Application of SOX13 as a gastric cancer marker

Abstract

The present invention relates to the application of SOX13 as a gastric cancer biomarker. By constructing ferroptosis-resistant strains, the present invention uses a microarray to screen out SOX13 as an important regulatory molecule for gastric cancer ferroptosis sensitivity, and discovers that the anti-influenza drug zanamivir can specifically target the SOX13 protein. The present invention effectively verifies that the SOX13 gene is involved in the regulation of gastric cancer ferroptosis sensitivity, providing theoretical data for the ferroptosis sensitivity and chemosensitivity of gastric cancer cells. In addition, zanamivir can specifically target the SOX13 protein and reverse gastric cancer ferroptosis resistance, providing a theoretical basis for the treatment of ferroptosis resistance and chemoresistance of gastric cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of SOX13 as a gastric cancer biomarker. Background Art

[0002] Gastric cancer is the fifth most common cancer globally and the fourth leading cause of cancer-related deaths. Despite significant progress in gastric cancer treatment, the prognosis of patients with advanced gastric cancer remains poor. Cisplatin-based chemotherapy remains the first-line adjuvant or neoadjuvant treatment for advanced gastric cancer, but the understanding of chemotherapy resistance remains an obstacle to clinical efficacy. Cancer immunotherapy, which uses immune cells to enhance their functions to eliminate cancer, including immune checkpoint inhibitors, has become an effective treatment for various cancers. However, only a small proportion of gastric cancer patients (about 15%) respond to immunotherapy due to resistance. Therefore, more research should be conducted to clarify the mechanisms of chemical and immune resistance.

[0003] Ferroptosis is considered a common non-apoptotic cell death pathway due to abnormal metabolism leading to excessive lipid peroxidation. Ferroptosis is related to the efficacy of many anti-cancer therapies, including radiotherapy, chemotherapy, targeted therapy, and immunotherapy.

[0004] Although a small number of studies have been conducted to detect and verify molecules related to the ferroptosis sensitivity of gastric cancer cells, only a few molecular mechanisms have been revealed and translated into clinical applications so far. Increasing evidence suggests that enhancing ferroptosis sensitivity can be used as a potential therapeutic means and has important clinical value. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art and provide the application of SOX13 as a gastric cancer biomarker. The present invention constructs a ferroptosis-resistant strain, uses a chip to screen out SOX13 as an important regulatory molecule for the ferroptosis sensitivity of gastric cancer, and discovers that the anti-influenza drug zanamivir can specifically target the SOX13 protein.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first purpose of the present invention is to provide the application of SOX13 as a gastric cancer biomarker, and the amino acid sequence of the biomarker SOX13 is shown in SEQ ID NO.1.

[0008] In an embodiment of the present invention, the biomarker SOX13 is the SOX13 protein.

[0009] In an embodiment of the present invention, the gastric cancer is stage IIIA-C gastric cancer.

[0010] The second object of the present invention is to provide the use of a substance that inhibits the expression of the marker SOX13 gene in the preparation of a product for treating gastric cancer.

[0011] In one embodiment of the present invention, there is provided the use of a substance that inhibits the expression of the marker SOX13 gene in the preparation of a product for treating gastric cancer by enhancing ferroptosis sensitivity.

[0012] The third object of the present invention is to provide the use of an anti-influenza drug in the preparation of a product for treating gastric cancer.

[0013] In one embodiment of the present invention, the anti-influenza drug is a substance that inhibits the expression of the marker SOX13 gene.

[0014] In one embodiment of the present invention, the anti-influenza drug is zanamivir, and its chemical structure is shown as follows:

[0015]

[0016] The fourth object of the present invention is to provide a kit for treating gastric cancer, and the kit includes the anti-influenza drug zanamivir.

[0017] In one embodiment of the present invention, the kit includes the anti-influenza drug zanamivir and cisplatin.

[0018] In one embodiment of the present invention, the reagents in the kit are oral preparations, intravenous injection preparations or intraperitoneal injection preparations.

[0019] Compared with the prior art, the advantages of the present invention are as follows: Currently, the ferroptosis sensitizers invented mainly include drugs related to regulating cell metabolism, which may have disadvantages such as affecting normal human functions and high prices; The technology of regulating gene expression with siRNA / shRNA as a medium has disadvantages such as low human safety, immature technology and high prices. Zanamivir, as an anti-influenza drug that has been widely used for a long time, has a simple preparation process and has been tested for safety for a long time, and has great application prospects.

[0020] The present invention effectively verifies that the SOX13 gene is involved in the regulation of ferroptosis sensitivity in gastric cancer, provides theoretical data for the ferroptosis sensitivity and chemosensitivity of gastric cancer cells. In addition, zanamivir can specifically target the SOX13 protein and reverse ferroptosis resistance in gastric cancer, providing a theoretical basis for the treatment of ferroptosis resistance and chemoresistance in gastric cancer cells. Description of the Drawings

[0021] Figure 1 Parental or Erastin treated with different concentrations of ferroptosis inducer Erastin or RSL3 resisCell viability diagrams of SNU-668 cells, where n = 3 biological replicates (mean ± standard deviation), * represents p < 0.05 (two-way ANOVA);

[0022] Figure 2 Parental or RSL3 treated with different concentrations of the ferroptosis inducer Erastin or RSL3 resis Cell viability diagrams of SNU 484 cells, where n = 3 biological replicates (mean ± standard deviation), * represents p < 0.05 (two-way ANOVA);

[0023] Figure 3 Parental or Erastin treated with different concentrations of the apoptosis inducer doxrubicin or gemcitabine resis Cell viability diagrams of SNU-668 cells, where n = 3 biological replicates (mean ± standard deviation), * represents p < 0.05 (two-way ANOVA);

[0024] Figure 4 Parental or RSL3 treated with different concentrations of the apoptosis inducer doxrubicin or gemcitabine resis Cell viability diagrams of SNU484 cells, where n = 3 biological replicates (mean ± standard deviation), * represents p < 0.05 (two-way ANOVA);

[0025] Figure 5 Detection of SOX13 mRNA relative expression in parental gastric cancer cells and ferroptosis-resistant strain RSL3 resis SNU-484 and Erastin resis Diagram of relative expression of SOX13 mRNA in SNU-668;

[0026] Figure 6 Detection of relative expression of SOX13 protein in parental gastric cancer cells and ferroptosis-resistant strain RSL3 resis SNU-484 and Erastin resis Diagram of relative expression of SOX13 protein in SNU-668;

[0027] Figure 7 Diagram of the expression of SOX13 protein analyzed by immunohistochemistry in 109 gastric cancer patients who received postoperative cisplatin chemotherapy and adjacent non-cancerous tissues;

[0028] Figure 8 Diagram of the expression of SOX13 protein analyzed by immunohistochemistry in 109 gastric cancer patients who received postoperative cisplatin chemotherapy and adjacent non-cancerous tissues;

[0029] Figure 9 Schematic diagram of immunohistochemical analysis of SOX13 protein expression in 109 gastric cancer patients who received postoperative cisplatin chemotherapy and adjacent non-cancerous tissues;

[0030] Figure 10 Grouping of 109 gastric cancer patients who received postoperative cisplatin adjuvant chemotherapy according to high and low SOX13 expression groups, and survival analysis using Kaplan-Meier survival curve and log-rank test;

[0031] Figure 11 Grouping of 52 gastric cancer patients who received preoperative cisplatin neoadjuvant chemotherapy according to high and low SOX13 expression groups, and detecting the relationship between SOX13 expression and Tumor regression Grade using chi-square test;

[0032] Figure 12 Detecting the relative expression of SOX13 mRNA after knocking out SOX13 in ferroptosis-resistant strains RSL3 resis SNU-484 and Erastin resis Schematic diagram of the relative expression of SOX13 mRNA after knocking out SOX13 in SNU-668 cells;

[0033] Figure 13 Detecting the protein expression of SOX13 after knocking out SOX13 in ferroptosis-resistant strains RSL3 resis SNU-484 and Erastin resis Schematic diagram of the protein expression of SOX13 after knocking out SOX13 in SNU-668 cells;

[0034] Figure 14 For parental gastric cancer cells and ferroptosis-resistant strains RSL3 resis SNU-484 and Erastin resis Schematic diagram of cell viability after knocking out SOX13 in SNU-668, treating the cells with the ferroptosis inducer Erastin or RSL3 for 24 h, with or without the apoptosis inhibitor Z-VAD-FMK, the pyroptosis inhibitor NSA, and the ferroptosis inhibitor Fer-1;

[0035] Figure 15 For ferroptosis-resistant strain RSL3 resis SNU-484 and Erastin resis Schematic diagram of cell viability after knocking out SOX13 in SNU-668, treating the cells with different concentrations of cisplatin for 24 h, with or without the ferroptosis inhibitor Fer-1;

[0036] Figure 16 For ferroptosis-resistant strain Erastin resisSchematic diagram of detecting the level of cellular lipid peroxidation after knocking out SOX13 in SNU-668, treating the cells with the ferroptosis inducer Erastin or RSL3 for 24 h, with or without adding the ferroptosis inhibitor Fer-1;

[0037] Figure 17 is the ferroptosis-resistant strain RSL3 resis Schematic diagram of detecting the level of cellular lipid peroxidation after knocking out SOX13 in SNU-484, treating the cells with the ferroptosis inducer Erastin or RSL3 for 24 h, with or without adding the ferroptosis inhibitor Fer-1;

[0038] Figure 18 is to detect the ferroptosis-resistant strain RSL3 by flow cytometry resis SNU-484 and Erastin resis Schematic diagram of the percentage of apoptotic cells in SNU-668 cells after knocking out SOX13;

[0039] Figure 19 is to detect the ferroptosis-resistant strain RSL3 by flow cytometry resis SNU-484 and Erastin resis Schematic diagram of the percentages of G1 and G2 / M phase cells in SNU-668 cells after knocking out SOX13;

[0040] Figure 20 Flow chart for screening compounds specifically targeting the SOX13 protein;

[0041] Figure 21 are the effects of the top 40 candidate compounds with the highest scores on the sensitivity of Erastin resis on SNU-668 cells to RSL3;

[0042] Figure 22 Coomassie blue staining shows the purity of the recombinant SOX13 protein;

[0043] Figure 23 SPR analysis of the binding results between the SOX13 protein and zanamivir;

[0044] Figure 24 SPR analysis of the binding results between the SOX13 protein and several other small molecules (T0278, T4706, T2720, and T0853L);

[0045] Figure 25 Chemical structure of Zanamivir;

[0046] Figure 26Schematic diagram of the molecular docking analysis of the interaction between Zanamivir and SOX13, where 1 represents Zanamivir;

[0047] Figure 27 Relative expression schematic diagram of SOX13 protein detected by Western Blot technology in Erastin resis treated SNU-668 cells;

[0048] Figure 28 Melting curve of SOX13 protein in CETSA in Erastin resis or DMSO-treated SNU-668 cells;

[0049] Figure 29 Schematic diagram of lipid peroxidation measured by lipid peroxidation C11-BODIPY assay and flow cytometry histogram after pretreating Erastin resis SNU-668 with Zanamivir for 12 h, then exposing to Erastin or RSL3 for 24 h;

[0050] Figure 30 Schematic diagram of lipid peroxidation measured by lipid peroxidation C11-BODIPY assay and flow cytometry histogram after pretreating RSL3 resis SNU-484 with Zanamivir for 12 h, then exposing to Erastin or RSL3 for 24 h;

[0051] Figure 31 Schematic diagram of the SOX13 protein level in Erastin resis treated SNU-668 cells detected by western blot in the presence or absence of Zanamivir and treated with CHX; resis

[0052] Figure 32 Schematic diagram of the SOX13 protein level in RSL3 resis treated SNU 484 cells detected by western blot in the presence or absence of Zanamivir and treated with CHX; resis

[0053] Figure 33 Western blot analysis diagram of IP using anti-Flag antibody after transfecting SNU-668 cells or SNU-484 cells with the required plasmid, treating with Zanamivir for 24 h 36 h after transfection;

[0054] Figure 34 To detect the apoptotic cell percentage of zanamivir against ferroptosis-resistant strains RSL3 resis SNU-484 and Erastin resis SNU-668 by flow cytometry;

[0055] Figure 35 To detect the apoptotic cell percentage of zanamivir against ferroptosis-resistant strains RSL3 resis SNU-484 and Erastin resis SNU-668 for the percentages of G0 / G1, S, and G2 / M phase cells by flow cytometry. Specific implementation mode

[0056] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0057] The first object of the present invention is to provide the application of SOX13 as a gastric cancer biomarker, and the amino acid sequence of the biomarker SOX13 is shown in SEQ ID NO.1.

[0058] In an embodiment of the present invention, the biomarker SOX13 is SOX13 protein.

[0059] In an embodiment of the present invention, the gastric cancer is stage IIIA-C gastric cancer.

[0060] The second object of the present invention is to provide the application of a substance that inhibits the expression of the biomarker SOX13 gene in the preparation of a product for treating gastric cancer.

[0061] In an embodiment of the present invention, the application of a substance that inhibits the expression of the biomarker SOX13 gene in the preparation of a product for treating gastric cancer by enhancing ferroptosis sensitivity.

[0062] The third object of the present invention is to provide the application of an anti-influenza drug in the preparation of a product for treating gastric cancer.

[0063] In an embodiment of the present invention, the anti-influenza drug is a substance that inhibits the expression of the biomarker SOX13 gene.

[0064] In an embodiment of the present invention, the anti-influenza drug is zanamivir, and its chemical structure is shown as follows:

[0065]

[0066] The fourth object of the present invention is to provide a kit for treating gastric cancer, and the kit comprises an anti-influenza drug zanamivir.

[0067] In one embodiment of the present invention, the kit comprises an anti-influenza drug zanamivir and cisplatin.

[0068] In one embodiment of the present invention, the reagents in the kit are oral preparations, intravenous injection preparations or intraperitoneal injection preparations.

[0069] In the following examples, the amino acid sequences are as follows:

[0070] The amino acid sequence of SOX13, and the sequence is as shown in SEQ ID NO.1:

[0071]

[0072]

[0073] Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0074] The present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0075] Example

[0076] An application of SOX13 as a gastric cancer marker, and the specific steps are as follows:

[0077] S1. Detect the expression of SOX13 mRNA in a sample

[0078] Spread the samples (the samples include ferroptosis inducer-resistant gastric cancer cells and control gastric cancer cells) on a 6-well cell plate, place it in a cell incubator at 37 °C and 5% CO2 for culture. After the cells grow confluently, extract the total RNA of each cell, and detect the SOX13 gene of the sample by real-time PCR technology; at the same time, extract the total protein of the cells of each sample, and detect the protein expressed by the SOX13 gene in different samples by Western Blot technology.

[0079] The results are as Figure 5 、 Figure 6 、 Figure 12 and Figure 13 shown. It can be seen that compared with the control gastric cancer cells and adjacent cancer tissues, the expression of SOX13 in ferroptosis-resistant gastric cancer cells and gastric cancer tissues is significantly up-regulated.

[0080] S2. Detect and analyze the expression of SOX13 protein in gastric cancer specimens

[0081] In cohort 1, gastric cancer specimens and adjacent normal tissues were collected from 109 patients who underwent gastrectomy at Fudan University Shanghai Cancer Center from 2008 to 2012. None of the patients received preoperative radiotherapy or chemotherapy. The follow-up data of the patients were complete. According to the AJCC-TNM staging system (8th edition), the patients had stage IIIA-C gastric cancer. All patients received postoperative cisplatin-based adjuvant chemotherapy. The clinical information is shown in Table 1.

[0082] Table 1 Clinical information of 109 gastric cancer patients

[0083]

[0084]

[0085] In cohort 2, tissue specimens were collected from 52 gastric cancer patients who received cisplatin-based neoadjuvant chemotherapy at Yijishan Hospital Affiliated to Wannan Medical College from March 2019 to November 2020. All specimens were obtained by gastroscopic biopsy before chemotherapy. The tumor tissues were immediately frozen in liquid nitrogen after being taken out and stored at -80°C. The normal tissues were all obtained 3 cm away from the tumor. The clinical data are shown in Table 2.

[0086] Table 2 Clinical information of 52 gastric cancer patients

[0087]

[0088] Each sample was paraffin-embedded and frozen-sectioned, and immunohistochemistry was used to observe the distribution of SOX13 in human gastric cancer tissues and normal tissues; and the correlation between SOX13 mRNA expression and the efficacy of postoperative adjuvant chemotherapy and preoperative neoadjuvant chemotherapy for gastric cancer was further compared;

[0089] Combined with the clinical survival follow-up data, taking the "death" of the patient directly or indirectly due to gastric cancer as the end event, the 5-year cumulative survival probability was calculated; according to the real-time PCR expression data of SOX13 mRNA in each sample, the samples were divided into a high-expression group and a low-expression group, and the survival curve was drawn with the follow-up time as the x-axis and the cumulative probability as the y-axis to analyze the correlation between SOX13 mRNA expression and the clinical prognosis of glioma patients.

[0090] The results are as Figures 7 to 11 shown. It can be seen that immunohistochemical analysis showed that the expression of SOX13 was significantly correlated with poor survival rate and cisplatin resistance. In Figure 8 , the expression levels of SOX13 and SCAF1 in gastric cancer tissues were significantly higher than those in normal tissues; Figure 7 , the proportion of SOX13 with strong positive (+++) was 23.85%, and the proportion of SCAF1 with strong positive (+++) was 47.71%;Figure 9 Figure 1 shows representative immunohistochemical images of SOX13 and SCAF1 in gastric cancer tissues and normal tissues; Figure 10 Figure 2 shows that in 109 patients with stage IIIA-C gastric cancer who received postoperative cisplatin-based adjuvant chemotherapy, high expression of SOX13 / SCAF1 was associated with shorter disease-free survival time and overall survival time; Figure 11 Figure 3 shows that in tissue specimens of 52 gastric cancer patients who underwent cisplatin-based neoadjuvant chemotherapy, the high expression rate of SOX13 / SCAF1 was 33.33% in patients with TRG ≤ 2 and 73.68% in patients with TRG ≥ 3. Using the chi-square test, p = 0.006, which was statistically significant, thus demonstrating that high expression of SOX13 was associated with poor efficacy of cisplatin chemotherapy.

[0091] S3. Construction of ferroptosis-resistant gastric cancer cell lines

[0092] After treating gastric cancer cell line SNU-484 with low-concentration ferroptosis inducer RSL3 (0.5 μM) for 28 days, and then treating it with high-concentration RSL3 (1 μM) for 28 days, the ferroptosis-resistant cell line RSL3 was obtained. resis SNU-484.

[0093] After treating gastric cancer cell line SNU-668 with low-concentration ferroptosis inducer Erastin (2 μM) for 28 days, and then treating it with high-concentration ferroptosis inducer Erastin (5 μM) for 28 days, the ferroptosis-resistant cell line Erastin was obtained. resis SNU-668.

[0094] S4. Sensitivity detection of the cells obtained in S3 to ferroptosis inducers and apoptosis inducers

[0095] Cell proliferation was detected using the Cell Titer-Glo luminescent method; after trypsinizing the cells in each experimental group in the logarithmic growth phase, they were resuspended in complete medium to form cell suspensions and counted; for parental cells SNU-484, SNU-668, and ferroptosis-resistant cell lines RSL3 resis SNU-484, Erastin resisSNU-668 was seeded at 5000 cells / well in six wells of a 96-well plate, with a culture system of 100 μL / well. During the seeding process, it was necessary to ensure that the number of cells added to each well was the same. Additionally, six blank wells with only 100 μl of complete medium were set as the baseline values. The plate was placed in a cell culture incubator at 37 °C and 5% CO2. Starting from the second day of culture, different concentrations of Erastin (0, 0.5, 1, 2, 5, 10 μM), RSL3 (0, 0.05, 0.1, 0.2, 0.5, 1, 2 μM), doxorubicin (0, 0.1, 0.5, 1, 5, 10 μM), or gemcitabine (0, 0.01, 0.05, 0.1, 0.5, 1, 5 μM) were added. 10 minutes before the end of the culture, CellTiter-Glo substrate (15 μl) was added to the wells without changing the medium. The plate was shaken on an oscillator for 10 minutes, and the OD value was measured at 490 / 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0096] The results are as Figures 1 to 4 shown. It can be seen that after treating parental or Erastin resis SNU-668 cells (A) or RSL3 resis SNU 484 (B) cells with different concentrations of ferroptosis inducer Erastin or RSL3 for 24 hours, the cell viability was detected. For Erastin resis SNU-668 cells or RSL3 resis SNU 484, the decrease in cell viability with the increase in the concentration of the ferroptosis inducer was less than that of the parental cells with the increase in the concentration of the ferroptosis inducer;

[0097] After treating parental or Erastin resis SNU-668 cells (C) or RSL3 resis SNU 484 (D) cells with different concentrations of apoptosis inducer doxorubicin or gemcitabine for 24 hours, the cell viability was detected. For Erastin resis SNU-668 cells or RSL3 resis SNU 484, the decrease in cell viability with the increase in the concentration of the apoptosis inducer was not significantly different from that of the parental cells with the increase in the concentration of the apoptosis inducer.

[0098] Therefore, the ferroptosis inducer had an effect on the cell viability of Erastin resis SNU-668 cells or RSL3 resis SNU 484. Ferroptosis inducer was used in subsequent experiments.

[0099] S5. Infect the sample gastric cancer cells

[0100] For RSL3 resis SNU-484, Erastin resis Perform lentiviral infection on SNU-668.

[0101] S6. Detect the cell viability of the infected cells in S5

[0102] Use the Cell Titer-Glo luminescent method to detect cell proliferation; after trypsinizing the cells in each experimental group in the logarithmic growth phase, resuspend them with complete medium to form a cell suspension and count; inoculate 6 wells at 5000 cells / well for the control lentivirus group and the shRNA-SOX13 lentivirus group in a 96-well plate, with a culture system of 100 μL / well. During the plating process, ensure that the number of cells added to each well is the same. Additionally, set 6 blank wells with only 100 μL of complete medium as the reference value; culture in a cell incubator at 37 °C and 5% CO2; starting from the second day of culture, add different concentrations of Erastin (2 μM), RSL3 (0.5 μM), or cisplatin (0, 5, 10, 15, 20, 25, 30, 35, and 40 μM), with or without the apoptosis inhibitor Z-VAD-FMK (10 μM), the pyroptosis inhibitor NSA (1 μM), and the ferroptosis inhibitor Fer-1 (1 μM). Add 15 μL of CellTiter-Glo substrate to each well 10 min before the end of culture, without changing the medium, shake on a shaker for 10 min, and detect the OD value with an ELISA reader at 490 / 570 nm.

[0103] The results are as Figure 14 and Figure 15 shown. It can be seen that after treating the parental or Erastin resis SNU-668 cells or RSL3 resis SNU 484 cells with different concentrations of the ferroptosis inducers Erastin, RSL3, and cisplatin for 24 hours and detecting the cell viability, the reduction in the cell viability of Erastin resis SNU-668 cells or RSL3 resis SNU 484 with the increase in the concentrations of Erastin, RSL3, and cisplatin is less than the reduction in the cell viability of the parental cells with the increase in the concentration of the ferroptosis inducer; knocking out SOX13 in Erastin resis SNU-668 cells or RSL3 resis SNU 484 can be found to significantly increase the reduction in cell viability with the increase in the concentrations of Erastin, RSL3, and cisplatin compared to the control group.

[0104] S7. Detect the level of cellular lipid peroxidation in the infected cells in S5.

[0105] One day before the experiment, seed 1×10 5 cells (transfected with the required vector) / well in a six-well culture dish. Treat the cells with Erastin (2 μM) or RSL3 (0.5 μM) for 24 h in the presence or absence of Ferrostatin-1 (1 μM). Harvest the cells by trypsinization, resuspend them in 500 μL of phosphate-buffered saline (PBS) containing 2 μM C11-BODIPY (581 / 591) (#D3861, Invitrogen), and incubate them in a tissue culture incubator at 37 °C for 30 minutes. Then resuspend the cells in 500 μL of fresh PBS, filter them through a 40-μm cell strainer, and analyze them using a flow cytometer (FACSuite, BD Biosciences) equipped with 488-nm laser excitation. Collect data from the FL1 channel (527 nm). Analyze at least 1000 cells under each condition. Use FlowJo version 7.6 software for data analysis.

[0106] The results are as Figure 16 and Figure 17 shown. It can be seen that knocking out SOX13 in Erastin resis SNU-668 cells or RSL3 resis SNU484 results in a significantly higher increase in the level of cellular lipid peroxidation in the SOX13-knockout group than in the control group under the action of ferroptosis inducers.

[0107] S8. Perform apoptosis experiments on the ferroptosis-resistant cells and parental cells obtained in S3.

[0108] Detect cell apoptosis using the AnnexinV-APC single-staining method. Seed three wells of each of the two experimental groups of cells in a six-well cell culture plate; when the cells are in good growth condition, digest them with trypsin and centrifuge. Wash them with PBS at room temperature (1500 rpm / min, centrifuge for 5 min); resuspend the cells in 500 μl of binding buffer, add 10 μl of Annexin-V medium buffer and 1 μl of AnnexinV fluorescent antibody, incubate at room temperature in the dark for 15 min and then centrifuge; after washing with PBS, resuspend the cells in 500 μl of binding buffer and detect them by flow cytometry and perform data analysis within 1 h.

[0109] The results are as Figure 18 shown. It can be seen that Erastin resis SNU-668 cells or RSL3resis There was no significant difference in the apoptosis rate of SNU 484 compared with parental cells, thus demonstrating Erastin resis SNU-668 cells or RSL3 resis The resistance of SNU484 to ferroptosis inducers compared with parental cells did not originate from differences in apoptosis rate.

[0110] S9. Perform cell cycle detection on the ferroptosis-resistant cells and parental cells obtained in S3

[0111] The cell cycle was detected using PI single staining method. Each of the two experimental groups of cells was seeded in 3 wells of a 6-well cell culture plate; the cells were treated with propidium iodide (PI, 50 mg / L) at 4 °C for 30 minutes in the dark. The cell cycle distribution was measured at 488 nm on an EPICS 752 flow cytometer (Coulter, Hialeah, FL) equipped with MPLUS software (Phoenix 140 flow Systems, San Diego, CA). The data were expressed as the percentage distribution of cells in the G0 / G1, S, and G2 / M phases of the cell cycle.

[0112] The results are as Figure 19 shown. It can be seen that Erastin resis SNU-668 cells or RSL3 resis There was no significant difference in the cell cycle of SNU 484 compared with parental cells, thus demonstrating Erastin resis SNU-668 cells or RSL3 resis The resistance of SNU 484 to ferroptosis inducers compared with parental cells did not originate from cell cycle disruption.

[0113] S10. Optimize the structure of SOX13 protein and predict small molecule compounds acting on SOX13

[0114] As Figure 20As shown, there is no crystal structure of the SOX13 protein in the Protein Data Bank (http: / / www.rcsb.org / pdb / home / home.do). Comparing with the AlphaFold database, there is such a structure AF-Q9UN79-F1 (https: / / alphafold.ebi.ac.uk / entry / Q9UN79), and this structure is downloaded for optimization. The sequence region with relatively high protein structure reliability (424 - 494) is intercepted, and this structure is optimized using kinetic software. In this experiment, the Desmond module in the Schrodinger software package is used for relevant calculations of molecular dynamics simulation. In the molecular dynamics simulation, first, the protein system to be simulated is embedded in a solvent box containing a three-point water model, and the distance between its boundary and the protein boundary is set to be no less than An appropriate number of Na + or Cl - is added to maintain the electrical neutrality of the system and make the solution maintain a physiological saline concentration of 0.15 M to complete the construction of the system. The constructed system is energy-minimized under the OPLS-2005 force field to eliminate unreasonable contacts between atoms in the system and prevent the collapse of the kinetic simulation process. After the energy minimization of the system is completed, the system is set to be simulated under the NPT ensemble, the temperature is set to 300 K, the pressure is set to 1.01325 bar, the SHAKE algorithm is used to restrict the stretching vibration of all hydrogen-containing atom chemical bonds in the system, the time step is set to 2 fs, and the total simulation time is 200 ns. The system is subjected to molecular dynamics simulation under the OPLS-2005 force field.

[0115] The Protein Preparation Wizard in the Schrodinger software package is used to prepare the protein structure, specifically including adjusting the protonation state of amino acid residues according to the set pH conditions, complementing hydrogen atoms and possibly missing protein structures, and saving the structure after preparation processing as a pdb file for subsequent molecular simulation research.

[0116] For the database to be screened, these structures are prepared through the LigPrep module in the Schrodinger software package to generate 3D conformations, and the protonation state at pH 7 is set. At the same time, their conformations are optimized under the OPLS-3e force field.

[0117] Molecular docking calculations are performed using the Glide module in the Schrodinger software package. The prepared protein structure is imported into the software, and the Receptor Grid Generation function in the Glide module is used to define the center of the active site based on the position of the ligand small molecule to generate the corresponding docking grid file.

[0118] Virtual screening process

[0119] The molecular docking process is divided into four rounds for screening:

[0120] In the first round, high-throughput virtual screening (HTVS) is carried out for rough screening, and 1% of the hit structures in the database are retained;

[0121] In the second round, standard precision (SP) screening is carried out, and 10% of the hit structures are retained;

[0122] Glide HTVS and SP use a series of hierarchical filters to search for the possible positions of ligands in the receptor binding site region. The shape and properties of the receptor are represented on the grid by different field groups, which provide a gradually more accurate scoring of the ligand poses. An exhaustive count of ligand torsions generates a set of ligand conformations, which are examined during the docking process. Given these ligand conformations, an initial screening is deterministically carried out over the entire space available to the ligand to find promising ligand poses. The poses selected by the initial screening are refined for the ligand in the torsional space of the receptor region using OPLS3 4 (Glide SP&XP) or OPLS2005 (GLIDE HTVS) with a distance-dependent dielectric model. Finally, a small number of poses are minimized within the region of the receptor, with full ligand flexibility (post-docking minimization or PDM).

[0123] In the third round, extra precision (XP) screening is carried out, and 10% of the hit structures are retained;

[0124] In the fourth round, the mode of action and binding energy are analyzed based on the final hit structures.

[0125] S11. Screen drugs that can both promote the anti-tumor activity of ferroptosis inhibitors and inhibit the expression of SOX13

[0126] Select the top 40 compounds with the highest scores in screening step S10. Use the Cell Titer-Glo luminescent method to detect cell proliferation; after trypsinizing the cells in each experimental group in the logarithmic growth phase, resuspend them with complete medium into cell suspensions and count; inoculate 6 wells at 5000 cells / well in a 96-well plate, with the culture system being 100 μL / well. During the plating process, ensure that the number of cells added to each well is the same. Additionally, set up 6 blank wells with only 100 μl of complete medium as the reference value; culture in a cell incubator at 37 °C and 5% CO2; starting from the second day of culture, after treating the cells with the top 40 compounds (10 μM) in step S10 for 12 hours, add RSL3 (0.5 μM) and treat for 24 hours. Add CellTiter-Glo substrate (15 μl) to the wells 10 minutes before the end of culture, without changing the medium, shake on an oscillator for 10 minutes, and detect the OD value with an ELISA reader at 490 / 570 nm.

[0127] The results are as Figure 21 shown. It can be seen that among the 40 compounds, T2529, T0278, T4706, T2720, and T0853L most significantly enhanced the killing effect of the ferroptosis inducer RSL3 on Erastin resis SNU-668 cells.

[0128] Transform the pET-28a(+) plasmid expressing His-tagged SOX13 into Escherichia coli BL21 Star(DE3) cells, and add 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce protein expression at 37 °C. Bacterial cells are lysed by sonication, and the His-tagged SOX13 fusion protein is purified on a His-binding column (Beyotime, Shanghai, China) and analyzed by Western blotting. As described previously, SPR analysis is performed using a Biacore X100 system (GE Healthcare LifeSciences, Marlborough, MA, USA). The SOX13 protein is immobilized on a CM7 chip (GE Healthcare Life Sciences) by amine coupling. Dilute the above compounds with running buffer and load the samples. Curve fitting is performed using Biacore analysis software to obtain the K d value of the protein.

[0129] The results are as Figure 22 shown, and relatively pure SOX13 protein is purified.

[0130] After treating the cells with the above compounds for 36 hours, detect Erastin by Western blotresis The expression of SOX13 in SNU-668 was detected, and the compounds with the most significant effects on SOX13 expression and ferroptosis sensitivity were screened out.

[0131] The results are as Figures 23 to 24 shown. It can be seen that among all the compounds, zanamivir has the lowest K d value with SOX13, which means that zanamivir has the strongest binding ability to SOX13 among all the compounds.

[0132] The results are as Figures 25 to 26 shown. It can be seen that Figure 25 is the chemical structure of zanamivir, Figure 26 and is the predicted binding mode diagram of zanamivir and SOX13 by computer.

[0133] S12. Detect the binding of the compounds screened in S11 to SOX13

[0134] To determine the intracellular targeting binding of zanamivir to SOX13, 70%–80% of Erastin resis SNU-668 cells in a 15-cm culture dish were treated with zanamivir or DMSO for 1 hour. The cells were harvested and washed once with PBS, then suspended in 1 ml of PBS (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors, and maintained with the same dose of zanamivir or DMSO as the initial treatment. The cell suspension was distributed into seven 0.2-mL PCR tubes at different designated temperatures. The samples were heated in a 96-well thermal cycler for 2 minutes at different designated temperatures. The tubes were immediately removed after heating and incubated at room temperature for 3 minutes. Three freeze-thaw cycles were performed in liquid nitrogen to lyse the cells. The tubes were shaken each time after thawing. The cell lysates were collected, and cell debris, as well as precipitated and aggregated proteins, were removed by centrifuging the samples at 20,000 g for 20 minutes at 4°C. The cell lysate samples were boiled with loading buffer at 95°C for 5 minutes and subjected to western blot analysis.

[0135] The results are as Figures 27 to 28 shown. It can be seen that Figure 27 shows that only zanamivir significantly reduces the expression of SOX13 protein among the compounds screened in S11. Figure 28 shows the quantification of SOX13 protein relative to temperature points based on western blot analysis. Zanamivir significantly enhanced the protein stability of SOX13 under various temperature conditions compared with the control group, thus demonstrating that zanamivir can bind to SOX13.

[0136] 70%–80% Erastin in 15-cm culture dishes resis SNU-668 cells were added with the protein synthesis inhibitor Cycloheximide (5 μM) and treated with zanamivir (10 μM) or DMSO for 0, 3, 6, and 12 hours. Cells were harvested at 0, 3, 6, and 12 h, washed once with PBS, and subjected to western blot analysis.

[0137] 70%–80% Erastin in 15-cm culture dishes resis SNU-668 cells were transfected with Flag-tagged SOX13. After 48 hours, they were treated with zanamivir (10 μM) for 12 hours, and Erastin was enriched using the Ubiquitin Enrichment kit (ThermoFisher Scientific, Waltham, MA). resis Endogenous ubiquitin proteins in SNU-668 cells. Briefly, whole cell extracts (1 mg) were incubated with polyubiquitin affinity resin at 4 °C for 4 hours. Then, the polyubiquitin affinity resin was washed three times with TBS and then eluted with 1× sample buffer. The eluted ubiquitinated proteins were separated by SDS-PAGE and immunoblotted with an anti-Flag antibody. Whole cell extracts were analyzed with an anti-β-Actin protein antibody as an internal control.

[0138] The results are as Figures 31 to 33 shown. It can be seen that under the condition of inhibiting endogenous protein synthesis, zanamivir significantly reduced the content of SOX13 protein, thus demonstrating that zanamivir reduced the stability of SOX13 protein. In addition, zanamivir significantly increased the ubiquitination degradation of SOX13.

[0139] Detection of the effect of zanamivir on the level of cellular lipid peroxidation in S13

[0140] One day before the experiment, 1×10 5Cells (transfected with the required vector) / well were seeded in six-well culture dishes. In the presence or absence of zanamivir (10 μM), cells were treated with Erastin (2 μM) or RSL3 (0.5 μM) for 24 h. Cells were harvested by trypsinization, resuspended in 500 μL of phosphate-buffered saline (PBS) containing 2 μM C11-BODIPY (581 / 591) (#D3861, Invitrogen), and incubated in a tissue culture incubator at 37 °C for 30 minutes. Then the cells were resuspended in 500 μL of fresh PBS, filtered through a 40-μm cell strainer, and analyzed using a flow cytometer (FACSuite, BD Biosciences) equipped with 488-nm laser excitation. Data were collected from the FL1 channel (527 nm). At least 1000 cells were analyzed for each condition. Data analysis was performed using FlowJo version 7.6 software.

[0141] The results are as Figure 29 and Figure 30 shown. It can be seen that pretreatment with zanamivir of ferroptosis-resistant cells Erastin resis SNU-668 cells or RSL3 resis SNU 484 resulted in a significantly higher increase in the level of lipid peroxidation in cells of the pretreatment with zanamivir group than in the control group under the action of ferroptosis inducers.

[0142] S14. Detection of the effect of zanamivir on apoptosis

[0143] The AnnexinV-APC single staining method was used to detect apoptosis. Cells in the two experimental groups were seeded in 3 wells each in a six-well cell culture plate; when the cells were in good growth condition, the cells were treated with zanamivir (10 μM) or DMSO for 24 hours, digested with trypsin, and centrifuged; washed with PBS at room temperature (1500 rpm / min, centrifuged for 5 min); resuspended the cells with 500 μl of binding buffer, added 10 μl of Annexin-V medium buffer and 1 μl of AnnexinV fluorescent antibody, incubated at room temperature in the dark for 15 min and then centrifuged; after washing with PBS, resuspended the cells with 500 μl of binding buffer, and detected by flow cytometry and analyzed within 1 h.

[0144] The results are as Figure 34 shown. It can be seen that zanamivir on Erastin resis SNU-668 cells or RSL3 resisThere was no effect on the apoptosis of SNU 484 cells, thus demonstrating that the ferroptosis-sensitizing effect of zanamivir on gastric cancer cells did not originate from increasing the apoptosis of gastric cancer cells.

[0145] S15. Perform cell cycle detection on the infected cells in S5

[0146] Detect cell apoptosis using PI single staining method. Each of the two experimental groups of cells was seeded in 3 wells of a 6-well cell culture plate; when the cells were in good growth condition, the cells were treated with zanamivir (0 μM, 5 μM, 10 μM, 20 μM) or DMSO for 24 hours, digested with trypsin, and centrifuged; the cells were treated with propidium iodide (PI, 50 mg / L) at 4°C for 30 minutes in the dark. The cell cycle distribution was measured at 488 nm on an EPICS 752 flow cytometer (Coulter, Hialeah, FL) equipped with MPLUS software (Phoenix140 flow Systems, San Diego, CA). The data were expressed as the percentage distribution of cells in the G0 / G1, S, and G2 / M phases of the cell cycle.

[0147] The results were as Figure 35 shown. It could be seen that zanamivir had no effect on the cell cycle of Erastin resis SNU-668 cells or RSL3 resis SNU 484 cells, thus demonstrating that the ferroptosis-sensitizing effect of zanamivir on gastric cancer cells did not originate from disturbing the cell cycle.

[0148] Result analysis: The expression of SOX13 in control gastric cancer cells, ferroptosis inducer-resistant gastric cancer cells, gastric cancer tissues, and adjacent normal tissues was detected using real-time PCR technology and Western Blot technology. The results were as Figure 5 、 Figure 6 、 Figures 7 - 9 shown. It could be seen that the expression of SOX13 was significantly upregulated in ferroptosis-resistant gastric cancer cells and gastric cancer tissues compared with control gastric cancer cells and adjacent tissues.

[0149] The protein expression of SOX13 was analyzed in 161 paraffin-embedded gastric cancer specimens using immunohistochemistry technology. Then, the Cell Titer-Glo luminescent method and cell lipid peroxidation level detection were used to explore the effects of SOX13 on the resistance of gastric cancer cells to ferroptosis inducers and cisplatin, and flow cytometry was used to analyze the effects of SOX13 on the apoptosis and cell cycle of gastric cancer cells.

[0150] Experiments have shown that compared with control gastric cancer cells and adjacent tissues, the expression of SOX13 in ferroptosis-resistant gastric cancer cells and gastric cancer tissues is significantly upregulated. Immunohistochemical analysis shows that the expression of SOX13 is significantly correlated with poor survival rate and cisplatin resistance. Knockdown of SOX13 can promote the sensitivity of gastric cancer cells to ferroptosis inducers and cisplatin, but has no effect on the cell cycle and apoptosis of gastric cancer cells. This invention clarifies the pathogenesis of gastric cancer ferroptosis and cisplatin resistance, which has important theoretical and practical significance. SOX13 plays an important role in gastric cancer ferroptosis and cisplatin resistance, thus providing a potential therapeutic target for the treatment of gastric cancer.

[0151] In addition, through computer-aided molecular docking technology, drugs that can bind to SOX13 were screened. The Cell Titer-Glo luminescent method was used to further screen drugs that enhance the killing effect of ferroptosis inducers. SPR was used to further screen the binding between SOX13 and drugs, and western blot was used to detect the effect of zanamivir on the protein stability of SOX13. The cell lipid peroxidation level was detected to verify the effect of zanamivir on the resistance of gastric cancer cells to ferroptosis inducers. At the same time, flow cytometry was used to analyze the effect of zanamivir on the apoptosis and cell cycle of gastric cancer cells. Experiments have shown that zanamivir can promote the sensitivity of gastric cancer cells to ferroptosis inducers, but has no effect on the cell cycle and apoptosis of gastric cancer cells. This invention has important theoretical and practical significance for the treatment of reversing gastric cancer ferroptosis resistance.

[0152] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of an anti-influenza drug in the preparation of a product for treating gastric cancer, characterized in that, The anti-influenza drug is a substance that inhibits the expression of the marker SOX13 gene, and the amino acid sequence of the marker SOX13 is shown in SEQ ID NO.1; The anti-influenza drug is zanamivir, and its chemical structure is shown as follows:

2. Use of the anti-influenza drug according to claim 1 in the preparation of a product for treating gastric cancer, characterized in that, The gastric cancer is stage IIIA-C gastric cancer.

3. Use of the anti-influenza drug according to claim 1 in the preparation of a product for treating gastric cancer, characterized in that, Application of a substance that inhibits the expression of the marker SOX13 gene in the preparation of a product for enhancing ferroptosis sensitivity to treat gastric cancer.

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