Application of substance for regulating protein gene expression in preparation of tumor chemotherapy drug sensitizer

Abstract

The invention discloses application of a substance for regulating protein gene expression in preparation of a tumor chemotherapy drug sensitizer, and belongs to the technical field of biology. The invention provides any one of the following applications of a substance for promoting non-classical ferroptosis: A1) preparing a tumor chemotherapy drug sensitizer; and A2) preparing a medicine for treating tumors. The substance for promoting non-classical ferroptosis is iFSP1 or a biological material for inhibiting or reducing or down-regulating expression of a coding gene of USP18 protein, the chemotherapeutic drug is cis-platinum, and the tumor is ovarian cancer. Experiments prove that ubiquitin specific protease 18 participates in a molecular mechanism of mediating cisplatin resistance of ovarian cancer. The iFSP1 drug inhibits the FSP1 function so as to significantly enhance the sensitivity of the USP18 high-expression ovarian cancer to cis-platinum chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a substance that regulates protein gene expression in the preparation of a tumor chemotherapy drug sensitizer. Background Technology

[0002] Ovarian cancer (OC) is the third most common and leading cause of gynecological cancer death among women worldwide. Based on histological subtypes, ovarian cancer is generally classified into epithelial and non-epithelial ovarian cancer. Epithelial ovarian cancer is more common clinically, accounting for approximately 90% of new cases, while the remaining 10% are non-epithelial ovarian cancers. Epithelial ovarian cancer is further divided into serous carcinoma, endometrial carcinoma, clear cell carcinoma, and mucinous carcinoma. Serous carcinoma includes high-grade serous carcinoma (HGSC) and low-grade serous carcinoma (LGSC). HGSCs are the most common type of ovarian cancer, exhibiting high genomic heterogeneity, a high recurrence rate, and often showing resistance to platinum-based chemotherapy. Most ovarian cancer patients are diagnosed at an advanced stage, lacking effective treatment strategies. Currently, the first-line chemotherapy regimen for ovarian cancer is platinum-based chemotherapy combined with other chemotherapeutic drugs, such as paclitaxel. Although cisplatin is the first-line platinum-based chemotherapy drug in clinical practice and is often effective in initial treatment, more than 70% of patients experience disease relapse due to the development of drug resistance. The emergence of cisplatin resistance severely limits its clinical efficacy and has become one of the major challenges in the treatment of ovarian cancer.

[0003] Cisplatin (DDP), or cis-dichlorodiaminoplatinum, is a platinum-containing potent anticancer drug widely used to treat various solid tumors, such as testicular cancer, ovarian cancer, head and neck cancer, bladder cancer, lung cancer, cervical cancer, melanoma, and lymphoma. Cisplatin has a simple structure and exerts its anticancer activity through multiple mechanisms. Excessive accumulation of intracellular reactive oxygen species (ROS) is a key factor contributing to its antitumor activity. Cisplatin promotes the production of superoxide anions (O2) through multiple pathways. -The generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) triggers various programmed cell death pathways through different mechanisms. However, tumor cells have evolved multiple adaptive mechanisms to resist cisplatin-induced oxidative stress and to escape or inhibit programmed cell death. Ferroptosis is a programmed cell death caused by excessive iron accumulation and lipid peroxidation damage. Ferroptosis is regulated in vivo by multiple metabolic processes, including redox homeostasis, amino acid metabolism, and lipid metabolism. The classical ferroptosis signaling pathway mainly depends on the activity of glutathione peroxidase 4 (GPX4). Dysregulation of ferroptosis is closely related to tumor chemotherapy resistance. Tumor cells enhance their antioxidant capacity by negatively regulating the ferroptosis process, thereby leading to chemotherapy resistance.

[0004] Ferroptosis suppressor protein 1 (FSP1) is a novel ferroptosis defense factor discovered in 2019 that functions independently of the classical GPX4 pathway. FSP1 is located on the cell membrane and reduces ubiquinone (CoQ) to dihydroubiquinone (CoQH2) via the mevalonate pathway. An FSP1 inhibitor (iFSP1) is a selective and effective inhibitor of FSP1. It directly inhibits the NAD(P)H-ubiquinone reductase activity by binding to the FSP1 protein on the cell membrane. 50 The value is 103 nM.

[0005] Ferroptosis is precisely regulated by a variety of post-translational modifications, among which ubiquitination is particularly important. Ubiquitin-specific proteases (USPs) are the most important class of deubiquitinating enzymes (DUBs) in cells. They regulate protein stability and function by removing ubiquitin chains from substrate proteins, thereby broadly affecting a variety of signaling pathways and physiological processes, including cell death. Summary of the Invention

[0006] The technical problem solved by this invention is how to solve the problem of cisplatin resistance.

[0007] To address the aforementioned technical problems, the first aspect of the present invention provides the application of a substance that promotes nonclassical ferroptosis in any of the following ways: A1) Preparation of tumor chemotherapy drug sensitizers; A2) Prepare drugs for treating tumors.

[0008] In the above text, the role of tumor chemotherapy drug sensitizers is to enhance the sensitivity of tumors to chemotherapeutic drugs or to reverse the drug resistance of chemotherapeutic resistant tumor patients.

[0009] In the above-described application, the tumor chemotherapy drug is a drug that generates ROS.

[0010] In the above-described application, the drug that generates ROS is a platinum-based chemotherapy drug; And / or, the platinum-based chemotherapy drug is cisplatin.

[0011] In the above-described application, the substance that promotes non-classical ferroptosis is an FSP1 inhibitor.

[0012] In some implementations, the FSP1 inhibitor may be iFSP1 (CAS: 150651-39-1), icFSP1, or viFSP1.

[0013] In the above-described applications, the substance that promotes non-classical ferroptosis is a biological material that inhibits, reduces, or downregulates the expression of the gene encoding the USP18 protein, or a biological material that inhibits, reduces, or downregulates the content or activity of the USP18 protein.

[0014] In the applications described above, the biomaterial is any one of the following: B1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the USP18 protein, or nucleic acid molecules that inhibit, reduce, or downregulate the activity or content of the USP18 protein. B2) Genes that express the nucleic acid molecules described in B1) B3), an expression cassette containing the gene described in B2). B4) a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3). B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4). Modifications to the nucleic acid molecules described in B6) and B1), or pharmaceutically acceptable salts thereof.

[0015] In the above-described application, the nucleic acid molecule is an shRNA or siRNA that targets the gene encoding the USP18 protein.

[0016] In some embodiments, the nucleotide sequence of the shRNA encoding the gene targeting the USP18 protein is the RNA encoded by the gene obtained by annealing SEQ ID No. 5 and SEQ ID No. 6.

[0017] In some embodiments, the nucleotide sequence of the siRNA encoding the gene targeting the USP18 protein is SEQ ID No. 3 or SEQ ID No. 4.

[0018] In a second aspect, the present invention provides the use of the substance that promotes non-classical ferroptosis and cisplatin described in the first aspect in the preparation of a drug for treating tumors.

[0019] In some embodiments, the application is the use of iFSP1 and cisplatin in the preparation of drugs for treating tumors.

[0020] The aforementioned treatment of tumors involves inhibiting tumor cell proliferation, survival, and / or metastasis.

[0021] The tumor mentioned above is ovarian cancer.

[0022] The tumor mentioned above is a cisplatin-resistant tumor.

[0023] As mentioned above, the tumor cells of the tumor can highly express the USP18 protein-coding gene.

[0024] In the above text, USP18 can be any of the following proteins: M1) is a protein with the amino acid sequence of SEQ ID No. 2; M2) is a protein derived from M1) with the same function obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 2; Proteins M3 and M1 show more than 80% identity; A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of M4 (M1), M2, or M3).

[0025] The aforementioned 80% or more identity can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0026] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0027] The term "modifier" refers to a product obtained by modifying the double-stranded RNA molecule. Various modification methods can be used, including one or more combinations selected from ribose modification, base modification, and phosphate backbone modification. The term "pharmaceutically acceptable salt" refers to a salt suitable for contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, within the bounds of reliable medical judgment, and with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in SM Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1.

[0028] Thirdly, the present invention provides a product for improving the sensitivity of ovarian cancer to cisplatin chemotherapy, comprising the substance described in the first aspect.

[0029] Fourthly, the present invention provides an antitumor drug comprising cisplatin and the substance described in the first aspect.

[0030] In some embodiments, the active ingredient of the antitumor drug may consist only of the substance and cisplatin, and may also contain other biological or non-biological components. Other active ingredients can be determined by those skilled in the art based on the efficacy of the antitumor drug.

[0031] In some embodiments, the substance and cisplatin in the antitumor drug may be packaged separately.

[0032] Experiments of this invention demonstrate that: 1. The expression level of USP18 in cisplatin-resistant ovarian cancer tissues was significantly higher than that in cisplatin-sensitive tissues; overexpression of USP18 could reduce the cisplatin IC50 of ovarian cancer cells. 50 The value increases, while knocking down USP18 can make cisplatin ICs more effective. 50The value is reduced, thus reducing drug resistance. USP18 drives ovarian cancer cells to resist cisplatin-induced ROS-mediated non-canonical ferroptosis by regulating the FSP1 pathway.

[0033] 2. iFSP1 has a chemosensitizing effect and can inhibit the function of the USP18 / FSP1 pathway in a dose-dependent manner; combined use increases the sensitivity of ovarian cancer cells to cisplatin in vitro; the tumor volume in the combination therapy group was significantly reduced compared with the single drug group.

[0034] 3. In clinical practice, for patients with high USP18 expression, the treatment regimen of cisplatin combined with iFSP1 is recommended. The level of USP18 expression is positively correlated with the effectiveness of the combined treatment regimen. Attached Figure Description

[0035] Figure 1 Cisplatin-induced ROS mediates drug resistance in ovarian cancer cells by upregulating USP18, and scavenging ROS can reverse this effect. A shows the proliferation of SKOV3 cells after treatment with solvent control, cisplatin, and cisplatin + N-acetylcysteine ​​(NAC) + mitochondrial-targeted antioxidant Mito-TEMPO, respectively, as detected by RTCA Software 1.2.1; B shows the expression of USP18 protein after treatment with solvent control, cisplatin, and cisplatin + N-acetylcysteine ​​(NAC) + mitochondrial-targeted antioxidant Mito-TEMPO, respectively, as detected by Western Blot; C shows the cell viability after overexpression of USP18 in OVCAR-3 cells and treatment with cisplatin or cisplatin + N-acetylcysteine ​​(NAC) + mitochondrial-targeted antioxidant Mito-TEMPO.

[0036] Figure 2 Immunohistochemical staining images of USP18 protein in tissues; A shows typical immunohistochemical staining images of USP18 in normal ovarian and ovarian cancer tissues (scale bar: 100 μm) and quantitative score comparison; B shows typical immunohistochemical staining images of USP18 in cisplatin-sensitive and drug-resistant ovarian cancer tissues and quantitative score comparison of USP18 expression levels.

[0037] Figure 3 Western blotting was used to detect the expression level of USP18 protein in cisplatin-sensitive and drug-resistant ovarian cancer tissues and SKOV3 cells. A shows the protein expression of USP18 in cisplatin-sensitive and drug-resistant patient tissues. B shows the expression of USP18 in wild-type SKOV3 cells and cisplatin-resistant SKOV3 cells.

[0038] Figure 4The effect of USP18 expression level on cisplatin sensitivity in ovarian cancer cells; A shows the transfection efficiency by Western blot after transient overexpression of USP18 in OVCAR-3 and A2780 cells; B shows the transfection efficiency by Western blot after transient knockdown of USP18 in CAOV3 and SKOV3 cells; C shows the effect of USP18 overexpression on cisplatin IC50 in OVCAR-3 and A2780 cells. 50 The effect of USP18 knockdown on cisplatin IC50 in CAOV3 and SKOV3 cells; D shows the effect of USP18 knockdown on cisplatin IC50 in CAOV3 and SKOV3 cells. 50 The impact of the value.

[0039] Figure 5 The effects of USP18 on the in vitro biological functions of ovarian cancer cells are shown in the following diagrams: A shows that overexpression of USP18 enhances the clonogenic ability of OVCAR-3 and A2780 cells; B shows that knockdown of USP18 inhibits the clonogenic ability of CAOV3 and SKOV3 cells; C shows that overexpression of USP18 enhances the invasion and migration abilities of OVCAR-3 and A2780 cells; D shows that knockdown of USP18 inhibits the invasion and migration abilities of CAOV3 and SKOV3 cells (scale bar: 500 μm); E shows that overexpression of USP18 promotes the proliferation of OVCAR-3 and A2780 cells; F shows that knockdown of USP18 inhibits the proliferation of CAOV3 and SKOV3 cells.

[0040] Figure 6 To investigate the effects of altering USP18 expression on in vitro ferroptosis-related biological functions and the ferroptosis bypass pathway, A2780 cells were shown to have reduced intracellular Fe... 2+ Lipid peroxide accumulation; B showed that knockdown of USP18 increased intracellular Fe in CAOV3 cells. 2+ Lipid peroxide accumulation (scale bar: 30 μm); C shows that the NADP+ / NADPH ratio increased after USP18 overexpression and decreased after USP18 knockdown. NADP+ / NADPH is an indicator of non-classical ferroptosis; D shows that the FSP1 protein expression level decreased after USP18 knockdown.

[0041] Figure 7 The inhibitory effect of USP18 gene knockdown on the growth and metastasis of ovarian cancer in vivo is shown in Figure A. Western blot analysis results of CAOV3 cells with stable USP18 knockdown are shown in Figures B and C. The effect of stable USP18 knockdown on the tumorigenicity of tumor cells derived from CAOV3 cells in mice is shown in Figure D. Representative images of lung metastases and the statistical results of the number of metastases in mice after stable USP18 knockdown are shown in Figure D. (Scale bar: 200 μm).

[0042] Figure 8 To treat transfected cells with iFSP1 drug to cisplatin IC50 50 The impact of the value.

[0043] Figure 9 The effect of cisplatin combined with iFSP1 on the survival rate of ovarian cancer cells in vitro.

[0044] Figure 10 The effects of cisplatin and iFSP1 treatment alone and in combination on the proliferation of CAOV3 and SKOV3 cells in vitro were investigated.

[0045] Figure 11 The effects of cisplatin and iFSP1 alone and in combination on the growth of CAOV3 subcutaneous tumors of ovarian cancer cells were investigated in vivo. A shows the tumor volume at the treatment endpoint after treatment with cisplatin and iFSP1 alone and in combination in mice. B shows the subcutaneous tumor growth curves after treatment with cisplatin and iFSP1 alone and in combination in mice. C shows the body weight curves of mice during treatment with cisplatin and iFSP1 alone and in combination in mice. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0049] Statistical analyses were performed using SPSS 26.0 and GraphPad Prism 9.0, specifically including: unpaired two-tailed t-tests for comparisons between two groups; and chi-square tests or two-way ANOVA for comparisons among multiple groups. Significance criteria: P < 0.05 (*) was statistically significant; P < 0.01 (**) was statistically significant; P < 0.001 (***) and P < 0.0001 (****) were highly statistically significant.

[0050] The human USP18 gene is located at position 22q11.21 of the human genome. The nucleotide sequence of the USP18 gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the USP18 gene is shown in SEQ ID No. 2.

[0051] Example 1: Effects of cisplatin treatment on the expression levels of ROS and USP18 in ovarian cancer cells. 1. Experimental Materials Cell lines: OVCAR-3 (purchased from Shanghai Qincheng Biotechnology Co., Ltd., catalog number: CL-0178), SKOV3 (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) human ovarian cancer cells; Reagents: Cisplatin (purchased from Qilu Pharmaceutical, National Drug Approval Number H20023461, concentration for treating SKOV3 cells: 20 μM, concentration for treating OVCAR-3 cells: 20 μM), NAC (purchased from MedChemExpress, catalog number HY-B0215, concentration for treating SKOV3 and OVCAR-3 cells: 10 μM), Mito-TEMPO (purchased from MedChemExpress, catalog number HY-112879, concentration for treating SKOV3 and OVCAR-3 cells: 10 μM), CCK-8 assay kit (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., catalog number C6005).

[0052] Plasmid: The CDS region (SEQ ID No. 1) of the USP18 gene was inserted into the fragment between the XhoI and KpnI recognition sites of CMV-MCS-3FLAG-SV40-NEOMYCIN (HC321 vector, also referred to as the Flag empty vector) to obtain the USP18 overexpression plasmid (referred to as USP18-Flag plasmid). The USP18 overexpression plasmid expresses the USP18 gene (constructed by Shanghai Jikai Gene Technology Co., Ltd.).

[0053] 2. Experimental Methods 1) Real-time cell proliferation detection: ① Take cells in the logarithmic growth phase, digest the cells and resuspend them in 10% serum medium, seed 1000 cells per well in an E-plate 96-well plate from Essen Biotech, USA, and incubate at room temperature for 30 minutes before placing them in an RTCA analyzer.

[0054] ② The cell adhesion time was set at 4 hours, during which no data was collected. Data was then collected every 15 minutes thereafter. After approximately 24 hours, the culture medium was replaced with one containing the drug, and the cells were cultured again, with data collected every 15 minutes in the same manner.

[0055] The above-mentioned drugs are cisplatin alone, or cisplatin, NAC and mito-TEMPO together.

[0056] ③Use RTCA Software 1.2.1 to standardize the cell index at the time of drug administration and analyze cell proliferation.

[0057] 2) Western blot analysis of USP18 protein expression levels: ① Extraction of cell proteins: After treating SKOV3 cells with different drugs for a period of time, cells were collected using a cell scraper, washed with PBS, and then lysed with RIPA lysis buffer containing a mixture of 1× protease inhibitors (purchased from Beijing Pulilai Gene Technology Co., Ltd., catalog number C1055). The cells were lysed on ice for 30 min, centrifuged at 12000 rpm for 20 min, and the supernatant was collected and recorded as protein.

[0058] ② Electrophoresis: Prepare a 10% SDS-PAGE gel, load 40 μg of protein into each well, and electrophore at 80V until the bromophenol blue reaches the bottom.

[0059] ③ Wet transfer method: Use rapid transfer buffer (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., product number WB4600) at 0.4A constant current for 30 min for transfer.

[0060] ④ Sealing: Seal with 5% skim milk at room temperature for 1 hour.

[0061] ⑤ Primary antibody incubation: USP18 rabbit monoclonal antibody (1:500, purchased from Wuhan Aibote Biotechnology Co., Ltd., catalog number A16739, overnight at 4℃); β-actin mouse monoclonal antibody (1:5000, purchased from Sigma-Aldrich, catalog number A5316, overnight at 4℃) were used as internal controls.

[0062] ⑥ Secondary antibody incubation: Rabbit secondary antibody (1:3000, purchased from Prometheus, USA, catalog number W4011, room temperature for 1 hour), mouse secondary antibody (1:2000, purchased from Prometheus, USA, catalog number W4021, room temperature for 1 hour).

[0063] ⑦ Detection and analysis: Development was performed using a high-sensitivity ECL chemiluminescence reagent kit (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., catalog number P2300), and signal acquisition was performed using an ImageQuant™ 800 imaging system.

[0064] 3) Cell survival rate was detected after transfection and drug treatment: OVCAR-3 cells were transfected with the empty Flag vector (referred to as the control group) and the USP18-Flag plasmid (referred to as the overexpression group).

[0065] ①Cultured cells in complete culture medium at 37℃ and 5% CO2 until the logarithmic growth phase, digested the cells and seeded into six-well plates, and cultured at 37℃ until the cell density reached 70-80% confluence.

[0066] ②Preparation of the transfection complex: Solution A: Take 125 μl of serum-free culture medium, add 2 μg of plasmid, mix gently, and let stand at room temperature for 5 min.

[0067] Solution B: Take 125 μl of serum-free culture medium, add 5 μl of Lipofectamine 2000 transfection reagent, mix gently, and let stand at room temperature for 5 min.

[0068] ③ Transfection complex formation: Mix equal volumes of solution A and solution B, mix gently, and let stand at room temperature for 15-20 minutes to obtain the transfection complex.

[0069] ④ Cell transfection: Remove the original culture medium from the cell culture wells, add 2 mL of fresh complete culture medium, add 250 μl of transfection complex dropwise to the wells, and gently shake the culture plate to mix evenly.

[0070] ⑤ Post-transfection treatment: Incubate at 37℃ and 5% CO2 for 6 hours, then replace with fresh complete culture medium and continue incubation for 48 hours for subsequent experiments.

[0071] ⑥ Drug treatment: After transfecting, each cell was seeded into a 96-well plate and cultured for 24 hours. Then, cisplatin, NAC and Mito-TEMPO were added, and the cells were cultured for another 48 hours at 37°C and 5% CO2.

[0072] ⑦ Cell viability assay: The CCK8 assay kit was used according to the instructions. After incubation at 37°C in the dark for 1 hour, the absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated as: (Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%.

[0073] The results are as follows Figure 1 As shown in Figure A, cisplatin treatment of SKOV3 cells led to increased intracellular ROS and decreased cell proliferation. However, cell proliferation recovered after clearing intracellular ROS with NAC and mito-TEMPO, but remained lower than the solvent control group. Representative Western blot images in Figure B show that cisplatin treatment of SKOV3 cells induced upregulation of USP18, but USP18 expression levels returned to the solvent control group after clearing intracellular ROS with NAC and mito-TEMPO. Figure C shows that compared to the solvent control group, overexpression of USP18 followed by cisplatin treatment increased cell viability in OVCAR-3 cells, while overexpression of USP18 followed by cisplatin treatment and simultaneous clearing of intracellular ROS with NAC and mito-TEMPO decreased cell viability. This indicates that cisplatin-induced increases in intracellular ROS further induce USP18 expression, protecting cells from ROS-induced cell death.

[0074] Example 2: Immunohistochemical staining of USP18 in ovarian cancer patients and normal ovarian tissues 1. Sample Source The tissue microarrays (Avilabio, Ova-481D163) used in this study included cancerous tissues and paired normal tissues from 49 ovarian cancer patients; the tissue microarrays (Avilabio, OC Ova01001 N158) used for normal ovarian tissues included 60 normal ovarian tissues; and the tissue microarray samples of ovarian cancer-sensitive and drug-resistant tissues were obtained from the Cancer Hospital of the Chinese Academy of Medical Sciences, Shenzhen Hospital, including 96 ovarian cancer tissues. This study was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences. All included specimens were pathologically confirmed, and the enrolled patients had not received preoperative radiotherapy, chemotherapy, immunotherapy, or biotherapy. All patients had complete clinicopathological data and postoperative follow-up information.

[0075] 2. Immunohistochemical staining steps 1) Place the paraffin-embedded pathological sections (i.e., the aforementioned tissue microarrays) in a 65°C oven for 4-6 hours to allow the paraffin to completely melt.

[0076] 2) After the temperature drops to room temperature, place the solution in xylene for dewaxing for 20 minutes, and then rehydrate with a gradient concentration of alcohol solutions: 100% ethanol for 30 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 60% ethanol for 5 minutes.

[0077] 3) Add 16 ml of EDTA antigen retrieval solution (50×) to 784 ml of deionized water to prepare 1× EDTA antigen retrieval solution. Place the prepared EDTA antigen retrieval solution in a microwave oven and boil it on high for 10 minutes. Then place the pathological slides in the boiled antigen retrieval solution and perform antigen retrieval on high for 20 minutes. Then allow it to cool naturally at room temperature. Wash three times with PBS solution for 5 minutes each time.

[0078] 4) Incubate with endogenous peroxidase inhibitor (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number PV-9000) at room temperature in the dark for 20 min. Wash three times with PBS solution on a shaker, 5 min each time.

[0079] 5) Wipe the pathological sections clean, block them with sheep serum (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number ZLI-9056) at room temperature in the dark for 20 minutes, and then wash them with PBS solution.

[0080] 6) Dilute the USP18 primary antibody (purchased from ABclonal, catalog number A16739) with PBS solution at a ratio of 1:100. Add 50-100 μL of antibody to each pathological slide and incubate overnight at 4°C in a humidified chamber.

[0081] 7) The next day, place the pathological sections at room temperature. After they have returned to room temperature, wash them with PBS solution on a shaker for 5 minutes each time, for a total of 3 times.

[0082] 8) Use reaction enhancement solution (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number PV-9000), incubate at room temperature for 20 min, and then wash slowly three times with PBS solution on a shaker for 5 min each time.

[0083] 9) Add secondary antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number PV-9000), incubate at room temperature for 20 min, and then wash three times with PBS solution on a shaker for 5 min each time.

[0084] 10) Use DAB chromogenic solution to develop the pathological sections under a microscope. Pay attention to the chromogenic intensity. Once the chromogenic intensity is satisfactory, stop the chromogenic process with PBS solution.

[0085] 11) Stain the nuclei with hematoxylin solution in a humidified chamber for about 5 minutes, then rinse the back of the slide with tap water to remove the hematoxylin solution from the slide.

[0086] 12) Differentiate using 1% hydrochloric acid and 75% ethanol solution for 2 seconds, then quickly place in tap water to re-blue for about 20 minutes. During this period, observe the blue staining of the cell nuclei under a microscope. Once satisfied, perform gradient alcohol dehydration.

[0087] 13) The gradient alcohol dehydration process is as follows: 60% ethanol for 5 min, 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 100% ethanol for 20 min, xylene solution for 20 min.

[0088] 14) Air dry the sections at room temperature, mount them with neutral resin, wipe the neutral resin off the coverslip, and observe the staining effect under a microscope.

[0089] 3. Methods for evaluating immunohistochemical results 1) Interpretation method: Two experienced pathologists were invited to interpret the immunohistochemical results. The expression of the target indicators was evaluated based on the intensity of cell staining and the proportion of positively stained cells to all cells. Five high-power fields were randomly selected from each pathological slide.

[0090] 2) Scoring criteria: ①Scores are based on the percentage of positively stained cells: 0%-4% is 0 points; 5%-25% is 1 point; 26%-50% is 2 points; 51%-75% is 3 points; and 76%-100% is 4 points.

[0091] ②Score based on staining intensity: 0 points for no staining; 1 point for light yellow staining; 2 points for brownish-yellow staining; 3 points for brownish-red staining.

[0092] ③ Comprehensive score calculation: The final immunohistochemical score is the product of the scores obtained based on the staining range and staining intensity. 0 points is negative (-); 1-4 points is weakly positive (+); 5-8 points is positive (++); 9-12 points is strongly positive (+++).

[0093] ④ Expression level grading: The median of all immunohistochemical scores was used as the cutoff value. Scores above the median were considered high expression, and scores below the median were considered low expression.

[0094] The experimental results are as follows: (1) Comparison of USP18 expression in ovarian cancer tissue and normal ovarian tissue ( Figure 2 A): In ovarian cancer tissues, 49 cases (60.5%) showed high expression and 32 cases (39.5%) showed low expression; in normal ovarian tissues, 7 cases (8.4%) showed high expression and 76 cases (91.6%) showed low expression, and the difference was statistically significant (p<0.0001).

[0095] (2) Analysis of cisplatin resistance-related expression ( Figure 2 B): In the cisplatin-sensitive tissue group, 27 cases (42%) showed high expression and 37 cases (58%) showed low expression; in the cisplatin-resistant tissue group, 21 cases (66%) showed high expression and 11 cases (34%) showed low expression. The difference was statistically significant (p=0.0304). This suggests that high USP18 expression is positively correlated with cisplatin resistance.

[0096] Compared to normal ovarian tissue, ovarian cancer tissue has a higher USP18 staining score; compared to cisplatin-sensitive patients, cisplatin-resistant patients have a higher USP18 staining score.

[0097] The above results indicate that USP18 is specifically highly expressed in ovarian cancer tissues, and that the expression level of USP18 is positively correlated with cisplatin resistance. Therefore, USP18 can serve as a diagnostic biomarker and efficacy prediction indicator for ovarian cancer. USP18 expression detection can be used for: auxiliary pathological diagnosis of ovarian cancer, prediction of chemotherapy sensitivity, and screening for the development of targeted therapies.

[0098] Example 3: Western Blot analysis of USP18 protein expression levels in cisplatin-sensitive and drug-resistant ovarian cancer tissues and SKOV3 cells. 1. Tissue sample processing: cisplatin-sensitive tissues (n=4) and cisplatin-resistant tissues (n=4) from clinical ovarian cancer were collected. An appropriate amount of tissue was added to RIPA lysis buffer containing a mixture of 1× protease inhibitors (purchased from Beijing Pulilai Gene Technology Co., Ltd., catalog number C1055). After grinding, the tissues were lysed at 4°C for 30 min, centrifuged at 12000 rpm for 20 min, and the supernatant was collected and recorded as protein.

[0099] 2. Cell sample processing: SKOV3 wild-type cells (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) and purchased SKOV3 cisplatin-resistant cell line (SKOV3-R) (preservation institutions: ATCC; HTB-77 ECACC; 91091004; Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; China Center for Type Culture Collection). Cells were collected using a cell scraper, washed with PBS, and then lysed with the above-mentioned RIPA lysis buffer. The cells were lysed on ice for 30 min, centrifuged at 12,000 rpm for 20 min, and the supernatant was collected and recorded as protein.

[0100] The remaining experimental steps are the same as in Example 1.

[0101] Representative Western blot images show that USP18 was present in cisplatin-resistant tissues ( Figure 3 A) and SKOV3 resistant cells ( Figure 3 The expression in B) was significantly higher than that in the sensitive group and wild-type cells.

[0102] Example 4: Knockdown of USP18 enhances the sensitivity of ovarian cancer cells to cisplatin 1. Experimental Materials Cell lines: OVCAR-3 (purchased from Shanghai Qincheng Biotechnology Co., Ltd., catalog number: CL-0178), A2780 (preserved in our laboratory), CAOV3 (purchased from Shanghai Qincheng Biotechnology Co., Ltd., catalog number: QC-0055), SKOV3 (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) human ovarian cancer cells; Plasmid: The CDS region (SEQ ID No. 1) of the USP18 gene was inserted into the fragment between the XhoI and KpnI recognition sites of CMV-MCS-3FLAG-SV40-NEOMYCIN (HC321 vector, also referred to as the Flag empty vector) to obtain the USP18 overexpression plasmid (referred to as USP18-Flag plasmid). The USP18 overexpression plasmid expresses the USP18 gene (constructed by Shanghai Jikai Gene Technology Co., Ltd.).

[0103] siRNA: siNC (negative control), siUSP18-1 (RNA encoded by SEQ ID No. 3), siUSP18-2 (RNA encoded by SEQ ID No. 4); synthesized by Wuhan Jintuosi Biotechnology Co., Ltd. siUSP18-1: 5'-CCAGGGAGTTATCAAGCAA-3' (SEQ ID No. 3); siUSP18-2: 5'-CATCCGGAATGCTGTGGAT-3' (SEQ ID No. 4); Reagents: Cisplatin (purchased from Qilu Pharmaceutical, National Drug Approval Number H20023461; concentration gradients for treating OVCAR-3 and A2780 cells were 0, 2.5, 5, 10, 20, 25, 30, 40, 50 μM; concentration gradients for treating CAOV3 and SKOV3 cells were 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 μM), CCK-8 assay kit (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., catalog number C6005).

[0104] 2. Experimental Methods 1) Cell transfection: In OVCAR-3 and A2780 cells, the empty Flag vector (referred to as the control group) and the USP18-Flag plasmid (referred to as the overexpression group) were transfected, respectively; in CAOV3 and SKOV3 cells, siNC (referred to as the siNC group), siUSP18-1 (referred to as the siUSP18-1 group), and siUSP18-2 (referred to as the siUSP18-2 group) were transfected, respectively.

[0105] ①Cultured cells in complete culture medium at 37℃ and 5% CO2 until the logarithmic growth phase, digested the cells and seeded into six-well plates, and cultured at 37℃ until the cell density reached 70-80% confluence.

[0106] ②Preparation of the transfection complex: Solution A: Take 125 μl of serum-free culture medium, add 2 μg of plasmid or 5 μl of 20 μM siRNA solution, mix gently, and let stand at room temperature for 5 min.

[0107] Solution B: Take 125 μl of serum-free culture medium, add 5 μl of Lipofectamine 2000 transfection reagent, mix gently, and let stand at room temperature for 5 min.

[0108] ③ Transfection complex formation: Mix equal volumes of solution A and solution B, mix gently, and let stand at room temperature for 15-20 minutes to obtain the transfection complex.

[0109] ④ Cell transfection: Remove the original culture medium from the cell culture wells, add 2 mL of fresh complete culture medium, add 250 μl of transfection complex dropwise to the wells, and gently shake the culture plate to mix evenly.

[0110] ⑤ Post-transfection treatment: Incubate at 37℃ and 5% CO2 for 6 hours, then replace with fresh complete culture medium and continue culturing for 48 hours for subsequent experiments. Extract total cell protein and detect the protein expression level of USP18 using Western blot to verify whether the transfection was successful.

[0111] 2) Cisplatin treatment: After transfection, cells were seeded into 96-well plates and cultured for 24 h. Cisplatin was then added at gradient concentrations of 0, 2.5, 5, 10, 20, 25, 30, 40, 50 μM or 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 μM (each concentration was used in 3 replicates). Cells were then cultured at 37 °C and 5% CO2 for another 48 h.

[0112] 3) Cell viability assay: The CCK8 assay kit was used according to the instructions. After incubation at 37°C in the dark for 1 hour, the absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated as: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0113] 4) IC 50 Value calculation: Dose-response curve fitting was performed using GraphPad Prism 9.0 software. IC50 was calculated using nonlinear regression analysis. 50 value.

[0114] The results are as follows: Figure 4A shows the USP18 protein expression level in OVCAR-3 and A2780 cells after transfection, detected by Western blot using Flag M2 antibody (1:500, purchased from Sigma-Aldrich, catalog number F1804-1MG). The USP18 protein expression level was higher in the USP18-Flag transfection group, indicating successful transfection. Figure 4B shows the USP18 protein expression level in CAOV3 and SKOV3 cells after transfection, detected by Western blot using USP18 antibody (1:500, purchased from Wuhan Aiboteke Biotechnology Co., Ltd., catalog number A16739). The USP18 protein expression level was lower in the siUSP18-1 and siUSP18-2 transfection groups, indicating successful transfection.

[0115] Overexpression of USP18 to cisplatin IC 50 The effect of the value is as follows Figure 4As shown in Figure C, it can be seen that OVCAR-3 cells: control group IC 50 The concentration was 8.432 μM, and the IC50 of the overexpression group was [missing value]. 50 The concentration was 12.28 μM (p < 0.01). A2780 cells: Control group IC50 50 The concentration was 2.789 μM, and the IC50 of the overexpression group was [missing value]. 50 The value was 5.619 μM (p < 0.0001).

[0116] The effects of knocking down USP18 are as follows: Figure 4 As shown in Figure D, it can be seen that CAOV3 cells: siNC group IC 50 The IC is 11.24 μM, siUSP18-1 group. 50 The concentration was 4.275 μM (p < 0.001), and the siUSP18-2 group IC... 50 The concentration was 3.877 μM (p < 0.001). SKOV3 cells: siNC group IC50 50 13.65μM, siUSP18-1 group IC 50 The concentration was 5.644 μM (p < 0.05), and the siUSP18-2 group IC... 50 The value was 6.652 μM (p < 0.001).

[0117] The above results indicate that USP18 overexpression can significantly increase the IC50 of ovarian cancer cells against cisplatin. 50 USP18 knockdown significantly reduced the IC50 value of ovarian cancer cells, thus decreasing their sensitivity to cisplatin; USP18 knockdown significantly reduced the IC50 value of drug-resistant cells. 50 The value increases the sensitivity of ovarian cancer cells to cisplatin. Therefore, USP18 can serve as a key regulator of cisplatin resistance in ovarian cancer.

[0118] Example 5: Effects of USP18 on the in vitro biological function of ovarian cancer cells. 1. Experimental Materials Cell lines: OVCAR-3, A2780, CAOV3, SKOV3 human ovarian cancer cells Plasmids: Control Flag empty vector, USP18-Flag overexpression vector (constructed by Shanghai Jikai Gene Technology Co., Ltd.); siRNA: siNC (negative control), siUSP18-1, siUSP18-2 (synthesized by Wuhan Jintuosi Biotechnology Co., Ltd.) Main reagents: Crystal violet staining solution, Matrigel (purchased from Corning Incorporated, USA, catalog number 354234) Main consumables: Transwell chamber (8μm pore size, Corning Incorporated, USA, catalog number 3422) 2. Experimental Methods 1) Cloning experiment ① Cell transfection: Overexpression group: OVCAR-3 / A2780 transfected with control Flag (referred to as control cells) or USP18-Flag plasmid (referred to as overexpression cells).

[0119] Knockdown group: CAOV3 / SKOV3 transfected with siNC or siUSP18-1 or siUSP18-2.

[0120] ② After 48 hours of transfection, the cells were digested and seeded into 6-well plates.

[0121] ③ Incubate at 37℃ for 10-14 days, changing the culture medium every 3 days.

[0122] ④ Stain with 0.5% crystal violet (prepared with methanol) for 20 minutes.

[0123] 2) Transfer experiment ① Cell treatment is the same as ①-③ in the clonogenic experiment.

[0124] ② Transwell chamber hydration: Add 100 μL of blood-free and antibiotic-free culture medium to the upper chamber of the Transwell chamber and incubate at 37°C for 30 min.

[0125] ③ Culture the cells to be tested to the logarithmic growth phase, digest the cells, prepare a cell suspension with serum-free culture medium, add it to EP tubes, and wash twice with PBS.

[0126] ④ Discard the upper chamber culture medium, add 100 μL of cell suspension to the upper chamber of the Transwell, and add 600 μL of 20% / 30% serum-containing culture medium to the lower chamber. Incubate at 37°C for 12-24 hours.

[0127] ⑤ After migration is complete, remove the small chamber, discard the liquid in the upper chamber, and fix with 4% paraformaldehyde for 15 minutes.

[0128] ⑥ Stain with 0.5% crystal violet for 20 minutes.

[0129] ⑦ Wipe away the cells in the upper chamber with a cotton swab.

[0130] ⑧ Remove the microporous filter membrane with a blade and seal it.

[0131] 3) Invasion experiment: ① Transwell chamber pretreatment: Melt Matrigel on ice, dilute to 4% with serum-free medium, add 100 μL to the upper chamber of Transwell, and incubate at 37°C for 1 h to allow the gel to solidify. After the incubation, remove any unbound gel.

[0132] ②The remaining steps are the same as those in the transfer experiment.

[0133] 4) Real-time cell proliferation detection: ① Cell transfection: Overexpression group: OVCAR-3 / A2780 transfection of control Flag or USP18-Flag plasmid.

[0134] Knockdown group: CAOV3 / SKOV3 transfected with siNC or siUSP18-1 or siUSP18-2.

[0135] ② 24 hours after transfection, the cells were digested and resuspended in 10% serum medium. 1000 cells were seeded into each well of an E-plate 96-well plate from Essen Biotech Inc., USA. After standing at room temperature for 30 minutes, the cells were placed into an RTCA assay instrument.

[0136] ③ No data was collected during the 4-hour cell adhesion period. Data was then collected every 15 minutes thereafter, for a total of 96 hours.

[0137] ④ The cell proliferation was analyzed by standardizing the cell index at the time of adhesion completion (4h) using RTCA Software 1.2.1.

[0138] The results are as follows: Clonal formation ability results as follows Figure 5 As shown in Figures AB, compared with control cells, OVCAR-3 cells overexpressing USP18 showed increased clonogenic ability (p<0.0001); A2780 cells overexpressing USP18 showed increased clonogenic ability (p<0.0001); compared with siNC cells, CAOV3 cells knocked down with USP18 showed decreased clonogenic ability (p<0.0001); and SKOV3 cells knocked down with USP18 showed decreased clonogenic ability (p<0.0001).

[0139] Invasion migration ability results as follows Figure 5 As shown in CD, compared with control cells, OVCAR-3 cells overexpressing USP18 showed increased invasion, migration, and formation abilities (p<0.0001); A2780 cells overexpressing USP18 showed increased invasion and migration abilities (p<0.0001); CAOV3 cells knocked down by USP18 showed decreased invasion and migration abilities (p<0.0001); and SKOV3 cells knocked down by USP18 showed decreased invasion, migration, and formation abilities (p<0.0001).

[0140] Cell proliferation results as follows Figure 5 As shown in Figure EF, compared with control cells, OVCAR-3 and A2780 cells overexpressing USP18 showed significantly enhanced in vitro proliferation capacity, while CAOV3 and SKOV3 cells knocked down by USP18 showed significantly weakened in vitro proliferation capacity.

[0141] Example 6: Effects of altered USP18 expression on in vitro ferroptosis-related biological functions and the ferroptosis bypass pathway. 1. Experimental Materials Cell lines: A2780, CAOV3, SKOV3 human ovarian cancer cells Reagents: FerroOrange iron ion probe (purchased from Beiren Chemical Technology Co., Ltd., catalog number F374), Liperfluo lipid peroxide probe (purchased from Beiren Chemical Technology Co., Ltd., catalog number L248), NADP+ / NADPH assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0179).

[0142] Antibody: FSP1 rabbit monoclonal antibody (1:500, purchased from Wuhan Sanying Biotechnology Co., Ltd., catalog number 20886-1-AP) 2. Experimental Methods 1) Transfect A2780 cells with the empty Flag vector plasmid and the USP18-Flag overexpression plasmid, and transfect CAOV3 cells with siNC and siUSP18 (the method is the same as in Example 3).

[0143] 2) Fe² + and lipid peroxide detection ① Cell treatment: 48 h after transfection, add FerroOrange (1 μM, incubate at 37℃ in the dark for 1 h) and Liperfluo staining (10 μM, incubate at 37℃ in the dark for 1 h).

[0144] ② Confocal microscopy observation (FerroOrange: excitation / emission = 561 / 570-620 nm; Liperfluo: excitation / emission = 488 / 500-550 nm) and quantification of fluorescence intensity using ImageJ software.

[0145] 3) NADP+ / NADPH ratio detection: Follow the instructions in the kit to lyse the cells and separate the NADP+ and NADPH components. Measure the absorbance at OD450nm using a microplate reader and calculate the NADP+ / NADPH ratio.

[0146] 4) Transfect CAOV3 and SKOV3 cells with siNC and siUSP18 (method as in Example 3). After about 48 hours of transfection, cells were collected and total cell protein was extracted for Western blot analysis to detect the expression level of FSP1 protein (method as in Example 2).

[0147] The results are as follows: Fe² + and results of lipid peroxide levels, as follows Figure 6As shown in Figures AB, it can be seen that the fluorescence intensity of FerroOrange in A2780 cells overexpressing USP18 is decreased (p<0.001), and the fluorescence intensity of Liperfluo is decreased (p<0.0001); while the fluorescence intensity of FerroOrange in CAOV3 cells knocked down by USP18 is increased (p<0.0001), and the fluorescence intensity of Liperfluo is increased (p<0.0001).

[0148] The NADP+ / NADPH ratio results are as follows: Figure 6 As shown in Figure C, the ratio of the overexpression group increased (p<0.05), while the ratio of the knockdown group decreased (p<0.05).

[0149] Western blot results are as follows Figure 6 As shown in Figure D, knockdown of USP18 revealed a significant decrease in the expression of FSP1 protein, a key molecule in the ferroptosis bypass pathway.

[0150] The above results indicate that USP18 overexpression can reduce Fe² + Accumulation and lipid peroxidation ( Figure 6 A); Knockdown of USP18 significantly enhances ferroptosis sensitivity ( Figure 6 B); By examining the NADP+ / NADPH ratio, it was found that USP18 overexpression led to an increase in this ratio, suggesting that the FSP1 ferroptosis bypass pathway was inhibited; conversely, USP18 knockdown led to a decrease in the ratio, suggesting that this pathway was promoted. Figure 6 C). When USP18 was knocked down, FSP1 expression was suppressed, suggesting activation of the non-canonical ferroptosis pathway, which in turn promotes the process of ferroptosis. Figure 6 D).

[0151] Example 7: Inhibitory effect of USP18 gene knockdown on the in vivo growth and metastasis of ovarian cancer. 1. Experimental Materials 1) Laboratory animals: BALB / c female nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., strain code 401).

[0152] 2) Cell line: CAOV3 human ovarian cancer cells.

[0153] 3) Viral vectors: USP18 knockdown lentivirus and control virus (constructed by Shanghai Jikai Gene Technology Co., Ltd.). siUSP18-1, also known as USP18-siRNA, was designed based on the nucleotide sequence of the USP18 gene, and its sequence is as follows: USP18-siRNA: 5'-CCAGGGAGTTATCAAGCAA-3' (SEQ ID No. 3); The shRNA designed for USP18-siRNA and the construction of an expression vector containing the Sh-USP18 gene (denoted as Lenti-KD-USP18) were performed by replacing the fragment between the restriction endonuclease Agel and EcoRI recognition sites of the GV344 vector (Gekcare Co., Ltd., which contains the following element sequence hU6-MCS-Ubiquitin-firefly_Luciferase-IRES-puromycin) with the Sh-USP18 gene sequence, while keeping the remaining nucleotide sequence unchanged. The Sh-USP18 gene is a DNA molecule obtained by reverse complementation of the sense and antisense strands of the Sh-USP18 gene. The 5'-CCGG-3' end of the sense strand is used to ligate to the sticky end 5'-GGCC-3' of the vector after digestion with the restriction endonuclease Agel, and the 5'-AATT-3' end of the antisense strand is used to ligate to the sticky end 5'-TTAA-3' of the vector after digestion with the restriction endonuclease EcoRI.

[0154] The sequence of the Sh-USP18 gene is as follows: Chain of Justice: 5'-ccggCCAGGGAGTTATCAAGCAActcgagTTGCTTGATAACTCCCTGGtttttg-3' (SEQ ID No. 5).

[0155] antisense chain: 5'-aattcaaaaaCCAGGGAGTTATCAAGCAActcgagTTGCTTGATAACTCCCTGG-3' (SEQ ID No. 6).

[0156] The Lenti-KD-USP18 vector was transduced into 293T cells and packaged to obtain USP18 knockdown lentivirus.

[0157] The GV344 vector was transfected into 293T cells, and the resulting control virus was packaged.

[0158] 2. Experimental Methods 1) Construction of stable transfected cell lines: CAOV3 cells were infected with a control virus and a constructed USP18 knockdown lentivirus to obtain a stable transfected control cell line CAOV3-Lenti-KD-NC and a stable transfected knockdown cell line CAOV3-Lenti-KD-USP18. Total cellular protein was extracted, and the expression level of USP18 was detected by Western blot to verify successful stable transfection.

[0159] ① Cell seeding: Prepare cells by culturing in complete culture medium at a density of 3-5 × 10⁶ cells / year. 4CAOV3 cell suspension was seeded into 24-well plates and cultured at 37°C for 16-24 hours until the cell confluence reached 20-30%.

[0160] ② Cell Infection: On the second day, cell growth density was observed under a microscope. Viral infection was initiated when the cell density reached approximately 30%. Based on the MOI of the infected cells and the viral titer, appropriate volumes of USP18 knockdown lentivirus and empty lentivirus (control virus) were added. The calculation formula was: Virus volume = (MOI × cell number) / viral titer. The infection system was prepared as follows: 1 ml complete culture medium + 40 μL infection reagent (25×) + virus volume. Cells were incubated at 37°C for 16 hours, then replaced with complete culture medium and incubated at 37°C with 5% CO2. A control group of uninfected cells was also established to observe cell growth.

[0161] ③ 48-72 hours after lentiviral infection, the infected cells and control cells were cultured in a medium containing an appropriate concentration of puromycin. The medium containing puromycin was changed every 3-4 days until the uninfected control cells were killed by puromycin and no more cells died in the infected group. Then the puromycin concentration was reduced to the maintenance concentration (1 / 2-1 / 4 of the screening concentration), and the infected cells were screened and expanded. The cells were collected for Western blot to identify the expression level of USP18 protein.

[0162] 2) Subcutaneous xenograft model: 100 μl of CAOV3-Lenti-KD-NC and CAOV3-Lenti-KD-USP18 cell suspensions were injected subcutaneously into the bilateral axillae of mice, containing 1×10⁻⁶ cells. 6 Cells were collected subcutaneously from both axillae of mice. The long diameter (a) and short diameter (b) of the tumor were measured every 3 days, and the tumor volume was calculated using the formula V = 0.5 × a × b². Mice were sacrificed on day 22, and the tumor tissue was completely dissected.

[0163] 3) Lung metastasis model: Mice were randomly divided into two groups of 10 each. 100 μl of CAOV3-Lenti-KD-NC or CAOV3-Lenti-KD-USP18 cell suspension, containing 1 × 10⁻⁶ cells, was inoculated into the mid-tail vein. 6 Each cell. Mice were sacrificed approximately 8 weeks later, and lung tissue was obtained from their lungs to observe the number of metastatic lesions on the lung surface.

[0164] The results are as follows: Western blot analysis was performed on the expression levels of USP18 protein in the stable control cell line CAOV3-Lenti-KD-NC and the stable knockdown cell line CAOV3-Lenti-KD-USP18. The results were obtained using a USP18 antibody. Figure 7 As shown in Figure A, the CAOV3-Lenti-KD-USP18 cells exhibited low USP18 expression levels, indicating successful construction of stably transfected cells. Subcutaneous tumor growth inhibition results are as follows... Figure 7 As shown in BC, it can be seen that the growth rate of subcutaneous tumors in mice was significantly inhibited after stable knockdown of USP18 (p<0.0001).

[0165] Lung metastasis suppression results as follows Figure 7 As shown in Figure D, lung metastases occurred in 6 mice (60%) inoculated with CAOV3-Lenti-KD-NC and in 1 mouse (10%) inoculated with CAOV3-Lenti-KD-USP18. The difference was statistically significant (p<0.05). This indicates that stable knockdown of USP18 significantly reduced the number of lung metastases in mice and significantly decreased the tumor metastasis ability.

[0166] The above results indicate that knocking down USP18 inhibits the proliferation and metastasis of human ovarian cancer tumors.

[0167] Example 8: Study on the enhancement of cisplatin chemosensitivity by iFSP1 I. Effect of iFSP1 on the cisplatin sensitivity of ovarian cancer cells overexpressing USP18 1. Experimental Materials Cell lines: OVCAR-3 and A2780 human ovarian cancer cells; Plasmids: Flag empty vector, USP18-Flag overexpression vector (constructed by Shanghai Jikai Gene Technology Co., Ltd.); Reagents: Cisplatin (purchased from Qilu Pharmaceutical, National Drug Approval Number H20023461, concentration gradients for treating OVCAR-3 cells were 0, 2.5, 5, 10, 20, 25, 30, 40, 50 μM, and concentration gradients for treating A2780 cells were 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 μM), iFSP1 (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number I414449, concentration used was 5 μM), CCK-8 assay kit (purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., catalog number C6005).

[0168] 2. Experimental Methods 1) Cell transfection: The Flag empty vector (designated as the control group) and the USP18-Flag plasmid (designated as the overexpression group) were transfected into OVCAR-3 and A2780 cells; subsequent experiments were performed 48 h after transfection. The method for detecting transfection efficiency was the same as in Example 4.

[0169] 2) Cisplatin treatment: After transfection, cells were seeded into 96-well plates and cultured for 24 h. Cisplatin was then added at gradient concentrations of 0, 2.5, 5, 10, 20, 25, 30, 40, 50 μM or 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50 μM (each concentration was used in 3 replicates). Control solvent or iFSP1 (5 μM) was added simultaneously. Cells were cultured at 37°C and 5% CO2 for another 48 h. Cells transfected with different concentrations of cisplatin and the Flag cell was transfected with an empty cell and the control solvent was designated as Flag + solvent control; cells transfected with different concentrations of cisplatin and the USP18-Flag cell was transfected with the control solvent was designated as USP18-Flag + solvent control; cells transfected with different concentrations of cisplatin and the USP18-Flag cell was transfected with iFSP1 was designated as USP18-Flag + iFSP1. 3) Cell viability assay: The CCK8 assay kit was used according to the instructions. After incubation at 37°C in the dark for 1 hour, the absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated as: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0170] 4) IC 50 Value calculation: Dose-response curve fitting was performed using GraphPad Prism 9.0 software. IC50 was calculated using nonlinear regression analysis. 50 value.

[0171] Experimental results are as follows Figure 8 As shown: OVCAR-3 cells: Control group (Flag + solvent control) IC 50 The concentration of USP18 overexpression was 10.12 μM (USP18-Flag + solvent control), and the IC50 concentration was [missing value]. 50 The concentration was 20.05 μM, and the concentration in the USP18 overexpression and iFSP1 addition group (USP18-Flag + iFSP1) was 14.8 μM. A2780 cells: Control group (Flag + solvent control) IC50 50 The concentration was 1.999 μM, and the IC50 value for the USP18 overexpression group (USP18-Flag + solvent control) was 1.999 μM. 50 The concentration was 7.097 μM, while the concentration of USP18 overexpression combined with iFSP1 (USP18-Flag+ iFSP1) was 1.741 μM.

[0172] The above results indicate that USP18 overexpression can significantly increase the IC50 of ovarian cancer cells against cisplatin. 50 The addition of iFSP1 reduces the IC50 of ovarian cancer cells to cisplatin, thus lowering their sensitivity. 50 The value restores the sensitivity of ovarian cancer cells to cisplatin.

[0173] II. Effects of cisplatin combined with iFSP1 on the survival rate of ovarian cancer cells in vitro 1) Cell culture and treatment: CAOV3 and SKOV3 cells were seeded into 12-well plates and cultured for 24 hours.

[0174] 2) Cells were treated with a combination of cisplatin and iFSP1 and cultured at 37°C and 5% CO2 for 48 h: Twelve different drug concentrations of cisplatin (0, 5, 10, or 20 μM) were prepared by combining it with 0, 5, or 10 μM iFSP1 in culture media. The drug-free groups received an equal volume of physiological saline + DMSO (purchased from Sigma-Aldrich, catalog number D1650). 3) Cell viability was detected using a CCK-8 assay kit (Beijing Xinsaimei Biotechnology Co., Ltd., catalog number C0043), and absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula: [(OD experiment - OD blank) / (OD control - OD blank)] × 100%.

[0175] Experimental results are as follows Figure 9 As shown, cisplatin and iFSP1 alone effectively reduced cell viability; the combination of cisplatin and iFSP1 exhibited a significant synergistic effect, which was dose-dependent, with lower cell viability at higher concentrations. With increasing concentrations of both drugs, the cell viability level in the cisplatin and iFSP1 combination therapy group significantly decreased compared to the cisplatin-only group.

[0176] III. Effects of in vitro treatment with cisplatin and iFSP1, alone and in combination, on the proliferation of CAOV3 and SKOV3 cells. 1. Experimental Methods 1) CAOV3 and SKOV3 cells were seeded at 1000 cells per well in E-plate 96-well plates from Essen Biotech Inc., USA, and cultured at 37°C and 5% CO2 for 24 h.

[0177] 2) After replacing the culture medium with the drug-containing medium, place the samples in an RTCA analyzer. The drug treatment groups are as follows: ① Cisplatin monotherapy group (Qilu Pharmaceutical, National Drug Approval Number H20023461, 10μM): cultured in cell culture medium with 10μM cisplatin added; ②iFSP1 monotherapy group (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number I414449, 5μM): cultured in cell culture medium with 5μM of iFSP1 added; ③ Combined treatment group: cisplatin (10 μM) + iFSP1 (5 μM): cultured in cell culture medium supplemented with 10 μM cisplatin and 5 μM iFSP1; ④ Solvent control group: Equal volume of physiological saline + DMSO (purchased from Sigma-Aldrich, USA, catalog number D1650); 3) No data was collected during the 4-hour cell adhesion period. Data was then collected every 15 minutes thereafter, for a total of 96 hours.

[0178] 4) The cell proliferation was analyzed by standardizing the cell index at the time of successful adhesion (4h) using RTCA Software 1.2.1.

[0179] Experimental results are as follows Figure 10 As shown, compared with the control group, cisplatin or iFSP1 treatment alone significantly reduced the in vitro proliferation capacity of CAOV3 and SKOV3 cells, and the in vitro proliferation capacity was further reduced after combination therapy. Compared with the cisplatin monotherapy group, the cell proliferation capacity of the cisplatin and iFSP1 combination therapy group was significantly reduced.

[0180] IV. Effects of intracorporeal administration of cisplatin and iFSP1 drugs, alone and in combination, on the growth of subcutaneous tumors. 1. Experimental Materials 1) Laboratory animals: 40 female BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., strain code 401) 2) Cell line: CAOV3 human ovarian cancer cells 3) Medications: Cisplatin injection (Qilu Pharmaceutical, National Drug Approval Number H20023461); iFSP1 (Shanghai Aladdin Biochemical Technology Co., Ltd., I414449); 0.5% CMC-Na solution (mass-volume percentage, g:ml, solvent is water); physiological saline.

[0181] 2. Experimental Methods 1) Forty nude mice were randomly divided into four groups (n=10): solvent control group, cisplatin monotherapy group, iFSP1 monotherapy group, and cisplatin + iFSP1 combination therapy group.

[0182] 2) Tumor inoculation: Preparation of CAOV3 cell suspension (1×10⁻⁶) 6 (100 μL of cell suspension was injected subcutaneously into the left axilla of each mouse using cells / 100 μL PBS).

[0183] 3) Drug preparation: Cisplatin: Dissolve in physiological saline to a final concentration of 0.2 mg / mL; iFSP1: Dissolve in 0.5% CMC-Na to a final concentration of 4 mg / mL; 4) Administration method and dosage: Solvent control group: Intraperitoneal injection of 100 μL of normal saline (once every 3 days) + gavage administration of 100 μL of 0.5% CMC-Na solution (once a day); Cisplatin group: Intraperitoneal injection of cisplatin 1 mg / kg (once every 3 days); iFSP1 group: iFSP1 20 mg / kg by gavage (once daily); Combination therapy group: cisplatin 1 mg / kg (once every three days, intraperitoneal injection) + iFSP1 20 mg / kg (once daily, gavage); 3. Observation indicators 1) Tumor volume measurement (once every three days): Measure the major diameter (a) and minor diameter (b) with vernier calipers. The volume calculation formula is: V = ab² / 2.

[0184] 2) Weight monitoring (once every three days).

[0185] 3) Endpoint treatment: Mice were euthanized 18 days after treatment, tumor tissue was completely dissected, and the tumor diameter was measured and weighed.

[0186] The experimental results are as follows: Tumor growth inhibition results as follows Figure 11 As shown in A and 11B, the tumor volume in the combination therapy group was significantly smaller than that in the cisplatin monotherapy group (p<0.01) and the iFSP1 monotherapy group (p<0.01), and the tumor growth rate in the combination therapy group was significantly smaller than that in the cisplatin monotherapy group (p<0.05) and the iFSP1 monotherapy group (p<0.01).

[0187] Security assessment results such as Figure 11 As shown in Figure C, there was no significant difference in body weight between the mice in each drug administration group and the solvent control group (p>0.05).

[0188] The above results indicate that iFSP1 can reverse drug resistance in cisplatin-resistant ovarian cancer patients.

[0189] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Substances that promote nonclassical ferroptosis in any of the following applications: A1) Preparation of tumor chemotherapy drug sensitizers; A2) Prepare drugs for treating tumors.

2. The application according to claim 1, characterized in that: The tumor chemotherapy drug is a drug that generates ROS.

3. The application according to claim 2, characterized in that: The drug that generates ROS is a platinum-based chemotherapy drug; And / or, the platinum-based chemotherapy drug is cisplatin.

4. The application according to any one of claims 1-3, characterized in that: The substance that promotes nonclassical ferroptosis is an FSP1 inhibitor; And / or, the FSP1 inhibitor is iFSP1, icFSP1, or viFSP1.

5. The application according to any one of claims 1-3, characterized in that: The substance that promotes nonclassical ferroptosis is a biological material that inhibits, reduces, or downregulates the expression of the gene encoding the USP18 protein, or a biological material that inhibits, reduces, or downregulates the content or activity of the USP18 protein.

6. The application according to claim 5, characterized in that: The biomaterial is any one of the following: B1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the USP18 protein, or nucleic acid molecules that inhibit, reduce, or downregulate the activity or content of the USP18 protein. B2) Genes that express the nucleic acid molecules described in B1) B3), an expression cassette containing the gene described in B2). B4) a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3). B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4). Modifications to the nucleic acid molecules described in B6) and B1), or pharmaceutically acceptable salts thereof.

7. The application according to claim 6, characterized in that: The nucleic acid molecule is an shRNA or siRNA that targets the gene encoding the USP18 protein.

8. The use of the substance that promotes non-classical ferroptosis as described in any one of claims 1-7 and cisplatin in the preparation of a medicament for treating tumors.

9. The application according to any one of claims 1-8, characterized in that: The tumor is ovarian cancer.

10. A product for improving the sensitivity of ovarian cancer to cisplatin chemotherapy, comprising the substance described in any one of claims 1-8; Alternatively, an antitumor drug comprising cisplatin and the substance described in any one of claims 1-8.

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