Application of RECQL as a target in the preparation of products for the treatment and diagnosis of gastric cancer

Abstract

This application discloses the application of RECQL as a target in the preparation of products for the treatment and diagnosis of gastric cancer, belonging to the field of biomedical technology. Its key technical point lies in providing the application of RECQL as a target in the preparation of products for diagnosing the sensitivity of gastric cancer patients to oxaliplatin chemotherapy or for diagnosing the efficacy and prognosis of patients receiving oxaliplatin. This application, by detecting RECQL gene / protein expression, histone H1.2 binding status, and K637 site lactation level, can efficiently predict patient drug sensitivity, avoiding the toxic side effects and treatment delays of indiscriminate chemotherapy. Targeted methods such as downregulating RECQL activity, blocking related binding, or inhibiting lactation can effectively restore oxaliplatin-induced DNA damage, significantly reverse drug resistance, and improve chemotherapy efficacy. Simultaneously, the key target is clearly identified, facilitating new drug development, and the relevant effects have been verified through in vitro and in vivo experiments, inhibiting the proliferation of drug-resistant cells and reducing tumor volume, laying a solid foundation for clinical translation, and possessing both practical value and translational potential.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of RECQL as a target in the preparation of products for the treatment and diagnosis of gastric cancer. Background Technology

[0002] Gastric cancer (GC) is one of the most common malignant tumors of the digestive system. Due to the lack of obvious clinical symptoms in its early stages, most GC patients are diagnosed at an advanced stage. For advanced GC, platinum-based chemotherapy is a commonly used treatment. However, resistance to platinum-based drugs in advanced GC is a serious problem in current GC treatment, leading to poor patient prognosis. Therefore, elucidating the molecular mechanisms of platinum-based drug resistance in GC is crucial for developing new treatment strategies.

[0003] The mechanisms of chemotherapy resistance in tumors involve multiple aspects, including increased drug efflux, altered drug targets, enhanced DNA damage repair, senescence escape, epigenetic changes, tumor heterogeneity, changes in the tumor microenvironment, and epithelial-mesenchymal transition. Among these, platinum-based chemotherapy-induced DNA damage plays a crucial role in tumor treatment. DNA repair capacity significantly influences platinum resistance, and this capacity is determined by the expression levels of DNA damage and DNA repair-related genes and the activity of their encoded proteins. Currently, the specific molecular regulatory mechanisms of oxaliplatin resistance in gastric cancer (including potential key protein interaction pathways and post-translational modification regulation) remain unclear, and precise predictive biomarkers for drug sensitivity and effective targeted therapy strategies for reversing resistance are lacking. Summary of the Invention

[0004] The purpose of this application is to address the technical problems in the prior art, namely, the lack of a clear specific molecular regulatory mechanism for oxaliplatin resistance in gastric cancer, the absence of accurate predictive biomarkers for drug sensitivity, and the lack of effective targeted therapy strategies for reversing drug resistance.

[0005] To address the aforementioned technical problems, this application provides the following technical solution:

[0006] The application of RECQL as a target in the preparation of diagnostic products, which are used to diagnose the sensitivity of gastric cancer patients to oxaliplatin chemotherapy or to diagnose the efficacy and prognosis of patients receiving oxaliplatin.

[0007] Preferably, the diagnostic product assesses the sensitivity of gastric cancer patients to oxaliplatin chemotherapy by detecting the expression level of the RECQL gene / protein.

[0008] Preferably, the diagnostic product also includes other medically acceptable adjuvants.

[0009] This application also provides the application of RECQL as a target in the preparation of oxaliplatin resistance reversal products for gastric cancer, which restores the sensitivity of gastric cancer cells to oxaliplatin by downregulating the expression or activity of RECQL through gene silencing, protein degradation or functional inhibition.

[0010] Preferably, the RECQL is used in the preparation of oxaliplatin resistance reversal products for gastric cancer by binding to histone H1.2.

[0011] Preferably, the product also includes other medically acceptable adjuvants.

[0012] This application also provides the use of the combination of lactate inhibitors and oxaliplatin in the preparation of drugs for treating gastric cancer.

[0013] Compared with the prior art, this application has at least the following beneficial effects:

[0014] 1. This application can accurately determine the risk of oxaliplatin resistance in gastric cancer patients by detecting the expression level of RECQL gene / protein, the binding status of RECQL with histone H1.2, or the lactation level of RECQL K637 site, thus avoiding treatment delays and drug toxicity caused by blind chemotherapy and providing objective molecular basis for the selection of clinical chemotherapy regimens.

[0015] 2. By intervening in RECQL expression / activity, blocking RECQL binding to histone H1.2, inhibiting RECQLK637 lactation, or reducing lactate accumulation, the DNA damage effect induced by oxaliplatin can be restored, significantly improving the sensitivity of drug-resistant gastric cancer cells to oxaliplatin and improving the efficacy of chemotherapy.

[0016] 3. Based on the detection results of the above molecular markers, gastric cancer patients can be stratified to develop targeted combination therapy plans (such as oxaliplatin + RECQL inhibitor, oxaliplatin + lactate metabolism inhibitor, etc.) for drug resistance risk groups with high RECQL expression, positive RECQL binding to H1.2, or positive K637 site lactation, thereby improving the precision and individualization of treatment.

[0017] 4. This application clarifies the binding process of RECQL and histone H1.2, and the lactation modification of RECQL K637 site as a key regulatory node for oxaliplatin resistance in gastric cancer. This provides a clear and verifiable target for the development of specific targeted drugs and has important clinical translational value. Attached Figure Description

[0018] Figure 1 RECQL bioinformatics analysis is associated with poor prognosis in gastric cancer;

[0019] A. Expression of platinum resistance-related genes in 19 GEO datasets and TCGA-STAD dataset; B. Expression of RECQL in 19 GEO datasets and TCGA-STAD dataset; C. In the TCGA database, Kaplan-Meier survival curve analysis was used to explore the correlation between RECQL expression and overall survival (OS), disease-specific survival (DSS), disease-free survival (DFI), and progression-free survival (PFI) of gastric cancer patients.

[0020] Figure 2 RECQL is upregulated in gastric cancer tissues and is associated with poor prognosis;

[0021] A. Real-time quantitative polymerase chain reaction (qRT-PCR) detection of RECQL mRNA expression levels in chemotherapy-sensitive and drug-resistant gastric cancer tissues; B. Representative images of tissue microarrays (scale bar: 50 μm); C. Quantitative analysis of RECQL protein expression in gastric cancer tissues by immunohistochemistry; D. Relationship between RECQL expression in tissue microarrays and overall survival (OS) of gastric cancer patients; Univariate and multivariate Cox regression analyses of gastric cancer patients in EF tissue microarrays (*P<0.05, ***P<0.001).

[0022] Figure 3 RECQL enhances the resistance of gastric cancer cells to oxaliplatin;

[0023] AB, Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the protein and mRNA expression levels of RECQL in four gastric cancer cell lines (AGS, HGC27, SNU216, MKN45) and normal gastric mucosal cells (GES1); CD, qRT-PCR and WB were used to detect the transfection efficiency of RECQL small interfering RNA (siRNA); EF, qRT-PCR and WB were used to detect the transfection efficiency of RECQL overexpression; GH, Cell counting kit-8 (CCK8) assay was used to detect the effect of RECQL expression on the half-maximal inhibitory concentration (IC50) of oxaliplatin in gastric cancer cells; IJ, Clonogenic assay was used to detect the effect of RECQL expression on the sensitivity of gastric cancer cells to oxaliplatin (*P<0.05, **P<0.01, ***P<0.001, P>0.05).

[0024] Figure 4 RECQL reduces chemotherapy-induced DNA damage;

[0025] A. Volcano plot of changes in gene mRNA expression after RECQL knockdown; B. Gene set enrichment analysis (GSEA) based on transcriptome sequencing results; CF. Comet assay to detect the effect of RECQL on DNA damage.

[0026] Figure 5 RECQL affects the sensitivity of gastric cancer cells to oxaliplatin by binding to histone H1.2;

[0027] A. Immunoprecipitation (Co-IP) followed by mass spectrometry analysis to identify proteins binding to RECQL; B. Co-IP detection of the interaction between RECQL and histone H1.2; C. Immunofluorescence assay (IF) detection of the co-localization of RECQL and histone H1.2 in gastric cancer cells (scale bar: 50 μm); D. Western blotting detection of the effects of oxaliplatin and RECQL expression on histone H1.2 expression; E. Detection of oxaliplatin-induced DNA damage and oxaliplatin sensitivity in gastric cancer cells after overexpression of RECQL and / or knockdown of histone H1.2.

[0028] Figure 6 RECQL lactation modification promotes oxaliplatin resistance in gastric cancer cells;

[0029] A. Western blot (WB) detection of RECQL lactation modification level; B. Detection of DNA damage and sensitivity to oxaliplatin in gastric cancer cells after lactate treatment; F. Detection of RECQL lactation level and its binding ability to histone H1.2 after RECQLK637 site mutation; G. Detection of DNA damage and sensitivity to oxaliplatin in gastric cancer cells after overexpression of RECQL and RECQLK637R mutant.

[0030] Figure 7 The effects of RECQL expression and lactate on the sensitivity of gastric cancer cells to oxaliplatin in in vivo experiments;

[0031] A. Representative images of subcutaneous xenografts; B. Statistical analysis of the volume and weight of xenografts; C. Immunohistochemical (IHC) detection of the expression levels of RECQL, histone H1.2 and γ2HX in subcutaneous xenografts (scale bar: 50 μm) (*P<0.05, **P<0.01, ***P<0.001).

[0032] Figure 8 A schematic diagram illustrating how RECQL inhibits histone H1.2 degradation and promotes oxaliplatin resistance in gastric cancer. Detailed Implementation

[0033] This application provides the application of RECQL as a target in the preparation of diagnostic products, which are used to diagnose the sensitivity of gastric cancer patients to oxaliplatin chemotherapy. The sensitivity of gastric cancer patients to oxaliplatin chemotherapy is assessed by detecting the expression level of the RECQL gene / protein.

[0034] In another embodiment, the diagnostic product is used to diagnose the efficacy and prognosis of a patient receiving oxaliplatin.

[0035] The product also includes other medically acceptable adjuvants.

[0036] The application of RECQL as a target in the preparation of oxaliplatin resistance reversal products for gastric cancer involves downregulating the expression or activity of RECQL through gene silencing, protein degradation, or functional inhibition, thereby restoring the sensitivity of gastric cancer cells to oxaliplatin.

[0037] In one embodiment, the RECQL binding to histone H1.2 is used in the preparation of a product for reversing oxaliplatin resistance in gastric cancer.

[0038] In addition, this application also provides the use of the combination of lactate inhibitors and oxaliplatin in the preparation of drugs for treating gastric cancer.

[0039] The above content will be explained in conjunction with specific verification experiments:

[0040] I. Experimental Materials and Sources

[0041] 1. Bioinformatics Analysis

[0042] The expression and prognostic significance of the PRR gene were assessed using the Cancer Genome Atlas (TCGA) and multiple GEO microarray datasets (including GSE118897, GSE118916, GSE13195, GSE13861, GSE13911, GSE17154, GSE179252, GSE19826, GSE27342, GSE29272, GSE29998, GSE30727, GSE33335, GSE50710, GSE51575, GSE63089, GSE65801, GSE66229, and GSE79973). Survival analysis was performed using the survivalR package, and gene set enrichment analysis (GSEA) was conducted using the h.all.v2023.1.Hs.symbols.gmt gene set.

[0043] 2. Patient and tissue samples

[0044] This application collected cancerous and adjacent normal tissues from 200 patients with gastric cancer who underwent surgery at the Department of Gastrointestinal Surgery, Affiliated Hospital of Nantong University, between January and December 2015. Follow-up ended in December 2020, with a median follow-up time of 68 months and a range of 2–79 months. All patients were pathologically diagnosed with gastric cancer and had not received chemotherapy, radiotherapy, or targeted therapy prior to surgery. Before oxaliplatin chemotherapy, tumor tissue samples were obtained from 10 pairs of gastric cancer patients via gastroscopy to differentiate between oxaliplatin-resistant and oxaliplatin-sensitive groups. Treatment efficacy was assessed according to the Evaluation Criteria in Solid Tumor Treatment (RECIST). This study was approved by the Ethics Committee of Affiliated Hospital of Nantong University (Approval No.: 2023-K076-01), and all patients signed informed consent forms. This study strictly adhered to the ethical standards of the Declaration of Helsinki.

[0045] II. Verification Experiment

[0046] 1. Bioinformatics analysis showed that RECQL was associated with oxaliplatin resistance and poor prognosis in gastric cancer patients.

[0047] PRR genes were collected from the HGSOC-Platinum database and screened using specific inclusion and exclusion criteria to obtain 20 paired gastric cancer datasets (including 19 GEO datasets and the TCGA-STAD dataset) for PRR gene expression analysis. Results showed that compared with normal gastric mucosa tissue, 16 PRR genes were upregulated and 2 PRR genes were downregulated in gastric cancer tissue. Further analysis of the expression of these 18 genes and their correlation with overall survival (OS), disease-specific survival (DSS), disease-free survival (DFI), and progression-free survival (PFI) in gastric cancer patients revealed that ADAM17, NOTCH3, and RECQL were significantly associated with poor prognosis in gastric cancer patients. Figure 1 A).

[0048] In most of the selected datasets, RECQL expression was upregulated, and high RECQL expression was significantly associated with overall survival (OS), disease severity syndrome (DSS), and progression-free survival (PFI) in gastric cancer patients. Figure 1 B-1C). Although the difference was not statistically significant, patients with high RECQL expression had shorter DFI ( Figure 1 C).

[0049] 2. RECQL is upregulated in gastric cancer tissues and is associated with poor prognosis in gastric cancer.

[0050] Immunohistochemistry was used to detect RECQL expression in clinical samples. Results showed that RECQL expression was significantly higher in chemotherapy-resistant gastric cancer tissues than in chemotherapy-sensitive tissues. Figure 2 A). Meanwhile, RECQL expression was higher in 71% (142 / 200) of gastric cancer tissues than in normal gastric tissues ( Figure 2B-2C). Further analysis of the relationship between RECQL expression and clinicopathological features of gastric cancer patients revealed a positive correlation between RECQL expression and TNM stage (P<0.001) and depth of invasion (P<0.001) (Table 1). Gastric cancer patients with high RECQL expression had significantly shorter overall survival (OS). Figure 2 D). Furthermore, Cox multivariate analysis showed that high RECQL expression was an independent predictor of overall survival (OS). Figure 2 E-2F).

[0051] 3. RECQL enhances the resistance of gastric cancer cells to oxaliplatin.

[0052] To investigate the role of RECQL in platinum-based drug resistance in gastric cancer, the expression of RECQL in gastric cancer cell lines (HGC-27, MKN-45, AGS, SNU-216) and normal gastric mucosal cells (GES-1) was first detected by PCR. The results showed that RECQL expression was higher in AGS and MKN-45 gastric cancer cells than in normal gastric mucosal cells. Figure 3 A-3B). A RECQL-stable knockdown gastric cancer cell line was constructed using MKN-45 as a vector, and a RECQL-overexpressing gastric cancer cell line was constructed using HGC27 as a vector. Figure 3 C-3F). The effect of RECQL expression on oxaliplatin resistance in gastric cancer cells was verified using CCK8 and colony formation assays. Results showed that, compared with the control group, RECQL knockdown significantly reduced IC50 and inhibited proliferation; RECQL overexpression significantly increased IC50 and enhanced proliferation. Figure 3 G-3J).

[0053] 4. RECQL knockdown affects oxaliplatin resistance by inducing DNA damage.

[0054] To further elucidate the potential mechanism by which RECQL affects platinum-based drug resistance in gastric cancer, transcriptome sequencing was performed on gastric cancer cells with and without RECQL knockdown. The results showed that 173 genes were upregulated and 87 genes were downregulated after RECQL knockdown. Figure 4 A). Further GSEA enrichment analysis revealed that RECQL expression was significantly negatively correlated with "DNA double-strand break response" and "DNA double-strand break repair, recruitment of signaling proteins, and ATM-mediated phosphorylation," all of which are associated with DNA damage. Figure 4 B). Meanwhile, oxaliplatin exerts its anticancer effect by inducing DNA damage and apoptosis, while RECQL is associated with DNA damage. Therefore, RECQL may enhance oxaliplatin resistance by inhibiting oxaliplatin-induced DNA damage. Comet assays were performed on cells treated with oxaliplatin. The results showed that, compared with the control group, RECQL knockdown significantly prolonged the comet tail length, while RECQL overexpression significantly shortened the comet tail length. Figure 4 C-4D). Meanwhile, oxaliplatin treatment significantly increased γ2HX expression in the RECQL knockdown group, while significantly decreased γ2HX expression in the RECQL overexpression group. Figure 4 E-4F).

[0055] 5. RECQL induces DNA damage via histone H1.2.

[0056] To further investigate the mechanism by which RECQL knockdown leads to DNA damage, immunoprecipitation and mass spectrometry were used to identify proteins that bind to RECQL. The results showed that histone H1.2 can bind to RECQL. Figure 5 A-5C). Immunofluorescence assays also showed significant co-localization between RECQL and histone H1.2 (A-5C). Figure 5 D).

[0057] RECQL binds to histone H1.2, inhibiting H1.2 degradation and thus reducing DNA damage. The expression of histone H1.2 in gastric cancer cell lines treated with oxaliplatin was detected. The results showed that the expression level of histone H1.2 was significantly downregulated after oxaliplatin treatment, and this phenomenon was reversed after RECQL overexpression. Figure 5 E). Following RECQL overexpression, further knockdown of histone H1.2 reversed the comet tail shortening and γ2HX expression downregulation caused by RECQL overexpression. Figure 5 F-5I).

[0058] 6. RECQL lactation modification enhances RECQL-induced oxaliplatin resistance.

[0059] This application verified through specific validation experiments whether RECQL undergoes lactation modification and affects chemotherapy resistance in gastric cancer cells. Western blotting results showed that RECQL can undergo lactation modification (…). Figure 6 A). RECQL lactation modification enhances the binding of RECQL to histone H1.2 and inhibits histone H1.2 degradation ( Figure 6 B). Simultaneously, after RECQL undergoes lactation modification, DNA damage is further reduced, and drug resistance in gastric cancer cells is further enhanced. Figure 6 C-6E). Predicting the lactation site of RECQL using a website revealed that RECQL lactation modification may occur at the K637 site. Following the mutation at the RECQL K637 site, both the RECQL lactation level and its binding affinity to H1.2 were significantly reduced. Figure 6 F). Meanwhile, compared to RECQL overexpression, RECQLK637R overexpression did not significantly reduce DNA damage in gastric cancer cells or enhance oxaliplatin resistance. Figure 6 G-6J).

[0060] 7. In vivo experiments confirmed that RECQL induces DNA damage repair and enhances oxaliplatin resistance.

[0061] To investigate the effects of RECQL in vivo on cancer cell proliferation and oxaliplatin resistance, a cancer xenograft model was established and administered the drug via intraperitoneal injection. The results showed that, compared to the control group, the RECQL knockdown group had significantly smaller tumor size and weight, while the lactate treatment group had significantly larger tumor size and weight. Figure 7 (A-7C). Immunohistochemistry was used to detect the levels of γ2HX and histone H1.2 in tissues of each group. The results showed that the expression of γ2HX and histone H1.2 was significantly upregulated in the RECQL knockdown group, while the expression of γ2HX and histone H1.2 was significantly downregulated in the lactate treatment group. Figure 7 D).

[0062] III. Experimental Procedure

[0063] 1. Tissue microarray (TMA) and immunohistochemistry (IHC)

[0064] Tissue microarrays and immunohistochemical assays were performed according to established methods. Antibodies used included: anti-Ki67 antibody (28074-1-AP, Proteintech, China), anti-RECQL antibody (bs-19784R, Bioss, China), anti-histone H1.2 antibody (19649-1-AP, Proteintech, China), and anti-γ2HX antibody (bs-3185R, Bioss, China). Staining intensity was manually assessed by two experienced pathologists according to the following criteria: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Positive percentages were scored: 0 (<5%), 1 (5-25%), 2 (>25-50%), 3 (>50-75%), and 4 (>75%). The final immunohistochemical score was the product of the intensity score and the staining proportion score. Based on the scores, RECQL expression was categorized as high expression (score ≥3) and low expression (score ≤2).

[0065] 2. Cell Culture and Reagents

[0066] Human normal gastric mucosal cell line GES-1 was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.; human gastric cancer cell line MKN45 was purchased from Shanghai Beina Biotechnology Co., Ltd.; human gastric cancer cell lines HGC-27 and AGS were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai); and human gastric cancer cell line SNU216 was purchased from Guangzhou Saiku Biotechnology Co., Ltd. All cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. Oxaliplatin was purchased from Shanghai Selleck Biotechnology Co., Ltd.; and lactate was purchased from Sigma-Merck, Inc. (USA).

[0067] 3. Cell transfection

[0068] Cell transfection was performed according to the previously described method. RECQL and histone H1.2 small interfering RNA (siRNA) were purchased from Beijing Qingke Biotechnology Co., Ltd.; the RECQL overexpression plasmid was purchased from Suzhou Jima Gene Co., Ltd. Cells were transfected in 6-well plates using jetPRIME transfection reagent from Polyplus, France. After plasmid transfection, stable transfected cell lines were selected using G418 (Beijing TransGen Biotech Co., Ltd.).

[0069] 4. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0070] Total RNA was extracted and detected by qRT-PCR according to the previously described method. The primer sequences used are as follows: RECQL-F: 5'-CTCCGAGTTAAAGCTGATTTATGTG-3', RECQL-R: 5'-GGGAACTGCCGCTTTAAGA-3'; GAPDH-F: 5'-CATGTGGGCCATGAGGTCCACCAC-3', GAPDH-R: 5'-GGGAAGCTCACTGGCATGGCCTTCC-3'.

[0071] 5. Western blotting (WB)

[0072] Total protein extraction and Western blot analysis were performed according to the previously described methods. The antibodies used included: anti-RECQL antibody (bs-19784R, Bioss, China), anti-histone H1.2 antibody (19649-1-AP, Proteintech, China), anti-γ2HX antibody (bs-3185R, Bioss, China), and anti-GAPDH antibody (60004-1-Ig, Proteintech, China).

[0073] Cell Counting Kit-8 (CCK8)

[0074] 6. Following the previously described method, detect and calculate the half-maximal inhibitory concentration (IC50) using a CCK8 assay. Brief steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 5 Cells were seeded in 96-well plates and different concentrations of oxaliplatin were added. After culturing for 48 hours, CCK8 solution was added, and after incubation for 2 hours, the absorbance was measured.

[0075] 7. Cloning experiment

[0076] Perform the clonogenesis experiment as described previously. Use 1×10 3 One gastric cancer cell line was seeded into a 6-well plate, with 4 μg / mL oxaliplatin added to each well. After 2 weeks of culture, the cells were stained with crystal violet, fixed with 4% paraformaldehyde, photographed, and the clone count was performed using ImageJ software.

[0077] 8. Comet Experiment

[0078] According to the manufacturer's instructions, use OxiSelect TM Comet experiments were conducted using a 96-well comet experiment kit (Cell Biolabs, USA). Images were taken using a Leica inverted fluorescence microscope (USA), and tail length was measured using comet evaluation software (CaspLab).

[0079] 9. Immunofluorescence assay (IF)

[0080] Immunofluorescence detection was performed according to the methods previously used by our research group. After treatment, gastric cancer cells were washed with PBS and incubated overnight at 4°C with anti-RECQL antibody (bs-19784R, Bioss, China). After washing with PBS, ABflo647-labeled goat anti-rabbit IgG (H+L) (AS060, ABclonal, China) was added and incubated at 37°C for 2 hours. Subsequently, anti-histone H1.2 antibody (19649-1-AP, Proteintech, China) was added and incubated overnight at 4°C. After washing with PBS, ABflo488-labeled goat anti-rabbit IgG (H+L) (AS053, ABclonal, China) was added and incubated at 37°C for 2 hours. After DAPI staining of cell nuclei for 10 minutes, images of stained cells were captured using a Zeiss LSM900 confocal microscope.

[0081] 10. Co-immunoprecipitation (co-IP)

[0082] Immunoprecipitation experiments were performed as previously described. Total cell lysate was incubated overnight at 4°C with 1 μg of primary antibody or negative control rabbit IgG. Then, 20 μL of Protein A+G agarose (Bioworld Technology, St. Paul, Minnesota, USA) was added, and incubation continued at 4°C for 2 hours. The agarose beads were washed four times with PBS to remove protein-antibody complexes, and collected by centrifugation. Proteins were identified by SDS-PAGE and Western blotting.

[0083] 11. Animal experiments

[0084] Twenty four-week-old male nude mice were purchased from the Experimental Animal Center of Nantong University and randomly divided into four groups of five mice each. Each mouse was injected subcutaneously with 1×10- ... 6 One tumor cell was observed, and the tumor volume was recorded every 3 days. When the tumor volume reached approximately 50 mm... 3 Oxaliplatin and lactate were administered intraperitoneally every 3 days. Four weeks later, nude mice were euthanized by cervical dislocation under deep anesthesia (isoflurane overdose, 5% for 1 minute), and subcutaneous tumor tissue was removed. This experiment was approved by the Laboratory Animal Ethics Committee of Nantong University.

[0085] 12. Statistical Analysis

[0086] Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 9.0 software. The t-test was used for comparisons between experimental groups, and the Kaplan-Meier method was used for prognostic analysis. All experiments were repeated at least three times.

[0087] In summary, this application found that RECQL is highly expressed in gastric cancer, and that RECQL binding to histone H1.2 can reduce oxaliplatin-induced DNA damage, thereby increasing the resistance of gastric cancer cells to oxaliplatin. This provides a potential new target for gastric cancer treatment and may offer new insights into the selection of chemotherapy regimens for gastric cancer.

Claims

1. The application of RECQL as a target in the preparation of diagnostic products, characterized by: The diagnostic product is used to diagnose the sensitivity of gastric cancer patients to oxaliplatin chemotherapy or to diagnose the efficacy and prognosis of patients receiving oxaliplatin.

2. The application of RECQL as a target in the preparation of diagnostic products according to claim 1, characterized in that: The diagnostic product assesses the sensitivity of gastric cancer patients to oxaliplatin chemotherapy by detecting the expression level of the RECQL gene / protein.

3. The application of RECQL as a target in the preparation of diagnostic products according to claim 1, characterized in that: The diagnostic products also include other medically acceptable adjuvants.

4. The application of RECQL as a target in the preparation of oxaliplatin resistance reversal products for gastric cancer, characterized by: By downregulating the expression or activity of RECQL through gene silencing, protein degradation, or functional inhibition, the sensitivity of gastric cancer cells to oxaliplatin can be restored.

5. The application of RECQL as a target according to claim 4 in the preparation of oxaliplatin resistance reversal products for gastric cancer, characterized in that: The RECQL is used in the preparation of oxaliplatin resistance reversal products for gastric cancer by binding to histone H1.

2.

6. The application of RECQL as a target according to claim 4 in the preparation of oxaliplatin resistance reversal products for gastric cancer, characterized in that: The product also includes other medically acceptable adjuvants.

7. Application of the combination of lactate inhibitors and oxaliplatin in the preparation of drugs for treating gastric cancer.

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