Use of a mimetic inhibitor of nna10-k148 in the preparation of a medicament for the treatment of prostate cancer

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a NAA10-K148 neosynthesis inhibitor in preparation of a preparation for treating prostate cancer, wherein the NAA10-K148 neosynthesis inhibitor refers to an RNA interference reagent for reducing neosynthesis level of the 148th lysine of NAA10; the amino acid sequence of NAA10 is shown as SEQ ID NO. 10. Through targeted NAA10-K148 neosynthesis modification, the application can effectively block the UBE2M-NAA10 oncogenic axis, reduce the NAA10 protein stability, inhibit the cancer-promoting function, and then significantly inhibit the proliferation, migration and invasion ability of prostate cancer cells. Therefore, the NAA10-K148 neosynthesis inhibitor can play a role in treating prostate cancer by specifically interfering with the modification site, and provides a brand-new treatment strategy for clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a mimetic inhibitor of NAA10-K148 in the preparation of a prostate cancer treatment agent. Background Technology

[0002] Currently, androgen deprivation therapy, targeting the androgen receptor signaling axis, is the main treatment for prostate cancer. The core of this therapy lies in inhibiting the growth of androgen-dependent prostate cancer cells by reducing androgen levels in the body or directly blocking androgen receptor function. Representative products include androgen receptor antagonists such as enzalutamide and CYP17 inhibitors such as abiraterone.

[0003] Enzalutamide competitively inhibits the binding of androgens to their receptors, blocking the nuclear translocation of androgen receptors and their downstream transcriptional activity; abiraterone, on the other hand, comprehensively blocks the production of androgens in the testes, adrenal glands, and tumor tissues by inhibiting the key androgen synthesis enzyme CYP17.

[0004] Although these therapies show good clinical efficacy in the early stages of treatment for most patients, significantly delaying disease progression and prolonging progression-free survival, the vast majority of patients eventually develop castration-resistant prostate cancer as treatment continues. At this stage, the tumor no longer relies on traditional androgen signaling pathways, exhibiting resistance to androgen deprivation therapy, and is often accompanied by increased invasiveness and distant metastasis. Current treatments have limited efficacy in the castration-resistant stage, and drug resistance is becoming an increasingly prominent issue, posing a significant challenge to clinical treatment.

[0005] This clinical situation highlights the urgent need to find and identify new therapeutic targets beyond the classic androgen receptor pathway. Exploring non-androgen receptor-dependent signaling pathways and their key regulators is of significant clinical and scientific value for developing novel treatment strategies, improving patient outcomes, and especially for the treatment of castration-resistant prostate cancer. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a mimetic inhibitor of NAA10-K148 in the preparation of a prostate cancer treatment agent, thereby solving the problems existing in the prior art.

[0007] The technical solution adopted in this invention is: This invention provides the application of a mimetic inhibitor of NAA10-K148 in the preparation of a prostate cancer treatment agent. The mimetic inhibitor of NAA10-K148 refers to an RNA interference agent that reduces the mimetic level of lysine at position 148 of NAA10. The amino acid sequence of NAA10 is shown in SEQ ID NO.10.

[0008] Preferably, the RNA interference reagent comprises any one of the following: A: Substances that reduce UBE2M expression levels; B: Substances that reduce RBX1 expression levels; C: Substances that reduce CUL4A expression; D: Substances that reduce NAA10 expression levels.

[0009] Preferably, the substance that reduces UBE2M expression includes any one of 1) to 5): 1) siRNA as shown in SEQ ID NO.7; 2) shRNA as shown in SEQ ID NO. 11; 3) shRNA as shown in SEQ ID NO. 12; 4) A recombinant vector containing the shRNA described in 2) or 3); 5) Produce lentiviral venom containing the shRNA described in 2) or 3).

[0010] Preferably, the preparation process of the lentivirus venom is as follows: The recombinant vector was transfected into HEK293T cells and cultured for 48-72 hours to obtain lentiviral venom.

[0011] Preferably, the substance that reduces RBX1 expression is the siRNA shown in SEQ ID NO.6.

[0012] Preferably, the substance that reduces CUL4A expression is the siRNA shown in SEQ ID NO.4.

[0013] Preferably, the substance that reduces NAA10 expression is the siRNA shown in SEQ ID NO.8.

[0014] Preferably, the formulation of the prostate cancer treatment agent is as follows: The liposome complex obtained by mixing the mimicry inhibitor with the transfection reagent is a preparation for treating prostate cancer.

[0015] Preferably, the formulation for treating prostate cancer also includes pharmaceutically acceptable excipients.

[0016] Preferably, the pharmaceutically acceptable excipients include at least one of lipid nanoparticles, protective agents, and isotonic modifiers.

[0017] Preferably, the lipid nanoparticles include at least one of SM-102, distearate phosphatidylcholine, and DMG-PEG2000.

[0018] Preferably, the protective agent includes at least one of sucrose and trehalose.

[0019] Preferably, the isotonic regulator includes at least one of sodium chloride, glucose, and glycerol.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention provides the application of a mimetic inhibitor of NAA10-K148 in the preparation of a prostate cancer treatment agent. The NAA10-K148 mimetic inhibitor is an RNA interference agent that reduces the mimetic level of lysine residue 148 of NAA10; the amino acid sequence of NAA10 is shown in SEQ ID NO. 10. This invention reveals for the first time that the NAA10 protein undergoes mimetic modification at the lysine residue 148, mediated by the UBE2M synergistic CRL4A-RBX1 E3 ligase complex, which is a key mechanism regulating the stability and function of the NAA10 protein. By targeting the mimetic modification of NAA10-K148, the UBE2M-NAA10 oncogenic axis can be effectively blocked, reducing the stability of the NAA10 protein and inhibiting its pro-cancer function, thereby significantly inhibiting the proliferation, migration, and invasion of prostate cancer cells. Therefore, the NAA10-K148 mimetic inhibitor can exert a therapeutic effect on prostate cancer by specifically intervening in this modification site, providing a novel treatment strategy for clinical practice. Attached Figure Description

[0021] Figure 1 UBE2M is highly expressed in prostate cancer patients; A: Analysis of the GEPIA2 dataset shows the expression of UBE2M in prostate cancer and normal tissues, presented as box plots; B: Western blot analysis of patients 1-3 showed that UBE2M expression was significantly higher in prostate cancer tissues compared with matched adjacent non-cancerous tissues. C: Western blot analysis of patients 4-6 showed that UBE2M expression was significantly higher in prostate cancer tissue compared with matched adjacent non-cancerous tissues; D: Statistical graph of protein blotting results.

[0022] Figure 2 UBE2M modulates the proliferation, migration, and invasion of prostate cancer cells; A: CCK-8 assay to evaluate the proliferation capacity of PC-3 cells after UBE2M knockdown; B: Colony formation assay to assess the proliferation capacity of PC-3 cells after UBE2M knockdown; C: Statistical results of the colony formation experiment; D: Transwell assay to evaluate the migration and invasion ability of PC-3 cells after UBE2M knockdown; E: Statistical results of the Transwell experiment; F: Western blot analysis of EMT marker expression levels in PC-3 cells after UBE2M knockdown; G: Statistical results of the F plot.

[0023] Figure 3 UBE2M interacts with NAA10 in prostate cancer cells; A: After enriching Flag-UBE2M with Flag magnetic beads in HEK293T cells, Western blot analysis detected the interaction between the two proteins. B: After enriching HA-NAA10 with HA magnetic beads in HEK293T cells, Western blot analysis detected the interaction between the two proteins. C: The endogenous interaction between UBE2M and NAA10 was detected in PC-3 cells using the UBE2M antibody; D: The endogenous interaction between UBE2M and NAA10 was detected in PC-3 cells using the NAA10 antibody; E: The proximity connection experiment demonstrated the interaction between UBE2M and NAA10 in PC-3 cells.

[0024] Figure 4 UBE2M regulates the expression of NAA10 in prostate cancer cells; A: NAA10 protein expression level was detected after UBE2M knockdown in PC-3 cells and NAA10 protein expression level was detected after UBE2M overexpression in DU145 cells; B: The expression level of UBE2M protein was detected after NAA10 knockdown in PC-3 cells and after NAA10 overexpression in DU145 cells. C: Western blot analysis of the expression levels of UBE2M, NAA10, and ADAM9 proteins in tumor tissue; D: Western blot statistical analysis of the expression levels of UBE2M, NAA10, and ADAM9 proteins in tumor tissues; E: CCK-8 assay to evaluate the proliferation capacity of DU145 cells under the regulation of UBE2M and NAA10; F: Transwell assays were used to evaluate the migration and invasion capabilities of DU145 cells under the regulation of UBE2M and NAA10. G: Statistical analysis of the migration and invasion capabilities of DU145 cells under UBE2M and NAA10 regulation using Transwell assays.

[0025] Figure 5 NAA10 undergoes UBE2M-dependent pseudomorphic modification; A: Western blot analysis of NAA10 mimicry modification levels when NEDD8 is overexpressed; B: Western blot analysis of NAA10 mimicry modification levels when NAA10 is overexpressed; C: Western blot analysis of the effects of UBE2M and UBE2F on NAA10 mimicry modification levels; D: Detection of NAA10 mimicry modification during UBE2M overexpression; E: Detection of NAA10 pseudomorphic modification when UBE2M is knocked down; F: Western blot analysis of NAA10 mimicry modification regulated by UBE2M and its catalytic inactivation mutant UBE2M-C111S.

[0026] Figure 6 CRL4A-RBX1 is an E3 ligase complex of NAA10; A: Western blot analysis of the interaction between NAA10 and NEDD8 after RBX1 knockdown; B: Western blot analysis of the interaction between NAA10 and NEDD8 after RBX1 overexpression; C: mimetic modification level of NAA10 under various Cullin knockdown conditions; D: Detection of NAA10 mimicry modification level after CUL4A overexpression; E: Prediction of mimetic modification sites on NAA10; F: The level of mimetic modification of NAA10 after site-directed mutagenesis.

[0027] Figure 7 The stability of NAA10 was enhanced for UBE2M; A: After overexpression of UBE2M in 293T cells, NAA10 ubiquitination was detected by HA magnetic bead immunoprecipitation. B: Treat cells with CHX for a specified time and measure NAA10 protein levels; C: Relative expression level of NAA10 after CHX treatment; D: HA magnetic bead immunoprecipitation showed that the HA-Ub modification of the NAA10 K148R mutant was increased compared with that of the NAA10 WT. E: Nucleocytoplasmic separation experiments showed the effect of UBE2M overexpression on the distribution of NAA10 protein; F: Relative expression levels of NAA10 in the cytoplasm and nucleus; G: Immunoprecipitation with HA magnetic beads showed that the interaction between the NAA10 K148R mutant and ADAM9 was weakened compared to NAA10 WT. Detailed Implementation

[0028] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0029] The inventive concept of this invention is as follows: Disorders of protein post-translational modifications are a key characteristic of cancer, with mimetic modifications receiving increasing attention. The mimetic cascade involves E1 activators, E2 conjugators, and E3 ligases. The E1 activator is NAE, the E2 conjugator is UBE2M or UBE2F, and the E3 ligase is the Cullin-RING ligase family.

[0030] Overactivation of the mimetic pathway is frequently observed in human cancers, accelerating the degradation of tumor suppressor factors mediated by tumor suppressor chains (CRLs) and promoting tumorigenesis. Notably, UBE2M, as a key node in this pathway, exhibits a strong correlation between its expression level and the overall degree of mimeticization. UBE2M is highly expressed in various malignant tumors, and its deficiency can effectively inhibit cancer cell proliferation by stabilizing tumor suppressor factors. However, the specific role and regulatory mechanism of UBE2M in the development and progression of prostate cancer remain not fully understood.

[0031] Interestingly, the pro-cancer role of mimetic modification in prostate cancer may be intertwined with other key signaling pathways. Acetylation, another important post-translational modification, is regulated by the acetyltransferase NAA10, also known as ARD1, which has been shown to activate androgen receptors through acetylation, thereby promoting prostate cancer progression. Although the role of NAA10 in various tumors is widely recognized, the upstream regulatory mechanisms of its stability and enzyme activity are not yet fully elucidated, particularly whether it is regulated by mimetic modification and the role of this modification in abnormal NAA10 expression and prostate cancer development. These remain research gaps in this field.

[0032] Currently, dysregulation of mimetic phenotypes is closely related to tumorigenesis, but its specific function and substrates in prostate cancer remain to be elucidated. Similarly, although the acetyltransferase NAA10 is known to be highly expressed in prostate cancer (PCa) and promote tumor progression, its own stability regulation mechanism remains a mystery. To address this, this invention discovers that UBE2M-mediated mimetic phenotypes are a novel modification for regulating NAA10 stability, and based on this, reveals a previously unknown PCa oncogenic axis, thus filling the aforementioned research gaps.

[0033] This invention is the first to demonstrate that UBE2M is a clinically significant oncogenic driver in prostate cancer. The results show that UBE2M expression was significantly upregulated in PCa tissue samples, and its expression level was positively correlated with the patient's clinical stage, suggesting a close relationship with disease progression. Further functional experiments confirmed that UBE2M can significantly promote the proliferation, migration, and invasion of PCa cells in vitro; simultaneously, in vivo experiments demonstrated that UBE2M can effectively drive tumor growth.

[0034] In terms of mechanism of action, this invention identified acetyltransferase NAA10 as a key downstream effector molecule of UBE2M. Studies revealed that UBE2M not only specifically interacts with NAA10 but also directly participates in regulating its protein stability. Further phenotypic rescue experiments confirmed that knocking down NAA10 eliminated the oncogenic phenotype induced by UBE2M overexpression. This result strongly suggests that NAA10 is a crucial functional substrate essential for UBE2M to exert its oncogenic function in PCa.

[0035] Most importantly, this invention reveals for the first time that NAA10 undergoes mimetic modification, catalyzed by the UBE2M synergistic CRL4A-RBX1 E3 ligase complex. Through site screening and identification, this invention further identifies the highly conserved lysine residue at position 148 as the major mimetic modification site on NAA10. This discovery adds a novel regulatory dimension to the complex post-translational regulatory network of NAA10—which is known to be modified by phosphorylation, acetylation, and hydroxylation. Functional studies confirm that this mimetic modification promotes the accumulation of NAA10 protein by enhancing its stability, thereby driving the aggressive phenotype of prostate cancer.

[0036] This invention reveals a novel mechanism of post-translational modification interaction in cancer: mimeticization drives tumor progression by stabilizing the acetyltransferase NAA10. Building on this discovery, this invention further raises the scientific question that mimetic modification of NAA10 may simultaneously regulate its acetyltransferase activity. In-depth exploration of the interaction between mimeticization and acetylation—that is, how one modification affects the activity of key regulators of another modification—will open new research directions for understanding the mechanisms of tumorigenesis.

[0037] Based on the above findings, targeting the UBE2M-NAA10 signaling axis may provide a new treatment strategy for prostate cancer patients with overactive mimetic pathways.

[0038] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0039] Explanation of relevant terms involved in this invention: Mimeticization: A process similar to ubiquitination that links NEDD8 to substrate proteins.

[0040] The amino acid sequence of UBE2M described in this invention is shown in SEQ ID NO.9, and the amino acid sequence of NAA10 is shown in SEQ ID NO.10.

[0041] SEQ ID NO.9: MIKLFSLKQQKKEEESAGGTKGSSKKASAAQLRIQKDINELNLPKTCDISFSDPDDLLNFKLVICPDEGFYKSGKFVFSFKVGQGYPHDPPKVKCETMVYHPNIDLEGNVCLNILREDWKPVLTINSIIYGLQYLFLEPNPEDPLNKEAAEVLQNNRRLFEQNVQRSMRGGYIGSTYFERCLK.

[0042] SEQ ID NO.10: MNIRNARPEDLMNMQHCNLLCLPENYQMKYYFYHGLSWPQLSYIAEDENGKIVGYVLAKMEEDPDDVPHGHITSLAVKRSHRRLGLAQKLMDQASRAMIENFNAKYVSLHVRKSNRAA LHLYSNTLNFQISEVEPKYYADGEDAYAMKRDLTQMADELRRHLELKEKGRHVVLGAIENKVESKGNSPPSSGEACREEKGLAAEDSGGDSKDLSEVSETTESTDVKDSSEASDSAS.

[0043] The abbreviations for this invention are as follows: PCa: Prostate cancer; NAA10: N-α-acetyltransferase 10; Co-IP: Co-immunoprecipitation; PLA: Proximity Linkage Technology; CRLs: Cullin-RING ligases.

[0044] Example 1: The application of NAA10-K148 mimicry inhibitors in the preparation of prostate cancer treatment agents, as detailed below: 1. Method.

[0045] (1) Tissue sample.

[0046] Six prostate cancer (PCa) patients undergoing prostate surgery at the Department of Urology, Shandong Provincial Hospital affiliated with Shandong First Medical University, were treated with this invention, and matched normal tissue samples were collected. Informed consent was obtained from all patients, and all tissue samples collected during the surgery were confirmed by histopathological diagnosis.

[0047] (2) Tissue microarray and immunohistochemistry.

[0048] After antigen retrieval and blocking, the tissue was incubated overnight at 4°C with rabbit anti-UBE2M monoclonal antibody. Subsequently, the tissue was incubated with secondary antibody at room temperature for 1 hour. The tissue was then imaged and analyzed under a microscope.

[0049] (3) Xenograft mouse model.

[0050] Lentivirally infected PCa cells were suspended in 100 μL PBS / Matrigel and subcutaneously injected into 4-week-old male nude mice. Tumor volume was measured every 5 days. Mice were euthanized 20 days post-injection, and tumors were removed and weighed. For quantitative analysis, total protein was extracted from tumor tissue using RIPA lysis buffer, and the expression levels of target proteins were detected by Western blotting.

[0051] (4) Bioinformatics analysis.

[0052] Protein expression of UBE2M in clinical datasets was analyzed using the UALCAN database. Gene survival analysis of identified core oncogenes was performed using the GEPIA2 web server. This server is designed for large-scale expression profiling and interactive analysis.

[0053] (5) Plasmid transfection and RNA interference.

[0054] Plasmid transfection was performed using Lipofectamine 2000, and siRNA was transfected into cells using Lipofectamine RNAiMAX. All procedures were performed according to the instructions provided with the transfection reagents. The cell culture medium was changed 6 hours after transfection. The siRNA sequence is as follows: The siRNA targeting CUL1 is shown in SEQ ID NO.1, the siRNA targeting CUL5 is shown in SEQ ID NO.2, the siRNA targeting CUL2 is shown in SEQ ID NO.3, the siRNA targeting CUL4A is shown in SEQ ID NO.4, the siRNA targeting CUL4B is shown in SEQ ID NO.5, the siRNA targeting RBX1 is shown in SEQ ID NO.6, the siRNA targeting UBE2M is shown in SEQ ID NO.7, and the siRNA sequence targeting NAA10 is shown in SEQ ID NO.8.

[0055] SEQ ID NO. 1: 5'-GGTCGCTTCATAAACAACA-3'.

[0056] SEQ ID NO. 2: 5'-GTCTCACTTCCTACTGAACTG-3'.

[0057] SEQ ID NO. 3: 5'-GCCCUUACGUCAGUUGUAAAUUACA-3'.

[0058] SEQ ID NO. 4: 5'-GAAGCUGGUCAUCAAGAAC-3'.

[0059] SEQ ID NO. 5: 5'-AAGCCUAAAUUACCAGAAA-3'.

[0060] SEQ ID NO. 6: 5'-GACAACAGAGAGUGGGAAU-3'.

[0061] SEQ ID NO. 7: 5'-CAGAGGUCCUGCAGAACAA-3'.

[0062] SEQ ID NO. 8: 5'-CUGACUGCGCCUUCACGAUUU-3'.

[0063] (6) Lentiviral infection.

[0064] PC-3 cells were infected with a lentivirus carrying shRNA targeting UBE2M. Lentiviral cells were produced by co-transfecting a recombinant vector containing shRNA into HEK293T cells. Viral supernatant was collected at 48 h and 72 h post-transfection and filtered through a 0.45 μm filter. Stable UBE2M knockdown cells were then selected using 1 μg / mL puromycin. Finally, the established stable cell lines were maintained in medium containing 0.4 μg / mL puromycin. This invention provides two shRNAs targeting UBE2M, with sequences shown in SEQ ID NO.11 and SEQ ID NO.12.

[0065] shUBE2M#1, SEQ ID NO. 11: GCGGATCCAGAAGGACATAAA.

[0066] shUBE2M#2, SEQ ID NO. 12: CAAGGTGAAGTGTGAGACAAT.

[0067] (7) CCK-8 experiment.

[0068] Collect cells from each group and digest them with trypsin, then at 3 × 10⁻⁶. 3 Cells were seeded at a density of [number] cells / mL into 96-well plates and incubated at 37°C for 8 hours. After cell adhesion, CCK-8 reagent was added, and the 96-well plates were incubated for 1 hour. OD values ​​at 450 nm were measured using a microplate reader at 0, 24, 48, and 72 hours. Cell proliferation curves were then generated based on the recorded OD values. Each experimental group was set up in triplicate, and the experiment was repeated three times for validation.

[0069] (8) Colony formation experiment.

[0070] For the colony formation experiment, 5×10 3 PC-3 cells were seeded into 6-well plates and cultured for 2 weeks. The cells were then washed with PBS, fixed with 1 mL of 4% paraformaldehyde in each well for 30 min, and washed again. Subsequently, 1 mL of 0.01% crystal violet staining solution was added to each well, and staining was performed for 20 min. The experiment was repeated three times.

[0071] (9) Migration and invasion experiments.

[0072] Total 1×10 4 Cells were seeded in serum-free medium into the upper chamber of a 12-well Transwell plate. The lower chamber was filled with medium containing 20% ​​FBS, and the cells were incubated for 48 h. After incubation, the cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and stained with 0.01% crystal violet solution. The experiment was repeated three times. For the invasion assay, 1×10⁶ cells were seeded into the upper chamber of a 12-well Transwell plate. 4 One cell was seeded into the upper chamber of a pre-coated Matrigel. All other steps were the same as described above.

[0073] (10) Protein blot analysis.

[0074] Cells were lysed after rinsing with phosphate-buffered saline and then lysed with RIPA lysis buffer supplemented with a protease inhibitor mixture. The supernatant was collected, SDS-PAGE loading buffer was added, and the mixture was boiled at 98°C for 5 min. Proteins in the sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocking with PBST containing bovine serum albumin at room temperature for 1 h, the membrane was incubated overnight at 4°C with a specific primary antibody diluted in PBST, followed by incubation at room temperature with a secondary antibody in PBST for 2 h. Protein bands were visualized using a chemiluminescence kit.

[0075] (11) Co-immunoprecipitation experiment.

[0076] Cells were lysed using a lysis buffer containing a mixture of 50 mM Tris-HCl, 50 mM NaCl, 2 mM DTT, 0.2% NP-40, and a protease inhibitor. For immunoprecipitation involving UBE2M or NAA10 antibodies, A / G magnetic beads were incubated with the designated antibody at 4°C for 8 h. The beads were then mixed with cell lysis buffer and incubated overnight at 4°C. For immunoprecipitation using Flag or HA magnetic beads, the cell lysis buffer was directly mixed with the Flag or HA beads and incubated overnight at 4°C. After incubation, the beads were washed three times with cold lysis buffer. The antigen was eluted by boiling in SDS-PAGE loading buffer, followed by Western blot analysis using the designated antibody.

[0077] (12) Proximity connection experiment.

[0078] Perform the proximity ligation assay according to the manufacturer's instructions. Seed cells onto coverslips placed in 12-well plates. Remove the culture medium, wash cells three times with PBS, fix with paraformaldehyde, and permeabilize with Triton X-100. After blocking, incubate cells with primary antibody overnight at 4°C. Then add PLA probe, anti-rabbit PLUS, and anti-mouse MINUS, and incubate at 37°C for 1 h. After two washes, incubate cells with ligation reagent at 37°C for 30 min, and then amplify with polymerase solution. Finally, stain the cell nuclei with DAPI. Acquire images using an Olympus microscope.

[0079] (13) Statistics and repeatability.

[0080] Unless otherwise specified, statistical significance of in vitro and in vivo tests was assessed using unpaired two-tailed Student's t-test or Wilcoxon's two-tailed rank-sum test. Unless otherwise specified, comparisons between multiple groups were performed using one-way ANOVA, with Dunnett's post-hoc test used where appropriate. All statistical tests were performed using GraphPad Prism.

[0081] 2. Results.

[0082] (1) UBE2M is highly expressed in prostate cancer.

[0083] To investigate the expression characteristics of UBE2M in prostate cancer, this invention first assessed the transcriptional level of UBE2M through bioinformatics analysis. Mining the TCGA database using the GEPIA2 platform revealed that the mRNA level of UBE2M was significantly upregulated in tumor tissue compared to normal prostate tissue. Based on this, this invention further validated the protein expression of UBE2M in clinical samples. Western blotting of prostate cancer specimens and their paired adjacent normal tissues confirmed that the protein expression level of UBE2M in tumor tissue was also significantly higher than that in normal tissue. The above results are detailed below. Figure 1 .

[0084] In summary, UBE2M exhibits consistently high expression at both the transcriptional and protein levels in prostate cancer and may drive tumor progression.

[0085] (2) UBE2M promotes the malignant phenotype of prostate cancer cells in vitro and in vivo.

[0086] To investigate the biological function of UBE2M in prostate cancer, this invention conducted a study on the gene knockdown of UBE2M.

[0087] This invention first established stable UBE2M knockdown PC-3 cells, namely shUBE2M#1 and shUBE2M#2, using lentivirus-mediated shRNA. CCK-8 assays showed that UBE2M knockdown significantly inhibited cell proliferation. This finding was confirmed by colony formation assays, which showed a significant decrease in colony-forming ability after UBE2M depletion. Given the crucial roles of migration and invasion in prostate cancer progression, this invention next assessed migration and invasion capabilities using Transwell assays; UBE2M knockdown significantly impaired the migration ability of PC-3 cells. Similarly, Matrigel invasion assays showed that invasion ability was significantly inhibited, indicating that UBE2M promotes metastatic potential. At the molecular level, Western blot analysis of epithelial-mesenchymal transition (EMT) markers showed that UBE2M knockdown increased the expression of the epithelial marker E-cadherin while decreasing the levels of mesenchymal markers N-cadherin and Vimentin. These results are detailed in [link to results]. Figure 2 .

[0088] In summary, these findings suggest that UBE2M acts as a key oncogenic protein in prostate cancer, driving tumor growth and metastatic progression by enhancing proliferation, migration, invasion, and EMT.

[0089] (3) UBE2M interacts directly with NAA10.

[0090] To screen downstream effector molecules of UBE2M, this invention performed UBE2M pull-down combined mass spectrometry analysis in DU145 cells and identified NAA10 as a high-confidence candidate interacting protein.

[0091] To further verify the specific binding between the two, an exogenous co-immunoprecipitation experiment was performed in HEK293T cells. The results showed that Flag-UBE2M could effectively precipitate HA-NAA10. Conversely, HA-NAA10 could also recycle Flag-UBE2M, confirming a direct physical interaction between UBE2M and NAA10. This invention was subsequently further confirmed in PC-3 cells using an endogenous Co-IP experiment, demonstrating that endogenously expressed UBE2M and NAA10 can form a natural protein complex.

[0092] Furthermore, to visualize their interactions in situ within cells, this invention employs proximity-based detection technology. Results showed that in DU145 cells, antibodies targeting endogenous UBE2M and NAA10 produced significant PLA fluorescence signals, primarily localized in the cytoplasm. These results are detailed in [the table below]. Figure 3 .

[0093] In summary, the interactions between UBE2M and NAA10 are confirmed through exogenous binding, endogenous binding, and in situ localization, and this interaction mainly occurs in the cytoplasm.

[0094] (4) UBE2M positively regulates the expression of NAA10 in prostate cancer cells.

[0095] Having determined the physical interaction between UBE2M and NAA10, this invention next investigates whether UBE2M affects the expression of NAA10.

[0096] This invention found that knocking down UBE2M in PC-3 cells reduces NAA10 protein levels, while overexpression of UBE2M in DU145 cells increases NAA10 protein levels; conversely, neither knockdown nor overexpression of NAA10 significantly alters UBE2M expression, indicating that UBE2M acts upstream of NAA10 in a unidirectional regulatory manner. These results are shown in [see attached image]. Figure 4 A and B.

[0097] This invention further validated this regulatory relationship in vivo using xenograft tumor tissue. Consistent with in vitro data, UBE2M knockdown led to a significant reduction in NAA10 and its key downstream effector ADAM9. To determine whether NAA10 serves as a functional downstream effector of UBE2M, a complementation experiment was performed in DU145 cells. NAA10 knockdown significantly inhibited cell proliferation, and this effect could not be reversed by UBE2M overexpression. Similarly, Transwell assays showed that NAA10 knockdown inhibited migration and invasion, and UBE2M overexpression failed to restore these abilities in NAA10-deficient cells. The above results are detailed in [link to documentation]. Figure 4 D~G.

[0098] In summary, these results indicate that NAA10 is essential for promoting UBE2M-mediated proliferation, migration, and invasion of prostate cancer cells.

[0099] (5) UBE2M promotes the mimicry of NAA10.

[0100] Since UBE2M is a key E2 enzyme in the mimicry cascade reaction, this invention hypothesizes that it may mediate the mimicry of NAA10. To verify this, this invention first used MLN4924, a selective inhibitor of NEDD8-E1, to perform in vitro mimicry experiments to examine whether NAA10 was mimicked.

[0101] To verify whether NAA10 undergoes mimicry modification, this invention performed in vitro mimicry detection in HEK293T cells. The results showed that exogenous overexpression of NEDD8 resulted in a clear migration-lag band, corresponding to NEDD8 covalently bound to NAA10, i.e., the mimicry form of NAA10. Treatment with the mimicry E1 enzyme inhibitor MLN4924 significantly weakened this band signal. Conversely, under NAA10 overexpression conditions, the NEDD8-NAA10 conjugate was also detected, and its formation was effectively inhibited by MLN4924. These results indicate that NAA10 can undergo mimicry modification intracellularly and is a true mimicry substrate. See [link to results]. Figure 5 A and B.

[0102] To identify the specific E2 enzyme catalyzing NAA10 mimicry, a series of comparative experiments were conducted. Results showed that in 293T cells, overexpression of UBE2M significantly enhanced NAA10 mimicry levels, while overexpression of UBE2F had no such effect. Further verification indicated that intervening in UBE2M expression levels correspondingly regulated NAA10 mimicry: overexpression of UBE2M upregulated mimicry, while knockdown of UBE2M downregulated it. To clarify whether this modification depended on the catalytic activity of UBE2M, a catalytically inactivating mutant, UBE2M-C111S, was introduced. Results showed that wild-type UBE2M enhanced NAA10 mimicry, while the mutant UBE2M-C111S lost this function. In summary, the mimicry modification of NAA10 specifically depends on the catalytic activity of UBE2M. The above results are shown in […]. Figure 5 C~F.

[0103] (6) Identify the key mimicry site K148 in the CRL4A–RBX1 E3 ligase complex and NAA10.

[0104] To identify the E3 ligases catalyzing NAA10 mimicry, this invention first focused on the Cullin-RING ligase family. Given that RBX1 is a known interacting protein with UBE2M, this invention examined the function of RBX1. The results showed that knockdown of RBX1 significantly inhibited NAA10 mimicry, while overexpression of RBX1 enhanced its modification. Through screening Cullin family members, this invention further identified CUL4A as a key component: interference with CUL4A expression could correspondingly downregulate or upregulate NAA10 mimicry levels, indicating that the CRL4A-RBX1 complex is the major E3 ligase mediating NAA10 mimicry. The above results are detailed in […]. Figure 6 A~D.

[0105] To precisely locate the mimetic modification sites on NAA10, this invention combined bioinformatics prediction with site-directed mutagenesis for screening. Four high-confidence lysine residues, K105, K148, K179, and K183, were predicted using the NeddyPreddy algorithm and mutated to arginine, respectively. Wild-type and mutant HA-NAA10 were co-expressed with His-NEDD8 in 293T cells, and anti-HA pull-down assays were performed. The results showed that the K148 mutation significantly eliminated the mimetic modification of NAA10, while the K105, K179, and K183 mutations had no such effect. These results are shown in […]. Figure 6 E and F.

[0106] (7) UBE2M enhances the stability of NAA10.

[0107] Given that mimeticization often competes with ubiquitination to regulate protein stability, this invention investigates whether UBE2M-mediated mimeticization affects NAA10 turnover. Overexpression of UBE2M significantly reduced the polyubiquitination level of NAA10. CHX tracking experiments with actinomycin showed that UBE2M knockdown significantly shortened the half-life of NAA10, indicating that UBE2M enhances the stability of NAA10.

[0108] To determine whether this stability depends on mimicry, this invention compared ubiquitination levels in wild-type NAA10 and the mimicry-deficient K148R mutant. The K148R mutation resulted in a significant increase in NAA10 polyubiquitination, demonstrating that mimicry at the K148 position antagonizes ubiquitin-dependent NAA10 degradation. Next, this invention examined whether UBE2M affects the subcellular distribution of NAA10. Nucleocytoplasmic separation in 293T cells showed that UBE2M overexpression promoted nuclear accumulation of NAA10, suggesting that mimicry may also regulate its compartmentalization. Finally, since NAA10 is known to regulate ADAM9 expression in prostate cancer, this invention tested whether their interaction is affected by mimicry. Co-immunoprecipitation experiments showed that the binding of the NAA10-K148R mutant to ADAM9 was significantly reduced compared to wild-type NAA10, indicating that mimicry is necessary for the efficient formation of a complex between NAA10 and its key downstream effectors. The above results are presented in […]. Figure 7 .

[0109] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of NAA10-K148 mimicry inhibitors in the preparation of agents for treating prostate cancer, characterized in that, NAA10-K148 mimicry inhibitors are RNA interference agents that reduce the mimicry level of lysine at position 148 of NAA10. The amino acid sequence of NAA10 is shown in SEQ ID NO.

10.

2. The application as described in claim 1, characterized in that, The RNA interference reagent includes any one of the following: A: Substances that reduce UBE2M expression levels; B: Substances that reduce RBX1 expression levels; C: Substances that reduce CUL4A expression; D: Substances that reduce NAA10 expression levels.

3. The application as described in claim 2, characterized in that, Substances that reduce UBE2M expression include any one of 1) to 5): 1) siRNA as shown in SEQ ID NO.7; 2) shRNA as shown in SEQ ID NO. 11; 3) shRNA as shown in SEQ ID NO. 12; 4) A recombinant vector containing the shRNA described in 2) or 3); 5) Produce lentiviral venom containing the shRNA described in 2) or 3).

4. The application as described in claim 3, characterized in that, The preparation process of the lentivirus venom is as follows: The recombinant vector was transfected into HEK293T cells and cultured for 48-72 hours to obtain lentiviral venom.

5. The application as described in claim 2, characterized in that, The substance that reduces RBX1 expression is the siRNA shown in SEQ ID NO.

6.

6. The application as described in claim 2, characterized in that, The substance that reduces CUL4A expression is the siRNA shown in SEQ ID NO.

4.

7. The application as described in claim 2, characterized in that, The substance that reduces NAA10 expression is the siRNA shown in SEQ ID NO.

8.

8. The application as described in claim 1, characterized in that, The formulation of the prostate cancer treatment is as follows: The liposome complex obtained by mixing the mimicry inhibitor with the transfection reagent is a preparation for treating prostate cancer.

9. The application as described in claim 8, characterized in that, Preparations for treating prostate cancer also include pharmaceutically acceptable excipients.

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