Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum. Cell lines were identified by STR mapping and tested for mycoplasma contamination using a kit (#40612ES, Yeasen Biotechnology, Shanghai, China). Results were negative during cell passage and culture.

[0038] 2. siRNA transient knockdown experiment

[0039] DU145, PC3, and 22Rv1 cells were seeded in suitable culture plates and transfected after reaching the appropriate confluence. The transfection mixture was prepared by mixing Opti-MEM medium (Gibco, #31985088), Lipofectamine RNAiMAX transfection reagent (Invitrogen, #13778150), and UFSP2 siRNA at a ratio of 500 μL:6 μL:12 μL. After incubation at room temperature for 15 min, the mixture was added to the cells to achieve a final siRNA concentration of 80 nM. The siRNA used was designed based on the UFSP2 sequence and synthesized by RiboBio (Guangzhou, China), and the sequence is as follows:

[0040] si-UFSP2-1:5'-GAACAAGGATGCATACTAT-3';

[0041] si-UFSP2-2: 5'-GACGGGAACTGGCTAATCA-3';

[0042] si-UFSP2-3: 5'-GTCTAATGCTTATCACTTT-3'.

[0043] A negative control group, si-NC, was also set up.

[0044] 3. Plasmid overexpression experiment

[0045] To investigate the effect of UFSP2 overexpression on the proliferation of prostate cancer cells, plasmids containing the wild-type coding sequence of UFSP2 were used for transfection. The vector used was CMV enhancer-MCS-3FLAG-SV40-Puromycin. DU145, PC3, and 22Rv1 cells were seeded in suitable culture plates. After cell adhesion, the UFSP2 overexpression plasmid was transfected using standard liposome transfection methods. An empty vector was used as a MOCK control group. Cells were cultured further after transfection for subsequent proliferation assays.

[0046] 4. Lentiviral stable transfection experiment

[0047] The lentiviral vector hU6-MCS-hEF1 / HTLVp-firefly_Luciferase-T2A-Puromycin, containing UFSP2 shRNA or a negative control shRNA, was used. The shRNA target sequence is consistent with the target sequence of the aforementioned siRNA. The lentivirus was constructed by Genechem (Shanghai, China). Logarithmic growth phase prostate cancer cells were seeded into 6-well plates and infected when the cell confluence reached approximately 50%. During infection, the lentivirus was added to the cells at a multiplicity of infection (MOI) of 10, and Polybrene was added to a final concentration of 5 μg / ml to improve infection efficiency. After overnight culture, the medium was replaced with fresh complete medium. Puromycin was used for selection as needed to establish stable UFSP2 knockdown cell lines for subsequent clonogenic assays and protein detection.

[0048] 5. Immunoblotting analysis

[0049] Cells from each of the above treatment groups were collected, and total protein was extracted using lysis buffer. After centrifugation, the supernatant was used for protein quantification. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking at room temperature, anti-UFSP2 (ab185965) and anti-GAPDH (#5174) antibodies were added, and the membranes were incubated overnight at 4°C. The following day, after washing the membranes, the corresponding secondary antibodies were added, and chemiluminescence immunoassay was used to develop and record band changes to evaluate the transient and stable knockdown efficiency of UFSP2.

[0050] Immunoblotting test results Figure 1 As shown in Figure A, si-UFSP2-1, si-UFSP2-2, and si-UFSP2-3 can significantly reduce the expression level of UFSP2 protein in DU145, PC3, and 22Rv1 cells, indicating that UFSP2 knockdown is effective.

[0051] After stable knockdown of UFSP2, the expression of UFSP2 protein in DU145, PC3, and 22Rv1 cells was significantly decreased, such as Figure 1As shown in D, this indicates that the constructed stable knockdown cell line can be used for subsequent clonogenic experiments.

[0052] 6. CCK-8 cell proliferation experiment

[0053] DU145, PC3, and 22Rv1 cells transfected with siRNA or overexpression plasmids were seeded in 96-well plates at a density of 2000 cells per well, with multiple replicates per group. At 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h, CCK-8 assay solution (Yeasen Biotechnology, Shanghai, China) was added to each well. After incubation at 37°C for the appropriate time, absorbance was measured at 450 nm to plot cell growth curves, which were used to evaluate the effects of UFSP2 knockdown or overexpression on the viability and proliferation of prostate cancer cells. Experiments were independently repeated three times, and data are expressed as mean ± standard deviation.

[0054] The results of the CCK-8 experiment are as follows: Figure 1 As shown in Figure B, compared with the si-NC group, the proliferation curves of DU145, PC3, and 22Rv1 cells were significantly lowered after UFSP2 knockdown, while the proliferation curves of all cell lines were significantly higher after UFSP2 overexpression, suggesting that UFSP2 can promote the proliferation of prostate cancer cells. Figure 1 As shown in C.

[0055] 7. Cloning experiment

[0056] Stable UFSP2 knockdown-mediated 22Rv1, DU145, and PC3 cells were seeded at low densities in 6-well plates, with 1000 22Rv1 cells per well and 500 DU145 and PC3 cells per well. The cells were cultured in complete medium for approximately 10–14 days, with the medium being changed according to cell growth status. Once visible colony formation was observed, culture was terminated, cells were fixed with fixative and stained with crystal violet, photographed, and the colony count was recorded to evaluate the effect of UFSP2 on the long-term proliferative capacity of prostate cancer cells. The experiment was independently replicated three times, and data are expressed as mean ± standard deviation.

[0057] Cloning experiment results as follows Figure 1 As shown in F, the number of DU145, PC3, and 22Rv1 cell clones in the sh-UFSP2-1 and sh-UFSP2-2 groups was significantly reduced compared to the sh-NC group. Figure 1 E in the figure shows that inhibiting UFSP2 can significantly weaken the long-term proliferation and clonogenic ability of prostate cancer cells.

[0058] Example 2: UFSP2 as a target to inhibit prostate cancer migration

[0059] 1. Cell treatment

[0060] DU145, PC3 and 22Rv1 cells were seeded in suitable culture plates. After the cells adhered, they were transfected with si-UFSP2-1, si-UFSP2-2, si-UFSP2-3 and si-NC respectively using the method in Example 1. After transfection, the cells were cultured for 48 hours until migration experiments were performed.

[0061] 2. Transwell migration experiment

[0062] Transwell chambers were used to evaluate the migration ability of prostate cancer cells. Cells transfected for 48 hours were digested and resuspended in serum-free medium, with 1×10⁶ 22Rv1 cells loaded per well. 5 DU145 cells were loaded at 2 × 10⁶ per well. 4 3 × 10⁶ PC3 cells were loaded per well. 4 Cells were added to the upper chamber of a Transwell apparatus; serum-containing complete culture medium was added to the lower chamber as a chemotactic solution. After an appropriate incubation period, unmigrated cells from the upper chamber were wiped away. Cells that had migrated across the membrane to the lower surface were fixed and stained with crystal violet. The cells were photographed under a microscope, and the number of migrating cells was counted. The experiment was independently repeated three times, and the data are expressed as mean ± standard deviation.

[0063] Transwell experimental results are as follows: Figure 2 As shown in Figure A, compared with the si-NC group, the number of migrating DU145, PC3, and 22Rv1 cells treated with si-UFSP2-1, si-UFSP2-2, and si-UFSP2-3 was significantly reduced: the longitudinal migration rate of DU145 cells after treatment decreased to 34.14 ± 1.83% (**). p <0.01), 17.68±3.64% (** p <0.01) and 24.44±1.38% (** p <0.01); the longitudinal migration rates of PC3 cells decreased to 13.14±1.32% (**p<0.01), 16.95±1.32% (**p<0.01), and 36.65±6.01% (***) after treatment, respectively. p <0.001); the longitudinal migration rate of cells treated with 22Rv1 decreased to 19.44±3.62% (** p <0.01), 16.20±2.12% (** p <0.01) and 25.46±1.67% (** p <0.01), such as Figure 2 As shown in B in the diagram.

[0064] 3. Scratch healing test

[0065] DU145, PC3, and 22Rv1 cells, transfected for 48 h, were seeded into the wells of 6-well plates pre-installed with IBIDI scratch inhibitors (IBIDI, Martin Redd, Germany). After the cells had fully adhered, the IBIDI scratch inhibitors were carefully removed, and detached cells were gently washed with PBS. Fresh culture medium was then added, and the initial scratch condition was recorded at 0 h. Subsequent culture continued, and images were acquired at corresponding time points to record changes in scratch width. Scratch healing was observed in DU145 and PC3 cells at 24 h, and in 22Rv1 cells at 120 h, and the relative scratch healing rate was calculated. Experiments were independently repeated three times, and data are expressed as mean ± standard deviation.

[0066] Scratch healing test, such as Figure 2 As shown in Figure C, knocking down UFSP2 significantly reduced the scratch closure rate in all groups, resulting in a lower relative scratch healing rate compared to the control group. The lateral migration rate of DU145 cells after treatment decreased to 35.60 ± 3.83% (**). p <0.01), 35.40±3.93% (** p <0.01) and 43.52±5.84% (** p <0.01); the lateral migration rate of PC3 cells decreased to 33.58±1.89% after treatment (*** p <0.001), 18.28±1.22% (*** p <0.001) and 33.72±7.92% (** p <0.01); the transverse migration rate of cells treated with 22Rv1 decreased to 55.43±1.71% (*** p <0.001), 48.76±6.48% (*) p <0.05) and 55.75±6.19% (* p <0.05), such as Figure 2 As shown in D in the diagram.

[0067] The above results indicate that UFSP2 can promote the migration of prostate cancer cells, and targeting UFSP2 can significantly inhibit the migration ability of prostate cancer cells.

[0068] Example 3: UFSP2 as a target to inhibit in vivo prostate cancer tumor growth

[0069] 1. Source of laboratory animals

[0070] Animal experiments were approved by the Laboratory Animal Ethics Committee of Guangzhou Medical University, approval number GY2024-397. NOG mice used in the experiments were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. and housed at the Laboratory Animal Center of Guangzhou Medical University. All mice were randomly assigned to groups of seven after one week of acclimatization.

[0071] 2. Construction of subcutaneous xenograft model

[0072] 22Rv1 cells with stable UFSP2 knockdown and negative control cells were selected, and cell suspensions were prepared after digestion. 4 × 10⁻⁶ cells were then added to the suspension. 6 A subcutaneous prostate cancer xenograft model was established by subcutaneously injecting 7 cells into the right side of 4-5 week old male NOG mice. The groups were sh-NC group, sh-UFSP2-1 group, and sh-UFSP2-2 group, with 7 mice in each group.

[0073] Subcutaneous xenograft photos as shown Figure 3 As shown in A, compared with the sh-NC group, the tumor volume formed in the sh-UFSP2-1 group and the sh-UFSP2-2 group was significantly smaller.

[0074] 3. Tumor growth monitoring

[0075] After modeling, the long and short diameters of the tumor were measured every 3 days, and the tumor volume was calculated using a standard formula. The mouse weight was also recorded to evaluate tumor growth and the overall condition of the animal.

[0076] Tumor growth curve as shown Figure 3 As shown in B, after stable knockdown of UFSP2, the tumorigenesis rate of 22Rv1 cells in mice was significantly slowed down.

[0077] Mouse weight statistics results are as follows Figure 3 As shown in C, there is no significant difference in body weight among the groups, indicating that UFSP2 knockdown did not cause significant systemic toxicity under the experimental conditions.

[0078] 4. Sample collection and tumor weighing

[0079] Four weeks after inoculation, the mice were euthanized and the subcutaneous tumor tissue was removed. The tumor weight was recorded by photograph and weighed to compare the differences in tumor growth among different groups.

[0080] Endpoint tumor weighing results as follows Figure 3 As shown in D, the tumor weights in both the sh-UFSP2-1 and sh-UFSP2-2 groups were significantly lower than those in the sh-NC group, indicating that targeting UFSP2 can significantly inhibit the in vivo growth of prostate cancer tumors.

[0081] Example 4: UFSP2 as a target to inhibit in vivo metastasis of prostate cancer

[0082] 1. Animal model construction

[0083] Single-cell suspensions were prepared using DU145 cells with stable UFSP2 knockdown and negative control cells. 1×10⁻⁶ cells were then used to prepare the suspensions. 6 A hematogenous metastasis model of prostate cancer was established by injecting cells into 4-5 week old male NOG mice via the tail vein. The groups were sh-NC group, sh-UFSP2-1 group, and sh-UFSP2-2 group, with 7 mice in each group.

[0084] 2. Liveness detection

[0085] After modeling, mice were anesthetized at predetermined time points, and in vivo bioluminescence imaging was performed using the PerkinElmer IVIS Spectrum in vivo imaging system and Living Imaging software to dynamically monitor tumor burden and metastasis in the mice. Imaging records were completed on days 15, 30, and 45.

[0086] In vivo imaging results as follows Figure 4 As shown in Figure A, compared with the sh-NC group, the luminescence signal in mice in the sh-UFSP2-1 and sh-UFSP2-2 groups was significantly reduced, suggesting that UFSP2 knockdown can inhibit the spread of prostate cancer cells in vivo.

[0087] 3. Imaging of ex vivo organs and statistics of metastatic lesions

[0088] Forty-five days after modeling, mice were euthanized and subjected to final in vivo imaging. Subsequently, organs such as the lungs, liver, and kidneys were isolated for in vitro imaging, and the number of metastatic lesions in each organ was statistically analyzed to evaluate the effect of UFSP2 on the in vivo metastatic ability of prostate cancer.

[0089] Ex vivo imaging results as follows Figure 4 As shown in B, the UFSP2 knockdown group exhibits significantly reduced translocation signals in organs such as the liver, lungs, and kidneys.

[0090] Statistical results of metastatic lesions are as follows Figure 4 As shown in the CE diagram, the number of organ metastases in the sh-UFSP2-1 and sh-UFSP2-2 groups was significantly reduced compared to the sh-NC group, indicating that targeting UFSP2 can significantly inhibit in vivo metastasis of prostate cancer.

[0091] The above results indicate that UFSP2 promotes the metastasis of prostate cancer, and inhibiting UFSP2 expression can reduce the metastatic burden, further supporting the application value of UFSP2 as a therapeutic target for prostate cancer.

[0092] Example 5: UFSP2 is highly expressed in clinical tissues of prostate cancer and is positively correlated with proliferation-related molecules.

[0093] 1. Collection of clinical tissue samples

[0094] All clinical samples for prostate cancer were obtained from the remaining discarded materials after routine examinations by the Department of Urology and the Department of Pathology of Foshan First People's Hospital. The acquisition and use of all samples were approved by the Medical Ethics Committee of Foshan First People's Hospital, with ethics approval number L

[2026] No. 28, and informed consent was obtained from all subjects. Eight pairs of fresh prostate cancer tissue and paired adjacent normal tissue were selected for protein immunoblotting detection; another 20 pairs of paraffin-embedded prostate cancer tissue and paired adjacent normal tissue were selected for immunohistochemical staining and correlation analysis.

[0095] 2. Clinical tissue protein immunoblotting detection

[0096] Eight pairs of fresh prostate cancer tissues and paired adjacent normal tissue samples were collected, and total protein was extracted using conventional methods. Preferably, tissue samples were lysed using tissue lysis buffer, and the supernatant was collected after centrifugation for protein quantification. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking at room temperature, anti-UFSP2 (ab185965) and anti-GAPDH (#5174) antibodies were added, and the membrane was incubated overnight at 4°C. The following day, the membrane was washed, and the corresponding secondary antibodies were added. Chemiluminescence imaging was used to develop and record band changes to compare the protein expression differences of UFSP2 in prostate cancer tissues and paired adjacent normal tissues.

[0097] Immunoblotting results as follows Figure 5 As shown in Figure A, in eight pairs of prostate cancer tissues and their paired adjacent normal tissues, UFSP2 protein expression in tumor tissues was generally higher than in adjacent normal tissues; quantitative analysis further confirmed that the UFSP2 protein level was significantly elevated in tumor tissues, such as... Figure 5 As shown in B in the diagram.

[0098] 3. Immunohistochemical staining

[0099] Twenty pairs of paraffin-embedded tissue sections from prostate cancer and paired adjacent normal tissue sections were collected. After dewaxing, hydration, antigen retrieval, and blocking, UFSP2, c-Myc, and Cyclin D1 antibodies were added and incubated, respectively. Subsequently, the corresponding secondary antibodies were added, followed by DAB staining and hematoxylin counterstaining. Images were acquired under a microscope, and the differences in staining intensity of each protein between adjacent and cancerous tissues were compared. Representative images were acquired at 200× and 400× fields of view, respectively.

[0100] Immunohistochemical results such as Figure 5As shown in Figure C, UFSP2 staining is significantly enhanced in prostate cancer tissue compared to adjacent normal tissue; simultaneously, the staining intensity of c-Myc and Cyclin D1 in cancer tissue is also significantly higher than that in adjacent normal tissue, as shown in Figure C. Figure 5 As shown in D in the diagram.

[0101] 4. Statistical analysis of staining intensity

[0102] ImageJ software was used for semi-quantitative analysis of immunohistochemical staining results. Images of prostate cancer tissue and paired adjacent normal tissue sections acquired under the same staining conditions and magnification were analyzed. The positive staining intensities of UFSP2, c-Myc, and CyclinD1 were measured, and the expression intensity values ​​of each protein were obtained to compare the expression differences between cancer tissue and adjacent normal tissue.

[0103] Clinical tissue correlation analysis results as follows Figure 5 As shown in E, UFSP2 expression is positively correlated with c-Myc expression and also positively correlated with CyclinD1 expression, suggesting that UFSP2 may be closely related to the molecular network associated with prostate cancer proliferation.

[0104] 5. Correlation analysis

[0105] Based on expression data obtained from immunohistochemistry, Pearson correlation analysis was used to evaluate the correlation between UFSP2 and the expression of c-Myc and CyclinD1. Further analysis of the GEPIA 3.0 database was conducted to further validate the clinical sample results.

[0106] The results of the GEPIA 3.0 database analysis are as follows: Figure 5 As shown in F, UFSP2 is positively correlated with CyclinD1 expression, while showing a weak positive correlation with c-Myc expression, further supporting the association between UFSP2 and prostate cancer proliferative activity.

[0107] The above results indicate that UFSP2 is highly expressed in clinical prostate cancer tissues and is associated with increased expression of tumor proliferation-related molecules, further supporting the application value of UFSP2 as a therapeutic target for prostate cancer from the perspective of clinical samples.

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of UFSP2 expression inhibitors in the preparation of drugs for treating prostate cancer, characterized in that, The UFSP2 is a human UFM1-specific protease 2, with an NCBI Gene ID of 55325 and a protein RefSeq accession number of NP_060829.

2. The expression inhibitor is a small interfering RNA, specifically one of si-UFSP2-1: 5'-GAACAAGGATGCATACTAT-3', si-UFSP2-2: 5'-GACGGGAACTGGCTAATCA-3', and si-UFSP2-3: 5'-GTCTAATGCTTATCACTTT-3'.

2. The application of UFSP2 expression inhibitors in the preparation of drugs for the prevention of prostate cancer, characterized in that, The UFSP2 is a human UFM1-specific protease 2, with an NCBI Gene ID of 55325 and a protein RefSeq accession number of NP_060829.

2. The expression inhibitor is a small interfering RNA, specifically one of si-UFSP2-1: 5'-GAACAAGGATGCATACTAT-3', si-UFSP2-2: 5'-GACGGGAACTGGCTAATCA-3', and si-UFSP2-3: 5'-GTCTAATGCTTATCACTTT-3'.

3. The application according to claim 1 or 2, characterized in that, The drug can inhibit the proliferation, clone formation, and migration of prostate cancer cells.

4. The application according to claim 1 or 2, characterized in that, The drug can inhibit tumor growth, reduce tumor size, and slow down tumor formation.

5. The application according to claim 1 or 2, characterized in that, The prostate cancer referred to is advanced prostate cancer, metastatic prostate cancer, or castration-resistant prostate cancer.

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