Application of HOXB9 inhibitor in medicine for reversing oxaliplatin resistance of gastric cancer

Abstract

The invention relates to application of an HOXB9 inhibitor in a medicine for reversing oxaliplatin resistance of gastric cancer, and belongs to the field of biological medicine. The invention discloses for the first time: HOXB9 is remarkably high in expression and is positively correlated with poor prognosis in tissues of patients with gastric cancer, which are poor in curative effect after chemotherapy containing oxaliplatin; by constructing an oxaliplatin drug-resistant gastric cancer cell strain and a patient-derived organ model, the HOXB9 is proved to specifically mediate the survival of the drug-resistant cell strain by activating a focal adhesion signaling pathway (p-FAK / AKT), and the HOXB9 is irrelevant to cell proliferation. Aiming at the mechanism, shRNA nucleic acid molecules (SEQ ID NO: 1-2) targeting HOXB9 are provided, and drug-resistant cell strains can be selectively killed through lentiviral vector delivery, so that the drug-resistant cell IC50 is reduced by more than 50%, the apoptosis rate is increased by 3 times, and the chemotherapy resistance of organoids is reversed. The invention provides a brand-new target spot and a treatment scheme for overcoming the drug resistance of oxaliplatin for gastric cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of HOXB9 inhibitor in reversing oxaliplatin drug resistance of gastric cancer. BACKGROUND

[0002] According to research statistics, gastric cancer (GC) is the fifth most common malignant tumor disease in China, with more than 350,000 new cases each year, and is the third largest factor related to cancer deaths, with more than 250,000 new deaths each year. Systemic chemotherapy is the main treatment for metastatic gastric cancer, and the median overall survival (OS) of patients receiving conventional chemotherapy is about 12 months. Radical surgery is the main treatment for resectable gastric cancer. Several treatment methods have been established to reduce the risk of recurrence and improve long-term survival, including perioperative chemotherapy, adjuvant chemotherapy, and adjuvant radiotherapy. They are listed as the recommended treatment for currently resectable localized gastric cancer. Perioperative chemotherapy mainly uses platinum combined with fluorouracil chemotherapy to reduce tumor stage, facilitate local resection, and improve progression-free survival. Perioperative FLOT regimen (fluorouracil, folinic acid, oxaliplatin, and polyene paclitaxel), ECF / ECX regimen (epirubicin, cisplatin, and fluorouracil / capetichine). In addition, there are two-drug combination regimens: XELOX regimen (oxaliplatin + capetichine); SOX regimen (oxaliplatin + tegafur); PF regimen (cisplatin + fluorouracil), etc. Fluorouracil, platinum, taxane, and irinotecan are the main treatment drugs for advanced cancer, and the recommended chemotherapy regimen for patients with advanced gastric cancer is usually fluorouracil (fluorouracil, capetichine, and S-1) combined with platinum as the main treatment regimen for first-line patients. Platinum chemotherapy drugs are used in various chemotherapy regimens, and oxaliplatin is the main platinum drug for gastric cancer chemotherapy. However, some patients show drug resistance to chemotherapy drugs, leading to poor prognosis. In advanced patients, trastuzumab and some immune checkpoint inhibitors (such as nivolumab and pembrolizumab) show consistent and reliable efficacy in HER2-positive and PDL1-positive tumor patients, respectively, but immunotherapy patients still accompany chemotherapy. In addition, the exploration of immune checkpoint inhibitors (carfilzomib), anti-angiogenesis (apatinib), and chemotherapy drugs in the neoadjuvant treatment of gastric cancer is also ongoing, and chemotherapy still plays an important role in treatment strategies. These treatment methods inevitably face new challenges brought about by chemotherapy resistance.

[0003] In summary, in-depth study of the molecular mechanisms of gastric cancer chemotherapy resistance is of great clinical significance and scientific value for finding effective treatment targets and guiding treatment strategies. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a HOXB9 gene inhibitor, and the purpose of the present application is to target HOXB9 of the oxaliplatin-resistant gastric cancer to enhance the sensitivity to oxaliplatin.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0006] The present application discloses the application of the HOXB9 expression inhibitor in the preparation of a drug for reversing the oxaliplatin resistance of gastric cancer.

[0007] Further, the HOXB9 inhibitor is a molecule for inhibiting the expression or function of the HOXB9 gene.

[0008] Preferably, the HOXB9 inhibitor is a nucleic acid molecule, and the nucleic acid molecule is an shRNA targeting the HOXB9 gene.

[0009] Preferably, the target sequence of the shRNA is selected from the following sequences: SEQ ID NO: 1: GGCAAAGAGTAAAGATTAA; SEQ ID NO: 2: GGCTAGAAAGTACAAGAAA.

[0010] The present application also discloses a composition for reversing the oxaliplatin resistance of gastric cancer, which comprises oxaliplatin and the HOXB9 inhibitor according to any one of the above-mentioned embodiments.

[0011] Further, the HOXB9 expression inhibitor is an shRNA lentivirus vector constructed by the target sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] The present application also discloses a gastric cancer oxaliplatin resistance detection kit, which is characterized by detecting the expression level of HOXB9 in a sample, and high expression of HOXB9 indicates a risk of drug resistance.

[0013] Further, the sample is a gastric cancer tissue, blood or organoid model.

[0014] Compared with the prior art, the present application has the following beneficial effects.

[0015] The present application directly targets the drug-resistant gastric cancer cells (non-primary treatment cells) that progress after chemotherapy, and breaks through the bottleneck that the existing scheme is ineffective for drug-resistant patients. The transcriptome sequencing of the drug-resistant cell strain confirms that there is a significant difference between the drug-resistant cell strain and the parent cell (such as specific activation of focal adhesion pathways), and the HOXB9 inhibitor can selectively reverse the drug resistance phenotype of such cells, so that the IC50 of the drug-resistant cell strain returns to the sensitive level.

[0016] Target discovery by drug-resistant tissue tracing, and first confirmed HOXB9 in gastric cancer by regulating focal adhesion pathway (p-FAK / AKT) to maintain cytoskeleton stability, mediate drug-resistant cell lines to survive under chemotherapy pressure (not through the proliferation pathway). This mechanism has not been reported in solid tumors, which is a new action pathway.

[0017] In drug-resistant cell lines and organoid models, targeting HOXB9 can enhance the killing effect of oxaliplatin by more than 3 times, and combination therapy can eliminate the survival advantage of drug-resistant cell lines under continuous chemotherapy (70% reduction in clonogenicity). HOXB9 expression level can be used as a biomarker for drug resistance to guide precise drug use for patients who do not respond to chemotherapy; at the same time, it provides a gene therapy tool (shRNA lentivirus) based on the target and an organoid drug sensitivity screening model.

[0018] The present application first proposes to target HOXB9 of oxaliplatin-resistant gastric cancer cells with small molecule nucleic acid sequences as inhibitors to enhance the sensitivity of gastric cancer cells to oxaliplatin, overcoming the influence of oxaliplatin on gastric cancer cells during gastric cancer treatment. Some gastric cancer cells have acquired oxaliplatin resistance due to high expression of HOXB9 and affect the survival progress after drug resistance. Compared with traditional signal pathway inhibition, the present application plays a role from the expression regulation aspect, effectively enhancing the sensitivity of gastric cancer cells to oxaliplatin. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 HOXB9 expression is up-regulated in gastric cancer tissues that do not respond to chemotherapy, and is associated with poor prognosis. (A) RNA-seq gene expression consistency analysis chart of locally advanced gastric cancer tissue samples after neoadjuvant therapy (Pearson Correlation Coefficient analysis), (B) volcano plot of differentially expressed genes in two groups, HOX family members are labeled, (C) Heat map of the top 50 genes up-regulated in NHR compared to MjHR, and HOXB9 is labeled. (D) mRNA expression analysis of HOXB9 in TCGA dataset of gastric adenocarcinoma tumor tissues and normal tissues, p <0.001. (E) Kaplan-Meier curve of overall survival of gastric cancer patients, (F) Kaplan-Meier curve of overall survival classified by differentiation type, (G) Kaplan-Meier curve of overall survival classified by gastric cancer type, log-rank is used for comparison statistical analysis, p <0.05 has statistical significance. (H) Imaging results after immunohistochemical staining of HOXB9 expression in gastric cancer pathological tissue samples, the results are analyzed by IHC score, and a column chart is drawn (I), p <0.001, scale bar is 100 μm.

[0020] Figure 2 Overexpression of HOXB9 in gastric cancer cells reduced the sensitivity to oxaliplatin. (A) Colony formation assay images and relative quantification bar graph of AGS (AGS HOXB9) / HGC27 (HGC27 HOXB9) cells overexpressing HOXB9 and control Vector group, values are mean ± SD of three independent experiments; p <0.05; ns represents no significant difference. (B) Dose response curves and IC50 values of AGS HOXB9 / HGC27 HOXB9 cells and control cells, each curve represents the mean ± SD of 4 replicates for each condition. (C) Light microscope imaging and ATP energy bar graph of AGS HOXB9 and control cells in Matrigel matrix gel formation 3D cell sphere experiment, values are mean ± SD of three independent experiments; p <0.001. (D) Western blot detection of Caspase3 / Caspase8 protein expression in AGS HOXB9 cells and control cells, values are mean ± SD of three independent experiments; p <0.001. (E) Immunofluorescence detection of γ-H2AX expression in AGS HOXB9 cells and control cells treated with oxaliplatin, values are mean ± SD of three independent experiments; p <0.001; ns represents no significant difference. (F) Tumor entity of tumor-bearing mice, (G) Tumor growth curve, (H) Volume of isolated tumors, (I) Weight of tumors. (J) Tumor entity of AGS HOXB9 and control tumor-bearing mice after oxaliplatin treatment, (K) Tumor growth curve, (L) Volume of isolated tumors, (M) Weight of tumors. p <0.01, p <0.001; ns represents no significant difference.

[0021] Figure 3 Knockdown of HOXB9 in gastric cancer drug-resistant cell lines improves sensitivity to oxaliplatin. (A) Dose response curves and IC50 values of oxaliplatin-resistant gastric cancer cell lines and corresponding control parental cells, each curve represents the mean ± SD of 4 replicates for each condition. (B) mRNA expression level bar graph of HOXB9 during the establishment of gastric cancer drug-resistant cell lines, values are mean ± SD of three independent experiments; p <0.05, p<0.01; ns represents no significant difference. (C) Western blot analysis of HOXB9 protein expression level. (D) Dose response curves and IC50 values of HOXB9 knockdown resistant cell lines (AGS OR shHOXB9 / HGC27 OR shHOXB9) and corresponding control NC cells, each curve represents the mean of 4 replicates ± SD. (E) Images and (F) relative quantification results of colony formation assay of AGS OR shHOXB9 / HGC27 OR shHOXB9 and corresponding control NC cells, values are the mean of three independent experiments ± SD. p <0.05, * p <0.001, ns represents no significant difference. (G) Flow cytometry analysis of apoptosis rate of AGS OR shHOXB9 / HGC27 OR shHOXB9 and corresponding control NC cells, (H) statistical analysis of column chart with total apoptosis rate = Q2 + Q4, values are the mean of three independent experiments ± SD. p <0.001. (I) Optical microscope imaging of Matrigel matrix gel forming 3D cell spheres of AGS OR shHOXB9 / HGC27 OR shHOXB9 and control cells, scale bar is 100 μm, and (J) column chart of ATP energy detection, values are the mean of three independent experiments ± SD. p <0.001.

[0022] Figure 4 HOXB9 regulates focal adhesion signaling pathway in gastric cancer cells. (A) KEGG enrichment analysis of differentially expressed genes of AGS HOXB9 cells and control Vector cells, AGS OR cells and control AGS PC cells, (B) Venn diagram of intersection analysis of differentially expressed genes of AGS OR vs AGS PC and AGS HOXB9 vs AGS Vector, and Venn diagram of intersection analysis of up-regulated differentially expressed genes of the two, (C) KEGG enrichment analysis of commonly up-regulated differentially expressed genes. (D) QPCR analysis of mRNA expression level of focal adhesion signaling pathway related molecules in AGS HOXB9 cells (control Vector cells), AGS OR cells (control PC cells), AGS OR shHOXB9 cells (control NC cells), values are the mean of three independent experiments ± SD. p <0.05, * p <0.01, * p<0.001, ns represents no significant difference. (E) Western blot detected the expression level of FAK, p-FAK in AGS HOXB9 cells (control Vector cells) and AGS OR HOXB9 cells (control AGS OR Vector cells), (F) Western blot detected the expression level of FAK, p-FAK in AGS OR shHOXB9 cells (control AGS OR NC cells), and the p-FAK / FAK ratio column chart, the value is the average of three independent experiments ± SD; p <0.05, p <0.01. (G) Immunofluorescence detected the expression of p-FAK, F-actin in AGS HOXB9 cells (control Vector cells) and the corresponding subcutaneous xenograft tumors after oxaliplatin treatment, and the relative mean fluorescence intensity column chart, (H) Immunofluorescence detected the expression of p-FAK, F-actin in AGS OR cells (control AGS PC cells) and the corresponding cells after oxaliplatin treatment, and the relative mean fluorescence intensity value column chart, (I) Immunofluorescence detected the expression of p-FAK, F-actin in AGS HOXB9 cells (control Vector cells) and the corresponding cells after oxaliplatin treatment, and the relative mean fluorescence intensity value column chart, the value is the average of three independent experiments ± SD; p <0.05, p <0.01, p <0.001, ns represents no significant difference. Scale bar, 20 μm.

[0023] Figure 5 HOXB9-dependent p-FAK activates AKT to affect the sensitivity of gastric cancer cells to oxaliplatin. (A) AGS HOXB9 cells (control Vector cells) / HGC27 HOXB9 cells (control Vector cells) colony formation experiment image and relative quantitative results column chart of colony formation experiment, the value is the average of three independent experiments ± SD; p <0.01, ns represents no significant difference. (B) Flow cytometry detected the apoptosis rate of AGS HOXB9 cells (control Vector cells) / HGC27 HOXB9 cells (control Vector cells) scatter plot, (C) Column chart for statistical analysis of total apoptosis rate = Q2+Q4, the value is the average of three independent experiments ± SD; p <0.05, p<0.001, ns represents no significant difference. (D) Western blot detection of AKT and p-AKT expression levels in AGS HOXB9 cells (control Vector cells) and AGS OR HOXB9 cells (control AGS OR Vector cells). (E) Western blot detection of AKT and p-AKT expression levels in AGS OR shHOXB9 cells (control AGS OR NC cells). (F) Western blot detection of AKT and p-AKT expression levels in AGS HOXB9 cells (control Vector cells) after the addition of Y15.

[0024] Figure 6 A gastric cancer organoid model was used to validate the effect of HOXB9 on the sensitivity of gastric cancer cells to oxaliplatin. (A) Imaging of gastric cancer organoid-derived tissue samples after HE staining, light microscopic imaging of organoids, and imaging of gastric cancer organoids after HE staining; (B) Immunohistochemical marker Ki67 in the source tissue samples and corresponding gastric cancer organoid samples; (C) qPCR was used to detect the mRNA expression level of HOXB9 in gastric cancer organoids. A bar chart was plotted with P1 as the control group for relative quantification. Values ​​are the mean ± SD of three independent experiments. * p <0.05,** p <0.01, ns represents no significant difference. (D) Dose-response curves and IC50 values ​​of gastric cancer organoids treated with oxaliplatin, each curve representing the mean ± SD of four replicates under each condition. (E, G) Dose-response curves and IC50 values ​​of P1 and P4 HOXB9 overexpression treated with oxaliplatin, each curve representing the mean ± SD of four replicates under each condition. (F, H) Light micrographs of gastric cancer organoids P1 HOXB9 (control P1 Vector) and P4 HOXB9 (control P4 Vector) overexpressing HOXB9. (I) Bar chart of relative cell viability, values ​​are the mean ± SD of three independent experiments; * p <0.05,** p <0.01, ns represents no significant difference. (J, L) Dose-response curves and IC50 values ​​of PC-P1 and PC-P2 HOXB9 knockdown after oxaliplatin treatment; each curve represents the mean ± SD of four replicates under each condition. (K, M) Light micrographs of gastric cancer organoids PC-P1 shHOXB9 (control PC-P1 NC) and PC-P2 shHOXB9 (control PC-P2 NC) with HOXB9 knockdown. (N) Bar chart of relative cell viability statistical analysis; values ​​are the mean ± SD of three independent experiments. p <0.01, *** p<0.001, ns represents no significant difference. DETAILED DESCRIPTION

[0025] The present application will be further described in the following detailed description with specific reference being made to the figures. It should be noted, however, that the following description is made for purposes of illustration only and not for purposes of limitation of the subject matter described above, which will be limited only by the claims and equivalents thereof.

[0026] Unless otherwise indicated, the reagents and materials used in the present application are commercially available.

[0027] I. Research methods and materials

[0028] 1. Cell lines and reagents. Two human gastric cancer cell lines AGS and HGC-27 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). AGS cells were cultured in DMEM / F-12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and HGC-27 cells were cultured in RPMI1640 (Gibco). Both media were supplemented with 10% fetal bovine serum (Gibco) and 100 U / mL penicillin / streptomycin (Gibco).

[0029] 2. Cell construction

[0030] To establish a stable oxaliplatin-resistant gastric cancer cell line, the parental gastric cancer cells were treated with gradually increasing concentrations of oxaliplatin for a long period of continuous drug treatment. Oxaliplatin (MCE, MedChemExpress, Cat No: HY-17371) was started at a concentration of 0.2 μM, and the cells were screened at each concentration until cell colonies were formed and after passage, the cells were restored to normal growth at that concentration, and then the concentration of oxaliplatin was increased for the next concentration of drug screening. The concentration of oxaliplatin was added from 0.2 μM to 8 μM, and the treatment lasted for 10 months, forming a stable growth of drug-resistant cell line, namely AGS OR cells and HGC-27 OR cells. CCK8 (Beiren Chemical Technology Co., Ltd., Cat No: CK04) was used to detect the IC50 of oxaliplatin on parental cells and the corresponding drug-resistant cell lines, and to evaluate the drug resistance of the drug-resistant cell lines.

[0031] Construction of AGS and HGC-27 cells overexpressing HOXB9 (AGS HOXB9 / HGC27 HOXB9) was performed using a GV341 vector (Ubi-MCS-3FLAG-SV40-puromycin) containing the human HOXB9 gene (NM_024017.5) CDS sequence recombinant lentivirus vector, and the packaged virus was obtained (Shanghai Jikai Gene Technology Co., Ltd.). The cells to be infected were seeded at an appropriate density (2 x 10 4The cells were cultured overnight in 24-well plates (1 x 105cells / well), and the virus was taken out from -80°C, thawed on ice, and the virus dosage was calculated using MOI = 10: virus amount (μL) = (MOI x cell number) / virus titer (TU / mL) x 1000. The old culture medium was discarded, and the cells were washed with PBS for 3 times, and fresh culture medium containing polybrene (25x stock solution) was prepared. The virus suspension of corresponding concentration was added, and after mixing, the cells were incubated at 37°C. 24 hours after infection, the virus-containing culture medium was removed, and fresh complete culture medium was added. 48-72 hours after infection, Puromycin was added for screening, and the drug-containing culture medium was replaced every 2-3 days. Screening was performed for about 2 weeks until all uninfected cells died, and the surviving positive cells were expanded and identified.

[0032] AGS OR and HGC-27 OR cells with HOXB9 knockdown (AGS OR shHOXB9 / HGC 27 OR shHOXB9) were constructed. The shRNA sequence (target sequence 1: GGCAAAGAGTAAAGATTAA; target sequence 2: GGCTAGAAAGTACAAGAAA) targeting the HOXB9 gene (NM_024017.5) was constructed into a recombinant lentivirus vector using the GV112 vector (hU6-MCS-CMV-puromycin), and the packaged virus was obtained (Shanghai Jikai Gene Technology Co., Ltd.).

[0033] 3. Gastric cancer tissue samples.

[0034] The gastric cancer tissue samples in this study were obtained after approval by the Ethics Committee of Liaoning Cancer Hospital. The primary tumor samples of all patients in this study were obtained from the biological sample bank of Liaoning Cancer Hospital.

[0035] The patients whose gastric cancer tissue samples were used in RNA-seq sequencing were treated in Liaoning Cancer Hospital between July 2020 and July 2021, and received at least two cycles of NACT and SOX regimen. SOX treatment included administration of oxaliplatin at a dose of 130 mg / m 2 for the first day, and S-1 at a dose of 60 mg twice a day for 2 weeks. According to the physical condition, serum tumor markers, and computed tomography results, clinical evaluation was performed every 2-3 cycles.

[0036] The paraffin sections of tissue samples used in immunohistochemical experiments were collected in Liaoning Cancer Hospital biological sample bank from 2011 to 2016, and were grouped according to the recurrence, with 49 cases of non-recurrence and 30 cases of recurrence, totaling 79 cases.

[0037] The tissue samples used for the establishment of gastric cancer organoids were postoperative tissue samples from 2024 to 2025, among which P1-P4 were untreated primary tumor tissue samples, and PC-P1 and PC-P2 were tumor tissue samples after neoadjuvant chemotherapy.

[0038] 4. RNA-seq sequencing analysis.

[0039] Sequencing of clinical gastric cancer tissue samples: The College of American Pathologists (CAP) system was used to grade the pathological response. The response was divided into four categories: TRG 0 means no residual cancer cells and complete response to treatment. TRG 1 means few cancer cells and almost complete response to treatment. TRG 2 indicates some residual tumor and partial response with clear regression; TRG 3 indicates poor response to extensive residual cancer and no obvious tumor regression. According to their response, patients were divided into two groups: the response group including patients with obvious histological response (MJHR; TRG 0-2) and the non-response group without histological response (NHR; TRG 3). The samples were sequenced using the Illumina Hiseq platform. Differential expression analysis was performed on the RNA-seq data of the two groups of clinical samples, with an absolute fold change greater than 2 and a P value less than 0.05 as the threshold for differentially expressed genes. Gene heat map, expression difference volcano plot were drawn using Novogene cloud platform (https: / / magic-plus.novogene.com / ).

[0040] Cell lines and sequencing of cell line samples: The AGS cell line stably resistant to oxaliplatin, the AGS cell line overexpressing HOXB9 constructed, and the control cell line were sequenced using the Illumina Hiseq platform for RNA-seq, and differential expression analysis was performed, with an absolute fold change greater than 2 and a P value less than 0.05 as the threshold for differentially expressed genes. Gene heat map, expression difference volcano plot, GO and KEGG enrichment analysis, Venn diagram, etc. were drawn using Novogene cloud platform.

[0041] 5. Public database analysis. TCGA database analysis of the expression of HOXB9 in gastric adenocarcinoma and control normal tissue, using GEPIA2 platform (http: / / gepia2.cancer-pku.cn / ), with 359 normal tissues and 339 tumor tissues. Survival analysis of gastric cancer patients using Kaplan-Meier Plotter platform, with data set (216417_x_at) for analysis (https: / / kmplot.com / analysis / ), respectively for total sample survival analysis, survival analysis according to Lauren classification, and survival analysis according to differentiation type.

[0042] 6. Immunohistochemistry (IHC).

[0043] Animal or human tumor tissue samples were fixed in 10 times 4% paraformaldehyde, and the fixed tissue samples were sequentially dehydrated by gradient ethanol (75%, 85%, 95%, 1 h each, anhydrous ethanol 1, anhydrous ethanol 2, 30 min immersion), and permeated by immersion in clearing agent (xylene 1, xylene 2, 10 min immersion each), and the permeated tissue samples were placed in melted paraffin (wax 1, wax 2, 1 h immersion each), and the paraffin-impregnated tissue blocks were placed in embedding molds, and melted paraffin was added, and cooled and solidified into wax blocks. The wax blocks were cut into 5 μm thick continuous sections using a microtome, and the sections were floated in a warm water bath (40-45°C) to flatten, and were picked up onto a non-stick glass slide (polylysine-coated), and were baked in a 60°C oven for 2 h. The tissue sections were subjected to immunohistochemical procedures, twice for 10 min in xylene, twice for 5 min in 100% ethanol, 5 min in 95% ethanol, 5 min in 85% ethanol, 5 min in 75% ethanol, 5 min in 50% ethanol, 1 min in distilled water, and were placed in PBS buffer. The sections were soaked in EDTA buffer at pH 9.0 for antigen retrieval, and were heated in a pressure cooker for 2 min, and were naturally cooled before continuing the experiment. The sections were removed and replaced with PBS, and subsequent experiments were performed using a universal two-step kit (mouse / rabbit enhanced polymer detection system) (Beijing Zhongshanjinqiao Biotechnology Co., Ltd., Catalog No. PV-9000). The kit blocked endogenous peroxidase, and the sections were washed 3 times for 3 min in PBS buffer, and 5% goat serum blocking solution (Beijing Solabio Technology Co., Ltd., Catalog No. SL038) was added and incubated at room temperature for 30 min. The primary antibody was used at the recommended ratio according to the antibody instructions, including HOXB9 (1:100; Thermo Fisher Scientific, Catalog No. PA5-40576) and ki67 (1:100; Aibotek Biotech Co., Ltd., Catalog No. A20018), and was incubated at 4°C overnight. The sections were washed 3 times for 3 min in PBS buffer, and the kit was added with reaction enhancer solution. The sections were washed 3 times for 3 min in PBS buffer, and the kit was added with enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer. The sections were washed 3 times for 3 min in PBS buffer, and DAB staining was performed using a DAB color reagent kit (Beijing Zhongshanjinqiao Biotechnology Co., Ltd., Catalog No. ZLI-9017), and was incubated at room temperature for 5-8 min. The sections were washed with tap water, and were stained with hematoxylin for 20 s, and were differentiated with hydrochloric acid alcohol for 2 s, and were washed with tap water to return to blue. The sections were sequentially dehydrated by gradient ethanol (50% ethanol for 5 min, 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min, 2 times), and were cleared (xylene 1, xylene 2, 10 min immersion each), and were mounted with neutral resin.

[0044] Imaging was performed using an optical microscope, including a low-power objective (10x objective) and a high-power objective (40x objective), and un-compressed images were saved with fixed exposure parameters. Image J software was used for IHC score analysis, which was reviewed and determined by two pathologists, and scored according to the intensity of cell staining in four levels: negative, 0 points; weakly positive, 1 point; positive, 2 points; strongly positive, 3 points. According to the percentage of positive cells, it was scored in four levels: 0%≤ percentage of positive cells≤25%, 1 point; 25%< percentage of positive cells≤50%, 2 points; 50%< percentage of positive cells≤75%, 3 points; 75%< percentage of positive cells≤100%, 4 points. The IHC score was: (cell staining intensity) x (percentage of positive cells). According to the IHC score, the column chart was drawn and statistical analysis was performed using Graphpad Prism 8 software.

[0045] 7. Western blotting experiment (WB).

[0046] Sample preparation: The target cell culture dish was washed twice with pre-cooled PBS, completely discarded, and RIPA lysis buffer containing protease inhibitors (Shanghai Biyun Tian Biotechnology Co., Ltd., Catalog No: P0013) was added (lysis on ice for 30 min). The lysis buffer was collected using a cell scraper, and centrifuged at 12000 rpm at 4°C for 15 min. The supernatant protein concentration was determined using a BCA kit (Shanghai Biyun Tian Biotechnology Co., Ltd., Catalog No: P0012), and adjusted to a uniform concentration. 6x SDS loading buffer sample buffer (Shanghai Biyun Tian Biotechnology Co., Ltd., Catalog No: P0015F) was added, and the protein was denatured by boiling at 95°C for 5-10 min.

[0047] SDS-PAGE electrophoresis: gel preparation, according to the molecular weight of the target protein, select the appropriate concentration of separation gel (10%, 12%). 10% separation gel formula: distilled water 4.0 mL, 30% acrylamide 3.3 mL, 1.5 M Tris-HCl (pH 8.8) 2.5 mL, 10% SDS 0.1 mL, 10% APS 0.1 mL, TEMED 0.005 mL Pour the separation gel to 1 cm from the comb, cover with anhydrous ethanol to flatten, after polymerization, pour off the anhydrous ethanol, pour the 5% concentrated gel and insert the comb. Load 20-50 μg of protein per well (phosphorylated protein can be increased to 30-50 μg). The electrophoresis conditions are as follows: concentrated gel 80V, enter separation gel 120V, bromophenol blue runs out of the bottom of the gel.

[0048] Transfer: After electrophoresis, the gel was equilibrated in transfer buffer for 15 min, and a "sandwich" structure was assembled: sponge pad-filter-paper-gel-membrane-filter-paper-sponge pad. The transfer was performed at 100 V under ice bath conditions (the time was adjusted according to the molecular weight of the protein: about 30 min for 22-36 kDa, about 60 min for 36-70 kDa, and about 90 min for 70-130 kDa).

[0049] Blocking: 5% skim milk or 5% BSA (phosphorylated protein) in TBST was used for blocking at room temperature for 1 h on a shaker.

[0050] Primary antibody incubation: The primary antibody was diluted with 1% BSA and incubated at 4°C overnight. TBST was used for washing 3 times, each for 10 min. The primary antibodies included HOXB9 (1:1000; Yano Biotech, Cat No: H00003219-M05), p-FAK (1:1000; MedChemExpress, Cat No: HY-P80460), FAK (1:1000; Aibotek, Cat No: A11195), Akt (1:2000; Wuhan Servicebio, Cat No: 10176-2-AP), and p-Akt (1:2000; Wuhan Servicebio, Cat No: 66444-1-lg).

[0051] Secondary antibody incubation: The HRP-labeled secondary antibody (1:10000; Abeam, Cat No: ab6721 / ab6789) was selected to match the species of the primary antibody. The secondary antibody was incubated at room temperature for 1 h, and TBST was used for washing 3 times, each for 10 min.

[0052] Exposure: The ECL luminescent substrate A and B (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, Cat No: 34580) were mixed in proportion, evenly coated on the surface of the membrane, incubated for 1 min, and the signal was collected using a chemiluminescence imaging system.

[0053] Image analysis: Image J was used to analyze the gray value of the image. The target protein band was normalized by taking the reference protein band as the standard. The analysis results of three independent experiments were used to draw a column chart and perform statistical analysis using Graphpad Prism 8 software.

[0054] 8. Drug-resistant clone formation experiment.

[0055] Logarithmic growth phase cells were taken, digested to make single cell suspension, counted, adjusted to 800 cells / mL with complete medium, 2 mL of cell suspension was added to each well, and 3 days later the corresponding drugs were added. Single drug treatment, oxaliplatin drug concentration was about 24h IC50 concentration corresponding to the target cells (AGS cells 5 μM, HGC27 cells 10 μM). Combination drug treatment, first incubate with the corresponding combination drug Y15 (MedChemExpress, HY-12444) at a concentration of 5 μM for 2 h, then add oxaliplatin, and the oxaliplatin drug concentration is the 24h IC50 concentration corresponding to the target cells. Incubate continuously for 14 days, replace the medium containing fresh drugs every 3 days and perform the above double drug treatment. After the experiment was terminated, the medium was discarded, washed twice with PBS, 1 mL of methanol was added, and fixed at room temperature for 30 min. After washing with PBS, 0.1% crystal violet staining solution (Shanghai Biyun Tian Biotechnology Co., Ltd., C0121) was added, and the solution was stained for 30 min in the dark. Slowly rinse with running water and air dry at room temperature.

[0056] Image J software was used for image analysis, and the total clone area of the control group was standardized, and the actual area of the other groups / the average area of the control group%=relative area%, i.e. relative area (%)=(treatment group average area / control group average area) x 100%. The analysis of the numerical results of three independent experiments was performed using Graphpad Prism 8 software for column chart drawing and statistical analysis.

[0057] 9. Drug IC50 detection experiment. Logarithmic growth phase cells were taken, digested to make single cell suspension, counted, adjusted to 800 cells / mL with complete medium, seeded in 96-well plates, 2500 cells / well, 7-8 concentration gradients were set for each group of cells, 5 replicates for each concentration, and 3 blank background control wells were set. Parental gastric cancer and overexpressed gene constructed gastric cancer cells, oxaliplatin concentration was 0, 1, 2, 10, 20, 100, 200 μM. Oxaliplatin-resistant gastric cancer cells, oxaliplatin concentration was 0, 4, 20, 40, 80, 120, 160, 240 μM. The next day, replace the medium with the corresponding concentration of drug after the cells adhere, incubate at 37°C, 5% CO2 for 24 or 72 h. CCK8 reagent was used to detect the absorbance value at 450 nm wavelength, 10 μL of reagent was added to each well, and the absorbance value was measured after 2 h of incubation. Cell survival rate (%)=(OD sample-OD blank) / (OD control-OD blank) x 100% (OD sample: absorbance of drug treatment group, OD control: absorbance of negative control group, OD blank: absorbance of blank well without cells). Independent repeat 4 times, analysis of numerical results, using Graphpad Prism 8 software for drug IC50 curve drawing and analysis.

[0058] 10. 3D cell spheroid experiment. Matrigel Basement Membrane Matrix (Xiamen Mod Base Biotechnology Co., Ltd., Cat No. 082706) was thawed at 4°C for 12 h. The consumables in contact with Matrigel were precooled at 4°C. 50 μL of Matrigel (8-11 mg / mL) was added to the precooled 96-well plate and incubated at 37°C for 30 min to make Matrigel gel. The logarithmic growth phase cells were collected to obtain cell pellets to be inoculated, and the cells were resuspended with complete medium, counted, and the cell density was adjusted to 3 x 10 4 6 x 105 / mL, and 50 μL of cell suspension was added to the well plate coated with Matrigel. The plate was incubated at 37°C for 30 min. Matrigel Basement Membrane Matrix was added to the precooled complete medium to a final volume of 10% to prepare a mixed culture medium. 100 μL of mixed culture medium was added to the incubated well, and the plate was incubated at 37°C for 9 days. On the 10th day, 10 μM oxaliplatin was added. The medium containing fresh drug was replaced every 2 days. During this period, imaging was performed using an optical microscope. The CellTiter-Glo® 3D Cell Viability Assay Kit (Promega, Cat No. G9681) was used to detect the cell viability of 3D cell spheroids using a microplate reader. Cell survival rate (%) = (sample luminescence value - blank luminescence value) / (control luminescence value - blank luminescence value) x 100%. Graphpad Prism8 software was used to plot and analyze the column chart.

[0059] 11. Immunofluorescence experiment. The corresponding gastric cancer cells were inoculated on the cell slides, and the number of cells inoculated was about 20,000 per slide. After the cells were in good condition, subsequent experiments were performed. The slides were gently washed with PBS buffer twice, 1 mL of 4% paraformaldehyde was added, and the slides were fixed at room temperature for 30 min. After fixation, the slides were washed with TBS buffer for 3 times, 10 min each time. 0.2% Triton-X100 permeabilization solution was prepared using TBS buffer. Add 1 mL of permeabilization solution, permeabilize at room temperature for 10 min. Wash the slides with TBS buffer for 3 times, 5 min each time. After discarding the TBS buffer, add 1 mL of 5% BSA blocking solution, block at room temperature for 30 min. Dilute the primary antibody with 1% BSA, and incubate the primary antibody at 4°C overnight. Wash the slides with TBST buffer for 3 times, 10 min each time. Add the secondary antibody (Thermo Fisher Scientific, Cat. No. A-11008) working solution in the dark, and incubate at room temperature for 2 h. Wash the slides with TBST buffer for 3 times, 10 min each time. Discard the TBST buffer, and add the DAPI (Thermo Fisher Scientific, Cat. No. D1306) working solution, stain the nucleus for 10 min, and wash the slides with TBST buffer for 2 times, 10 min each time. Rinse with ddH2O once, and mount the slides with an anti-quenching mounting medium (Thermo Fisher Scientific, Cat. No. P36961). Use a laser confocal microscope to image, and use ImageJ software to perform fluorescence statistical analysis, including focus statistics and average fluorescence intensity analysis. Then use Graphpad Prism 8 software to draw column charts and perform statistical analysis. The primary antibodies include: Anti-gamma H2A.X (phospho S139) (1:100; Abeam, Cat. No. ablll 174), Phospho-FAK (Tyr397) antibody (1:100; MedChemExpress, Cat. No. HY-P80460). F-actin uses Actin-Tracker Red-Rhodamine (microfilament red fluorescent probe) (Bi Yun Tian Biotechnology Co., Ltd., Cat. No. C2207S).

[0060] 12. Tumor-bearing mouse experiment. BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used, female mice, 4 weeks, 6 mice per group. After feeding for one week, subcutaneous injection of cells was performed at 5 weeks of age. The target cells and control cells were collected and counted, resuspended with PBS once, and resuspended with PBS containing 30% Matrigel, and aliquoted in sterile EP tubes, 10 7Cells were placed in an ice box for temporary storage and to avoid cell freezing injury. The skin was disinfected with 75% alcohol before injection, and the needle was inserted at a shallow angle of 15-30° in the right axillary subcutaneous site. After the needle traveled a certain distance under the skin, it was slowly injected. A visible bump was observed, and the needle was slowly pulled out and gently pressed to prevent backflow. The injection time was recorded. The state of the mice was observed daily, and the volume was measured every 3 days initially. When the volume reached 100 mm 3 After 12 days, the drug was administered intraperitoneally. The initial dose was 5 mg / kg, twice a week, and the dose was increased to 10 mg / kg twice a week from the third week. The experiment lasted for 61 days. Tumor diameter ≤20 mm, weight <10% of body weight, no ulceration or necrosis. The tumor volume and weight were measured. The survival curve was drawn, the final tumor volume was analyzed, and the final tumor weight was analyzed using Graphpad Prism 8 software.

[0061] 13. mRNA expression quantitative experiment.

[0062] RNA extraction: Collect cells and add 500 μL Trizol (Thermo Fisher Scientific, Cat: 15596018CN) to lyse cells at room temperature for 10 min. Add 0.1 mL chloroform, shake vigorously for 15 s, incubate on ice for 5 min, then centrifuge at 12,000 g at 4°C for 15 min. The sample is divided into three layers: upper aqueous phase (containing RNA), middle layer (containing DNA), and lower organic phase (containing protein). Carefully pipette the upper aqueous phase (not more than 80%) to a new tube, add 0.25 mL isopropanol, and mix gently. Incubate at room temperature for 10 min, then centrifuge at 12,000 g at 4°C for 10 min. Discard the supernatant and add 1 mL 75% ethanol (DEPC water) to wash the precipitate. Centrifuge at 12,000 g at 4°C for 5 min, discard the supernatant. After air-drying, dissolve the RNA with an appropriate amount of DEPC water.

[0063] Reverse transcription: After quantification of RNA, 1 μg per sample was reverse transcribed to cDNA using ABScript Neo RTMaster Mix for qPCR with gDNA Remover kit (Aibiotek Biotechnology Co., Ltd., Cat: RK20433), according to the reagent instruction. The reaction solution was prepared on a PCR reaction instrument for reverse transcription.

[0064] Quantitative Real-time PCR (qPCR): 2 μL cDNA product was taken for qPCR reaction. 2x Universal SYBR Green Fast qPCR Mix kit (Aibiotek Biotechnology Co., Ltd., Cat No: RK21203) was used to prepare the reaction solution according to the reagent instruction, and qPCR reaction was performed on the fluorescence quantitative PCR instrument. Graphpad Prism 8 software was used for analysis and statistical columnar chart drawing.

[0065] The relevant primers (Suzhou Hongxun Biotechnology Co., Ltd.) are as follows.

[0066] 14. Apoptosis experiment. The logarithmic growth phase of the cells to be tested was inoculated in a 6-well plate, 6x10 4 cells per well. When the cell density reached more than 70%, drug treatment was performed. Oxa1 platinum 10 μM was added and placed in a 37°C, 5% CO2 incubator for 24 h. The culture supernatant containing possible apoptotic cells was collected, and the adherent cells were washed once with pre-cooled PBS, 500 μL of non-EDTA trypsin was added for digestion, and when the cells were rounded, the culture medium was added to terminate the digestion and gently blow the single cell suspension, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with pre-cooled PBS and centrifuged at 1000 rpm for 5 min. Annexin V-FITC apoptosis detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd., Cat No: C1062M) was used for cell staining, 195 μL of Annexin V-FITC binding solution was added to gently resuspend the cells. 5 μL of Annexin V-FITC was added and mixed gently. 10 μL of propidium iodide staining solution was added and mixed gently. Incubate at room temperature (20-25°C) for 10-20 min in the dark, then place in ice bath. Aluminum foil can be used for light protection. Then, flow cytometry was used for detection. The results were statistically analyzed and columnar charts were drawn using Graphpad Prism 8 software. Combined drug treatment, first incubate with the corresponding combined drug Y15 (MedChemExpress company, Cat No: HY-12444) at a concentration of 5 μM for 2 h, then add oxa1 platinum.

[0067] 15. Gastric cancer organoid culture and construction experiment.

[0068] Gastric cancer organoid construction: Fresh gastric cancer tumor tissue (≤1 cm 3), keep active in pre-cooled sterile tissue preservation solution (Xiamen Mobio Biotech Co., Ltd., Cat. No. MB-0818L04S), the best within 12h. Remove necrotic tissue, fat and blood vessels, and retain the active tumor area for repeated washing with PBS containing double antibiotics (penicillin / streptomycin) for 3-5 times to remove blood and impurities. Sterile scissors cut the tissue into 1-2mm 3 chunks into tissue digestion solution (Xiamen Mobio Biotech Co., Ltd., Cat. No. MB-0818L05S) and shake at 37°C for 60 min, mix every 5-10 min, and monitor under a microscope until the tissue is fully dissociated. Add culture medium (Advanced DMEM / F12, Xiamen Mobio Biotech Co., Ltd., Cat. No. MB-0930L500) containing 10% FBS and 1% double antibiotics to terminate the reaction, filter out undigested lumps through a 100-μm cell strainer, centrifuge at 300g for 5 min, resuspend and wash twice with PBS to remove enzyme residues. Thaw Matrigel (Xiamen Mobio Biotech Co., Ltd., Cat. No. 082755) in advance (4°C overnight) and operate on ice to prevent solidification. Mix the cells and Matrigel at a ratio of 1:1 (density: 10,000 cells / 10 μL gel), add the mixture dropwise to the culture plate wells (50 μL per well), and incubate the gel drop at 37°C for 30 min. Add 500 μL of gastric cancer organoid culture medium (Xiamen Mobio Biotech Co., Ltd., Cat. No. MA-0807T008LP) to cover the surface of the solidified gel drop, and culture at 37°C, 5% CO2, and saturated humidity, with fresh medium changed every 2-3 days.

[0069] Passage of gastric cancer organoids: aspirate the culture medium, blow the matrix gel with pre-cooled PBS buffer, collect the organoid suspension, centrifuge and wash, then discard the PBS, add 1 mL of organoid digestion solution (Xiamen Mobio Biotech Co., Ltd., Cat. No. MB-0818L01S), and place it in an incubator for 10 min. Add the medium containing 10% FBS and 1% double antibiotics to neutralize the digestion solution. Then follow the primary steps for subsequent embedding, and the passage ratio is usually 1:4-1:8.

[0070] Construction of gastric cancer organoids overexpressing HOXB9 or knocking down HOXB9: dissociate the mature gastric cancer organoids into single-cell suspension, centrifuge at 300g for 5 min, collect the cells, and wash with pre-cooled medium. Plate the single-cell suspension in 24-well plates (2x10 5Cellular / well), add virus (the aforementioned overexpression HOXB9 or shRNA knockdown HOXB9 lentivirus, virus titer MOI is 10) - polybrene complex (containing 6 μg / mL polybrene to enhance the infection efficiency), 37 ℃ incubation for 24 h. Centrifugal collection of transfection cells, mixed with resuspension of Matrigel, plated into glue, according to the primary procedure for subsequent embedding to form gastric cancer organoids after gene editing. Add the culture medium containing puromycin (2 μg / mL) to screen positive stable strains, collect organoids for verification and subsequent experiments.

[0071] 16、gastric cancer organoids drug resistance IC50 detection experiment. Take the culture for 14 days, the diameter of 200 μm of organoids, with pre-cooled PBS to dissolve the Matrigel, centrifugal 300g 5min, collect organoids mass digestion, 37℃ 10min, the organoids dissociated into single cells or small cell clusters (<10 cells) organoids single cell resuspended in 50% Matrigel containing culture medium, 96-well plate each well added 5 μL containing 1500 cells of glue droplet, 37℃ solidification 30min, each hole added 50 μL gastric cancer organoids culture medium, culture 72h to make structure reconstruction. Set 6 concentration gradient of oxaliplatin drug condition medium (0, 5, 25, 50, 100, 400 μM), each well added 100 μL drug medium instead of the original liquid, and set blank solvent control group (containing drug dissolution solvent). Each concentration set 5 replicate wells. Drug treatment culture 72h. Each well added CCK-8 (10 μL / well), continue to cultivate 4h enzyme detector detection 450nm wavelength OD value. Using GraphPad Prism software, with drug concentration as the abscissa, survival rate as the ordinate to fit the dose-response curve, calculate the half inhibitory concentration (IC50).

[0072] 17. Paraffin embedding of gastric cancer organoids. Aspirate organoid culture medium, wash twice with PBS to remove residual culture medium, aspirate organoids into a centrifuge tube with pre-chilled PBS, centrifuge at 300g for 3 min, remove supernatant and retain the pellet. Add 4% paraformaldehyde and fix for 4h (4°C) to ensure organoid morphology is intact. After fixation, centrifuge to remove paraformaldehyde and wash twice with PBS. Pre-heat 3% agarose to complete dissolution and cool to 40°C for use, mix organoid suspension with agarose (1:1 ratio), transfer to a 1.5 mL EP tube, solidify at 4°C for 30 min to form a transparent gel block. Dehydrate by sequentially passing through gradient ethanol (75%, 85%, 95%, anhydrous ethanol 1, anhydrous ethanol 2, 30 min each) and permeabilize by sequentially passing through clearing agents (xylene 1, xylene 2, 10 min each), place the cleared tissue sample into melted paraffin (wax 1, wax 2, 1 h each), place the paraffin-impregnated tissue block into an embedding mold, add melted paraffin, and cool to solidify into a wax block. Cut the wax block into 5 pm-thick serial sections using a microtome, float the sections in a warm water bath (40-45°C) to flatten, pick up onto poly-L-lysine-coated slides, and bake the sections in a 60°C oven for 2h.

[0073] 18. HE staining.

[0074] De-waxing and hydration: place paraffin sections into xylene I and xylene II for 10 min each to remove paraffin. Dehydrate by sequentially passing through anhydrous ethanol I and II for 5 min each, 95%, 90%, 80%, and 70% ethanol for 5 min each, and distilled water.

[0075] Hematoxylin staining: immerse the sections into hematoxylin staining solution for 3 min, the nuclei are stained blue-purple, differentiate with 1% hydrochloric acid alcohol for 2 s to remove non-specific staining, rinse with running water, and return to blue with tap water.

[0076] Eosin staining: immerse the sections into eosin staining solution for 10 s, the cytoplasm is stained pink to red.

[0077] Dehydration and mounting: dehydrate by sequentially passing through 75%, 85%, and 95% for 2 min each, anhydrous ethanol I and II for 5 min each, xylene I and II for 5 min each, completely dehydrate and clear, mount with neutral balsam, avoid air bubbles, and image under a microscope.

[0078] 19. Continuous Oxaliplatin Treatment and Viability Determination of Gastric Cancer Organoids. Organoids cultured for 14 days with a diameter of 200 μm were collected. The matrix gel was dissolved in pre-cooled PBS, centrifuged at 300 g for 5 min, and the organoid clumps were collected and digested. The digestion was performed at 37°C for 10 min to dissociate the organoids into single cells or small cell clusters (<10 cells). Single organoid cells were resuspended in medium containing 50% Matrigel. 5 μL of gel droplets containing 1500 cells were added to each well of a 96-well plate, and the plates were cured at 37°C for 30 min. 50 μL of gastric cancer organoid culture medium was added to each well, and the plates were cultured for 72 h to allow for structural reconstruction. Oxaliplatin-containing medium (100 μM) was added, and treatment was continued for 7–14 days, depending on the specific characteristics of the organoids. Imaging was performed using an optical microscope. The plates were discarded at the experimental endpoint.

[0079] 20. Statistical Analysis. Independent experiments were conducted at least three times. Data are expressed as mean ± standard deviation (SD). All data were statistically analyzed using GraphPad Prism 8.0 software. The t-test was used to compare two independent groups. One-way ANOVA and Tukey's test were used for statistical analysis of data across multiple groups. p <0.05 is statistically significant.

[0080] II. Research Results.

[0081] 1. HOXB9 is associated with chemotherapy resistance in gastric cancer. Clinical tissue samples were collected from 19 patients with locally advanced gastric cancer who received neoadjuvant therapy with oxaliplatin. Nine of these were significantly reactive tissue samples (MjHR) after chemotherapy, and 10 were non-reactive tissue samples (NHR). Transcriptome sequencing analysis was subsequently performed, and the results showed a certain degree of consistency in gene expression among these clinical samples. Figure 1 A). Comparing differentially expressed genes between NHR and MjHR, 480 genes were upregulated in the NHR group, and 97 genes were downregulated in the NHR group. Figure 1 B). Combined with gene heatmap analysis, multiple HOX family genes were highly expressed in the NHR group, with HOXB9 showing significantly higher expression. HOXB9 expression was higher in multiple samples from the NHR group than in the MjHR group (B). Figure 1 B, C). TCGA dataset analysis showed that HOXB9 expression was significantly higher in gastric adenocarcinoma tumor tissues than in normal tissues (B, C). Figure 1 D). Analysis of overall survival of HOXB9 in gastric cancer patients using the Kaplan-Meier Plotter platform showed that high expression of HOXB9 was associated with lower survival in all gastric cancer patients. Figure 1 E). Further analysis of gastric cancer patients by differentiation type revealed that high expression of HOXB9 was associated with poor and moderately differentiated gastric cancer patients (E). Figure 1F). In different gastric cancer types analysis, we found that high expression of HOXB9 in intestinal type and mixed type of gastric cancer was associated with low survival rate. Figure 1 G). We collected gastric cancer pathological tissues for immunohistochemical staining, and the pathological tissues of patients with recurrence after chemotherapy had significantly higher expression of HOXB9 than those without recurrence. Figure 1 H. I). Therefore, HOXB9 affects the clinical progression of gastric cancer patients and may be related to the progression of chemotherapy.

[0082] 2. HOXB9 overexpression reduces the sensitivity of gastric cancer to oxaliplatin chemotherapy. To further confirm whether the increase of HOXB9 affects the progression of gastric cancer chemotherapy, we constructed AGS and HGC27 cell lines stably overexpressing HOXB9. In the drug resistance colony formation experiment using oxaliplatin, we found that the relative area of colonies formed by gastric cancer cells overexpressing HOXB9 was increased compared with the control group during continuous drug treatment, while in the case of drug withdrawal after 1 day of drug treatment, the relative area of colonies formed by gastric cancer cells overexpressing HOXB9 was significantly increased compared with the control group. Figure 2 A). Further verified by drug IC50 detection, the IC50 of AGS HOXB9 cells to oxaliplatin for 72h was significantly higher than that of the control group, and the IC50 of AGS HOXB9 cells to oxaliplatin was increased by about 3 times after the expression of HOXB9 was increased. Figure 2 B). In addition, in the 3D cell sphere formation experiment, we found that the volume of 3D cell spheres formed by AGS HOXB9 cells had no significant difference compared with the control group, but after oxaliplatin treatment, AGS HOXB9 cells had more 3D cell spheres left, and under high magnification, they formed more dense solid 3D spheres, while the control group was dissolved after oxaliplatin treatment, and ATP energy determination also showed that the survival rate of AGS HOXB9 cells after oxaliplatin treatment was significantly higher than that of the control group. Figure 2 C). Cell death after chemotherapy will activate the Caspase system. Western blot (WB) results showed that the expression of Caspase-3 and Caspase-8 in AGS cells overexpressing HOXB9 was reduced after treatment with different concentrations of oxaliplatin compared with the control group. Figure 2 D). And the DNA damage marker γ-H2AX, through immunofluorescence experiment showed that the intracellular nuclear damage of AGS HOXB9 cells induced by oxaliplatin was lower than that of the control group. Figure 2 E). In terms of in vivo experiments, we used BALB / c nude mice for tumor-bearing experiments, and the results showed that there was no significant difference in the volume and weight of subcutaneous tumors formed by AGS cells overexpressing HOXB9 and the control group. Figure 2F-I), but the tumors formed by HOXB9-overexpressing AGS cells were more resistant to oxaliplatin and were significantly larger in volume and weight than those formed by control cells in nude mice treated with oxaliplatin Figure 2 J-M). Thus, increased expression of HOXB9 in gastric cancer cells increased the resistance of gastric cancer cells to oxaliplatin, and this process also affected the activation of the Caspase family and weakened the damage to the nucleus.

[0083] 3. HOXB9 in gastric cancer cells is induced to increase during oxaliplatin treatment, and deletion of HOXB9 affects the survival of gastric cancer drug-resistant cell lines. To further investigate the role of HOXB9 in the process of oxaliplatin resistance in gastric cancer, we induced AGS and HGC27 cells with oxaliplatin at a concentration of 0.2 IC50 to establish gastric cancer oxaliplatin-resistant cell lines AGS OR and HGC27 OR. Subsequently, the oxaliplatin IC50 of the stable gastric cancer-resistant cell lines was verified Figure 3 A). HOXB9 expression was detected in the gastric cancer-resistant cell lines during the induction and establishment process, and it was found that the expression level of HOXB9 in gastric cancer cells gradually increased with the gradual increase of the induction concentration Figure 3 B). WB detection also showed that the expression of HOXB9 in the drug-resistant cell lines was increased compared with the parental cells Figure 3 C). After the oxaliplatin IC50 of the gastric cancer-resistant cell lines was stably increased by more than 8 times compared with the parental cells, HOXB9 was knocked down in the gastric cancer-resistant cell lines to construct stable cell lines AGS OR shHOXB9 (control group AGS OR NC) and HGC27 OR shHOXB9 (control group HGC27 OR NC). The IC50 of oxaliplatin for 24 h was detected in the gastric cancer-resistant cell lines with HOXB9 knocked down and the control cells, and the down-regulation of HOXB9 improved the sensitivity of the gastric cancer-resistant cell lines to oxaliplatin (the IC50 of AGS OR NC was 65.11 μM, the IC50 of AGS OR shHOXB9 was 9.745 μM, the IC50 of HGC27 OR NC was 93.01 μM, and the IC50 of HGC27 OR shHOXB9 was 23.59 μM) Figure 3 D). We performed continuous treatment of oxaliplatin on the gastric cancer-resistant cell lines with HOXB9 knocked down and the control cells, and the results showed that under drug-free conditions, the clonogenic ability of AGS OR cells with HOXB9 knocked down was not significantly different from that of the control group, but oxaliplatin significantly inhibited the clonogenic ability of AGS OR shHOXB9 cells. Under drug-free conditions, the clonogenic ability of HGC27 OR cells with HOXB9 knocked down was slightly lower than that of the control group, and continuous treatment with oxaliplatin also showed a significant inhibitory effect Figure 3E, F). Flow cytometry was used to detect the apoptosis of gastric cancer drug-resistant cell lines after oxaliplatin treatment. The results showed that the total apoptosis rate (Q2+Q4) of the gastric cancer drug-resistant cell line with HOXB9 knockdown was significantly increased, further proving that the deletion of HOXB9 improved the sensitivity of the gastric cancer drug-resistant cell line to oxaliplatin Figure 3 G, H). At the same time, similar results were also shown in the 3D cell spheroid experiment. We found that the spheroids formed by the gastric cancer drug-resistant cell line with HOXB9 knockdown were more easily dissolved after oxaliplatin treatment Figure 3 I). This is opposite to the phenomenon of the solid spheroids formed by the gastric cancer cell spheroids with HOXB9 overexpression. ATP energy assay also proved that the spheroid survival of the gastric cancer drug-resistant cell line with HOXB9 knockdown was inhibited Figure 3 J). These also indicated that the expression of HOXB9 was induced in gastric cancer cells during oxaliplatin treatment, and the deletion of HOXB9 could significantly enhance the sensitivity of the gastric cancer drug-resistant cell line to oxaliplatin.

[0084] 4. HOXB9 regulates focal adhesion signaling pathway in gastric cancer cells. To reveal how HOXB9 affects the progression of gastric cancer drug resistance, we further performed RNA-seq sequencing analysis on the gene-edited gastric cancer cells AGS HOXB9 and AGS Vector. In addition, we found in the previous results that the expression of HOXB9 in the gastric cancer drug-resistant cell line gradually increased with the increase of drug resistance. Therefore, we knocked down HOXB9 in AGS OR cells, combined with overexpression of HOXB9 in AGS OR cells, and control cells to find the effect of the continuous increase of HOXB9 in the gastric cancer drug-resistant cell line. We performed sequencing analysis on AGS HOXB9 cells and control cells, overexpression of HOXB9 in AGS OR and knockdown of HOXB9 in AGS OR and control drug-resistant cell lines, compared the gene expression differences between AGS OR cells and parental AGS PC cells, and found that the expression of related molecules in the ECM-receptor interaction, Focal adhesion, Cell adhesion molecules gene subset in AGS OR cells was increased. The related molecules in the Focal adhesion, ECM-receptor interaction subset in AGS HOXB9 cells were also activated compared with the control cells Figure 4 A). The intersection analysis of the differentially expressed genes of the two found that there were 2497 differentially expressed genes simultaneously. Further intersection analysis of the up-regulated differentially expressed genes of the two found that there were 880 differentially expressed genes simultaneously up-regulated Figure 4 B). KEGG enrichment analysis of the co-expressed genes up-regulated found that these up-regulated genes were significantly enriched in the Focal adhesion pathwayFigure 4 C). We also verified that HOXB9 expression in gastric cancer cells can regulate the expression of various focal adhesion signaling molecules. And the expression of focal adhesion signaling molecules is also up-regulated in gastric cancer drug-resistant cell lines. And after knocking down HOXB9 in gastric cancer drug-resistant cell lines, these focal adhesion signaling pathway molecules in the cells were also significantly down-regulated Figure 4 D). The key molecule in the focal adhesion signaling pathway is in the form of phosphorylated FAK. Therefore, we detected the expression level of FAK and p-FAK protein. The results showed that the expression of p-FAK in AGS HOXB9 cells was significantly increased, and the expression of HOXB9 in gastric cancer drug-resistant cell lines also significantly increased p-FAK Figure 4 E). And when HOXB9 in gastric cancer drug-resistant cell lines was knocked down, the expression of p-FAK was also inhibited Figure 4 F). Immunofluorescence detection of tumor tissue obtained from tumor-bearing mice found that the fluorescence intensity of p-FAK in the tumor tissue formed by AGS HOXB9 cells was significantly higher than that of the control group, and the expression of F-actin was also more than that of the control group. Similar phenomenon was also found in the tissues of mice after treatment Figure 4 G). At the same time, it was also found in cell detection that the fluorescence intensity of p-FAK in gastric cancer drug-resistant cell lines was higher than that of the parent cells before and after oxaliplatin treatment Figure 4 H). And we found that the fluorescence intensity of p-FAK in AGS HOXB9 cells was also significantly up-regulated compared with the control cells, and there was more abundant F-actin formation. After oxaliplatin treatment, p-FAK and F-actin in AGS HOXB9 cells remained at a higher level than the control cells Figure 4 I). Adhesion plaques can fix cytoskeletal proteins and stabilize cell morphological structure. In gastric cancer cells after oxaliplatin treatment, the cytoskeleton was destroyed, and the cells lost normal morphology. Gastric cancer cells overexpressing HOXB9 and gastric cancer drug-resistant cell lines showed more stable cytoskeletal structure and cell morphology. This also shows that HOXB9 in gastric cancer cells can regulate the focal adhesion signaling pathway and increase the expression of p-FAK, and stabilize the cytoskeletal morphology of cells after oxaliplatin treatment.

[0085] 5. Inhibition of p-FAK can enhance the sensitivity of gastric cancer cells with increased HOXB9 expression to oxaliplatin. We explored whether HOXB9 depends on the activation of p-FAK to play a role in oxaliplatin resistance. FAK inhibitor (Y15) combined with oxaliplatin was used to treat gastric cancer cells overexpressing HOXB9. Cloning experiments showed that after inhibiting FAK phosphorylation, the sensitivity of gastric cancer cells to oxaliplatin was increased, and there was no significant difference with the control group. The drug resistance of gastric cancer cells overexpressing HOXB9 treated with oxaliplatin alone was inhibited Figure 5A, B). In addition to the change in sensitivity of gastric cancer cells under the condition of continuous treatment with oxaliplatin drugs, we considered whether it was effective under the condition of 24h treatment. Flow cytometry detection found that the sensitivity of gastric cancer cells to oxaliplatin was improved after treatment with FAK inhibitors, and the drug resistance of gastric cancer cells overexpressing HOXB9 was also inhibited (Fig. 4A, B). Figure 5 B, C). Detection found that the activation of p-FAK in gastric cancer cells overexpressing HOXB9 further activated the PI3K / AKT signal, and the expression of p-AKT was significantly increased in gastric cancer cells overexpressing HOXB9 (Fig. 5B, C). Figure 5 D). While the content of p-AKT in gastric cancer drug-resistant cell lines was significantly reduced after knocking down HOXB9 (Fig. 5D). Figure 5 E). In addition, after adding FAK inhibitors to gastric cancer cells overexpressing HOXB9, the increased expression of p-AKT in cells was inhibited (Fig. 5E). Figure 5 F). This also indicates that the expression of HOXB9 in gastric cancer cells upregulates the expression of p-FAK, further upregulates p-AKT, and activates the PI3K / AKT signaling pathway, thereby increasing the drug resistance of gastric cancer cells to oxaliplatin treatment.

[0086] 6. Gastric cancer organoid model shows that HOXB9 affects the survival of gastric cancer organoids, and targeting HOXB9 can improve the sensitivity of oxaliplatin-resistant gastric cancer organoids to oxaliplatin. We successfully established 6 cases of gastric cancer organoid models, including 2 cases of gastric cancer organoids derived from gastric cancer tissue samples obtained after oxaliplatin treatment. After successful primary culture, the mature gastric cancer organoids were obtained by subculture, and optical microscopy was used for observation imaging. Combined with HE staining, the source gastric cancer tissue samples and the corresponding gastric cancer organoids were preliminarily identified (Fig. 6A). Figure 6 A). At the same time, we further identified these gastric cancer organoids by immunohistochemical staining of ki67 (Fig. 6B). Figure 6 B). Then, STR genotyping was performed. We detected the expression of HOXB9 at the RNA level in these gastric cancer organoids and found that the expression level of HOXB9 in gastric cancer organoids treated with drugs was higher than that in untreated gastric cancer organoids (Fig. 7C). Figure 6 C). We detected the IC50 of oxaliplatin in these gastric cancer organoids, and the results showed that the IC50 of gastric cancer organoids treated with drugs was significantly increased (Fig. 7D). Figure 6 D). We constructed gastric cancer organoids overexpressing HOXB9 in P1 and P4, and performed oxaliplatin IC50 determination, and found that the sensitivity of gastric cancer organoids overexpressing HOXB9 to oxaliplatin was reduced (Fig. 7D). Figure 6E, G). To consider whether the effect of continuous drug treatment is also affected, we performed continuous oxaliplatin treatment on gastric cancer organoids for 2 weeks, and found that gastric cancer cells overexpressing HOXB9 still remained after continuous treatment, and were transformed from the original cystic organoids into solid organoids (F, H). After cell viability assay, it was also verified that the cell survival rate of gastric cancer organoids overexpressing HOXB9 was higher than that of the control group (G, I). Figure 6 F, H) After cell viability assay, it was also verified that the cell survival rate of gastric cancer organoids overexpressing HOXB9 was higher than that of the control group (G, I). Figure 6 I). In addition, we constructed an HOXB9 knockdown organoid model for PC-P1, PC-P2 drug-treated gastric cancer organoids, and performed oxaliplatin IC50 determination, and the results showed that the sensitivity of the organoids after HOXB9 knockdown to oxaliplatin was enhanced (J, L). Figure 6 J, L). After continuous oxaliplatin treatment for 2 weeks, it was found that the control organoids remained more after continuous treatment, and some tended to be solid organoids, while the gastric cancer organoids after HOXB9 knockdown were more easily decomposed and dispersed by the drug (K, M). Figure 6 K, M). After cell viability assay, it was also verified that the cell survival rate of gastric cancer organoids after HOXB9 knockdown was reduced (N). Figure 6 N). The tumor organoid model is more similar to the actual state of human tumors, and the above phenomena also show that the expression change of HOXB9 affects the sensitivity of gastric cancer tumor tissue to oxaliplatin, and inhibiting HOXB9 can significantly improve the sensitivity of gastric cancer tissue to oxaliplatin, and targeting HOXB9 for gastric cancer tissue that has developed oxaliplatin resistance can effectively reduce its resistance to oxaliplatin, thereby providing a new treatment strategy for gastric cancer treatment and gastric cancer chemotherapy resistance.

[0087] The above only describes the preferred embodiments of the present application and is not used to limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a HOXB9 expression inhibitor in the preparation of a drug for reversing gastric cancer oxaliplatin drug resistance.

2. Use according to claim 1, characterized in that, The HOXB9 inhibitor is a molecule that inhibits the expression or function of the HOXB9 gene.

3. Use according to claim 2, characterized in that, The HOXB9 inhibitor is a nucleic acid molecule, which is an shRNA targeting the HOXB9 gene.

4. Use according to claim 3, characterized in that, The target sequence of the shRNA is selected from the group consisting of: SEQ ID NO: 1: GGCAAAGAGTAAAGATTAA; SEQ ID NO: 2: GGCTAGAAAGTACAAGAAA.

5. A composition for reversing gastric cancer oxaliplatin drug resistance, comprising oxaliplatin and the HOXB9 inhibitor of any one of claims 1-4.

6. The pharmaceutical composition of claim 5, wherein The HOXB9 expression inhibitor is an shRNA lentivirus vector constructed from the target sequence shown as SEQ ID NO: 1 or SEQ ID NO:

2.

7. A gastric cancer oxaliplatin drug resistance detection kit, characterized in that: Detecting the expression level of HOXB9 in the sample, and high expression of HOXB9 indicates a risk of drug resistance.

8. The method of claim 7, wherein, The sample is gastric cancer tissue, blood or organoid model.

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