Application of ADAMTS2 related to gastric cancer

Abstract

The invention relates to application of ADAMTS2 related to gastric cancer. The application comprises an application of the ADAMTS2 inhibitor in preparation of drugs for preventing, relieving and / or treating gastric cancer, an application of a reagent for detecting ADAMTS2 and / or COL3A1 in preparation of a kit or system for predicting gastric cancer prognosis, and a system or a prognosis instrument for predicting gastric cancer prognosis. The metalloproteinase ADAMTS2 is found to be remarkably and highly expressed in gastric cancer tissues and is associated with peritoneal metastasis and tumor dryness. Therefore, ADAMTS2 can be used as a diagnosis and prognosis marker and a treatment target of cervical cancer, and has a huge application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of the metalloproteinase ADAMTS2 in relation to gastric cancer. Background Technology

[0002] Gastric cancer (GC), a highly heterogeneous and aggressive malignant tumor, remains a major challenge in global public health. Data shows that in 2020, there were 19.3 million new cancer cases worldwide, with gastric cancer accounting for 5.6%; cancer-related deaths reached 9.9 million, with gastric cancer accounting for 7.7%, making it the fourth leading cause of cancer death after lung, colorectal, and liver cancer. Despite significant progress in basic research on gastric cancer in recent years, with researchers systematically elucidating its genetic basis and discovering several important breakthroughs, including predictive biomarkers, the complex pathogenic mechanisms and relatively low clinical efficacy of gastric cancer still require further breakthroughs. Notably, although some biomarkers have been used as targeted therapy strategies for advanced gastric cancer, the overall treatment effect is still far from expected, highlighting the urgent need to explore therapeutic targets for advanced gastric cancer.

[0003] In summary, gastric cancer patients currently face widespread clinical challenges with poor prognosis. The main reason is that most cases are diagnosed at an advanced stage, and existing treatments have limited efficacy for advanced-stage patients, resulting in a persistently low overall survival (OS) globally. Current research focuses on two main directions: developing novel genetic biomarkers for early diagnosis and exploring more effective targeted therapy strategies. Summary of the Invention

[0004] By integrating GEO databases (GSE122796, GSE172159, GSE111556), the inventors discovered that ADAMTS2 is significantly overexpressed in gastric cancer tissues and is associated with peritoneal metastasis and tumor stemness. Transcriptome sequencing and Co-IP mass spectrometry analysis showed that knocking down ADAMTS2 led to the synergistic downregulation of ECM-related genes (such as COL3A1 and FN1), with COL3A1 being a core pivot gene. Survival analysis showed that patients with high COL3A1 expression had significantly shorter overall survival, and spatial transcriptome localization revealed that COL3A1 was specifically enriched at the tumor invasion front. Based on AlphaFold structure prediction and molecular docking simulation, the A1062-A1073 domains of ADAMTS2 were found to be closely related to its catalytic activity. In a nude mouse xenograft model, the ADAMTS2 knockout group showed a significant reduction in tumor mass, a significant slowdown in tumor growth rate, and a significant decrease in the incidence and extent of peritoneal metastasis. The above results indicate that ADAMTS2 catalyzes COL3A1 maturation, constructs the ECM microenvironment, synergistically maintains tumor stem cell characteristics, and drives metastasis; inhibiting ADAMTS2 gene expression can significantly slow down the tumor metastasis rate. Based on this, the present invention provides the use of ADAMTS2 in gastric cancer.

[0005] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides the use of ADAMTS2 inhibitors in the preparation of medicaments for the treatment of gastric cancer.

[0006] In conjunction with the first aspect of the present invention, in some embodiments, the ADAMTS2 inhibitor is a CRISPR-Cas9 plasmid carrying ADAMTS2-targeting sgRNA.

[0007] In conjunction with the first aspect of the present invention, in some embodiments, the sgRNA sequence targeting the ADAMTS2 gene is selected from any one of SEQ ID NO:1 to 3.

[0008] In conjunction with the first aspect of the present invention, in some embodiments, the ADAMTS2 inhibitor is a reagent that targets and blocks the activity of the ADAMTS2 enzyme, and it works by specifically binding to the A1062-A1073 domains of ADAMTS2.

[0009] In conjunction with the first aspect of the present invention, in some embodiments, the treatment of gastric cancer includes at least one of (1) to (4): (1) Reduces the proliferative capacity of gastric cancer cells; (2) Reduce the collagen content of the gastric tumor matrix; (3) Reduce the incidence of peritoneal metastasis in gastric cancer; (4) Reduce the extent of gastric cancer metastases.

[0010] In conjunction with the first aspect of the invention, in some embodiments, the drug further comprises a COL3A1 expression inhibitor.

[0011] In a second aspect, the present invention provides the use of reagents for detecting ADAMTS2 and / or COL3A1 in the preparation of kits or systems for predicting the prognosis of gastric cancer.

[0012] In conjunction with the second aspect of the invention, in some embodiments: the reagent for detecting ADAMTS2 and / or COL3A1 includes at least one of the following four reagents: (1) Reagents for detecting the endogenous expression level of ADAMTS2 mRNA in gastric cancer cell lines by RT-qPCR; (2) Reagents for detecting the protein expression level of ADAMTS2 in gastric cancer cell lines by Western blot; (3) Reagents for RT-qPCR detection of endogenous expression levels of COL3A1 mRNA in gastric cancer cell lines; (4) Reagents for Western blot detection of COL3A1 protein expression level in gastric cancer cell lines.

[0013] Thirdly, the present invention provides a gastric cancer risk assessment or prognosis system, comprising: The acquisition module is configured to acquire the expression level of the ADAMTS2 gene in the subject's biological sample; An assessment module is configured to assess the risk or prognosis of a subject having gastric cancer based on the acquisition results obtained by the acquisition module. The acquisition module and the evaluation module are connected wirelessly and / or via wired means.

[0014] Fourthly, the present invention provides a gastric cancer risk assessment or prognosis instrument, including the above-mentioned gastric cancer risk assessment or prognosis system.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention is the first to demonstrate that ADAMTS2 is significantly highly expressed in gastric cancer tissues and is closely related to peritoneal metastasis, tumor stemness, and patient prognosis, providing a novel specific molecular target for gastric cancer treatment. This invention establishes a gastric cancer prognostic assessment system based on the expression levels of ADAMTS2 and / or COL3A1. By detecting their mRNA and protein expression using RT-qPCR or Western blot, it can effectively predict patient survival and provide a reliable basis for clinical prognosis. Attached Figure Description

[0016] Figure 1Bioinformatics analysis of the association between ADAMTS2 and peritoneal metastasis and tumor stemness in gastric cancer. A. Differentially expressed genes in gastric cancer; B. Heatmap of the top 10 genes with high and low expression in gastric cancer; C. Venn diagram of GSE122796, GSE172159, and GSE111556; D. GO analysis of pathways associated with common genes; E. Venn diagram of pathways significantly associated with common genes. P <0.05;** P <0.01; *** P <0.001.

[0017] Figure 2 High expression of ADAMTS2 in gastric cancer is associated with pathological stage and prognosis. A. Pan-cancer analysis showed significantly high expression of ADAMTS2 in most cancers; B. Compared with adjacent normal tissue, ADAMTS2 was significantly more expressed in gastric cancer tissue; C. Pathological staging results showed a positive correlation between ADAMTS2 expression level and pathological stage; D. Survival analysis showed that patients with high ADAMTS2 expression had worse survival curves; E. RT-qPCR was used to detect the endogenous expression level of ADAMTS2 mRNA in gastric cancer cell lines; F. Western blot was used to detect the protein expression level of ADAMTS2 in gastric cancer cell lines; G. Western blot quantification. P <0.05;** P <0.01; *** P <0.001.

[0018] Figure 3 Constructing a stable ADAMTS2 knockout cell line. A. RT-qPCR was used to detect the knockout efficiency of ADAMTS2 in SGC-7901 and AGS cells; B. Western blot was used to detect the knockout efficiency of ADAMTS2 in SGC-7901 and AGS cells; C. Statistical analysis of the Western blot was performed using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0019] Figure 4 Knockdown of ADAMTS2 inhibits tumor stemness in gastric cancer cells; A. Knockdown of ADAMTS2 inhibits tumor stemness in gastric cancer cells (50 μm); B. Statistical analysis using ImageJ software. *P<0.05; **P<0.01; ***P<0.001.

[0020] Figure 5Knocking down ADAMTS2 inhibited the proliferation of gastric cancer cells; among which: A. Knocking down ADAMTS2 significantly reduced the number of ADAMTS2 colonies; B. Statistical quantification was performed using ImageJ software. *P<0.05; **P<0.01; ***P<0.001.

[0021] Figure 6 Knocking down ADAMTS2 inhibits the migration ability of gastric cancer cells; A. Knocking down ADAMTS2 significantly reduced the migration rate of gastric cancer cells (100 μm); B. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0022] Figure 7 Knocking down ADAMTS2 inhibits the migration ability of gastric cancer cells; A. Knocking down ADAMTS2 significantly reduces the number of cells that can migrate across the cell membrane (100 μm); B. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0023] Figure 8 : Knockdown of ADAMTS2 inhibits tumor growth in nude mice; A. Subcutaneous xenografts in nude mice; B. Subcutaneous xenograft tissue in nude mice, with tumor quality statistics on the right; C. Growth curve of subcutaneous xenografts in nude mice; D. Immunohistochemical staining of subcutaneous xenografts with ADAMTS2 (100 μm), with statistical quantification using ImageJ software on the right; E. Sirius red collagen fiber staining (100 μm), with statistical quantification using ImageJ software on the right. * P <0.05;** P <0.01; *** P <0.001.

[0024] Figure 9 : Injection of Daqu (a type of Chinese liquor) into the serosal membrane of the stomach in nude mice; A. Five weeks after serosal injection, the peritoneal cavity of the nude mice was opened to observe intra-abdominal tumor growth and metastasis; B. The rate of peritoneal metastasis was statistically analyzed. P <0.05;** P <0.01; *** P <0.001.

[0025] Figure 10Transcriptome sequencing analysis; including: A. Knockdown of gene expression in ADAMTS2 gastric cancer cells; B. Screening for genes with significant high and low expression (screening criteria: |log2FC|>2, p<0.05); C. Screening for genes with significant low expression and performing GO / KEGG pathway enrichment analysis; D. Significantly low expression of extracellular matrix-related pathways; E. Significant downregulation of extracellular matrix tissue pathway (NES=-1.3, P<0.05); F. Significant downregulation of extracellular matrix degradation pathway (NES=-1.66, P<0.001); G. Significant downregulation of cell-to-cell adhesion pathway via plasma membrane adhesion molecules (NES=-1.29, P<0.05); H. Membrane adhesion mediated by membrane adhesion molecules (NES=-1.55, P<0.001). P <0.05;** P <0.01; *** P <0.001.

[0026] Figure 11 GSEA regulatory pathway enrichment analysis; among which: A. Extracellular matrix-related pathways were significantly downregulated; B. Extracellular matrix tissue pathways were significantly downregulated (NES = -1.3, P < 0.05); C. Extracellular matrix degradation pathways were significantly downregulated (NES = -1.66, P < 0.001); D. Cell-to-cell adhesion pathways via plasma membrane adhesion molecules were significantly downregulated (NES = -1.29, P < 0.05); E. Membrane adhesion mediated by membrane adhesion molecules was downregulated (NES = -1.55, P < 0.001). P <0.05;** P <0.01; *** P <0.001.

[0027] Figure 12 The analysis included: Co-IP mass spectrometry; A. Co-IP mass spectrometry analysis of the PPI network; B. Venn diagram of Co-IP and RNA-seq; C. Western blot verification of the molecular interaction between ADAMTS2 and COL3A1; D. Patients with high COL3A1 expression had worse survival curves. P <0.05;** P <0.01; *** P <0.001.

[0028] Figure 13 Mapping analysis of COL3A1 to the spatial transcriptome database GSM6177607; where: A. Left side: clinical sample slices of gastric cancer; right side: mapping of genes significantly downregulated by transcriptome sequencing; B. Quantitative map of the mapping of genes significantly downregulated by transcriptome sequencing; C. Localization of COL3A1 in gastric cancer tissue. P <0.05;** P<0.01; *** P <0.001.

[0029] Figure 14 Immunofluorescence staining for ADAMTS2 knockdown; including: A. SGC-7901 cells Collagen III immunofluorescence staining; B. AGS cells Collagen III immunofluorescence staining; C. SGC-7901 cells Collagen I immunofluorescence staining; D. AGS cells Collagen I immunofluorescence staining; E. SGC-7901 cells Fibronectin immunofluorescence staining; F. AGS cells Fibronectin immunofluorescence staining. Scale bar in the figure: 50 μm; * P <0.05;** P <0.01; *** P <0.001.

[0030] Figure 15 : COL3A1 recovery efficiency detection; including: A. RT-qPCR detection of COL3A1 recovery efficiency; B. Western blot detection of COL3A1 recovery efficiency; C. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0031] Figure 16 The tumor stemness of COL3A1-overexpressing gastric cancer cells was restored; specifically: A. The volume of tumor spheroids formed by COL3A1-overexpressing gastric cancer cells in a suspension culture system was significantly increased (50 μm); B. Statistical quantification was performed using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0032] Figure 17 The proliferation ability of gastric cancer cells overexpressing COL3A1 was restored; among which: A. The number of COL3A1-overexpressing colonies was significantly increased; B. Statistical quantification was performed using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0033] Figure 18 COL3A1 overexpression significantly restored the migration ability of gastric cancer cells; specifically: A. COL3A1 overexpression significantly increased the migration rate of gastric cancer cells (100 μm); B. Statistical quantification using ImageJ software. P<0.05;** P <0.01; *** P <0.001.

[0034] Figure 19 The invasive ability of gastric cancer cells overexpressing COL3A1 was significantly restored; specifically: A. The number of transmembrane cells with COL3A1 overexpression was significantly increased (100 μm); B. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0035] Figure 20 Constructing ADAMTS2 mutants ADAMTS2-MutA1062-A1073 and ADAMTS2-MutA41-A48; A. AlphaFold resolution of the ADAMTS2 3D conformation; B. Molecular docking simulation of ADAMTS2 and COL3A1 using GARMM; C. Western blot detection of wild-type and mutant plasmid expression efficiency, and simultaneous detection of COL3A1 protein expression level; D. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0036] Figure 21 The A1062-A1073 domain mutation caused ADAMTS2 to lose its ability to promote tumor stemness in gastric cancer cells; among which: A. The volume of tumor spheroids in gastric cancer cells with the A1062-A1073 domain mutation did not increase significantly (50 μm); B. Statistical quantification plot using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0037] Figure 22 The A1062-A1073 domain mutation caused ADAMTS2 to lose its ability to promote the proliferation of gastric cancer cells; among which: A. The number of gastric cancer cell colonies formed by the A1062-A1073 domain mutation did not increase significantly; B. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0038] Figure 23A. The A1062-A1073 domain mutation caused ADAMTS2 to lose its ability to promote gastric cancer cell migration; among them, the migration rate of gastric cancer cells with the A1062-A1073 domain mutation did not increase significantly (100 μm); B. Statistical quantification plot using ImageJ software. P <0.05;** P <0.01; *** P <0.001.

[0039] Figure 24 The A1062-A1073 domain mutation caused ADAMTS2 to lose its ability to promote gastric cancer cell invasion; among which: A. The number of gastric cancer cells with the A1062-A1073 domain mutation did not significantly increase (100 μm); B. Statistical quantification using ImageJ software. P <0.05;** P <0.01; *** P <0.001. Detailed Implementation

[0040] This invention screens the differentially expressed gene ADAMTS2 in gastric cancer by integrating the GEO databases (GSE122796, GSE172159, GSE111556), and verifies the clinical relevance of ADAMTS2 by combining survival analysis and pathological staging. RT-qPCR and Western blot were used to detect ADAMTS2 expression in gastric cancer cell lines; stable ADAMTS2 knockout and COL3A1 overexpression cell models were constructed, and their effects on tumor stemness, proliferation, migration, and invasion were evaluated through tumor spheroid formation, colony formation, wound healing, and Transwell invasion assays. Nude mouse xenograft and peritoneal metastasis models were established to verify in vivo function, and the molecular mechanism of the ADAMTS2 / COL3A1 axis was elucidated through transcriptome sequencing, Co-IP mass spectrometry, immunofluorescence, and protein structure prediction.

[0041] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture, construction of stable transfected cell lines, total protein extraction, protein sample preparation, RNA extraction and reverse transcription, molecular biological detection (such as transcriptome sequencing, qPCR, Western blot), Transwell invasion assay, colony formation assay, wound healing assay, tumor spheroid formation assay, animal model construction, subcutaneous tumor formation assay in nude mice, peritoneal tumor formation assay in nude mice, immunohistochemistry, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met the generally accepted quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental groups and control groups were set up according to the grouping methods commonly used in the art.

[0042] Table 1 Cell line information table

[0043] The ADAMTS2 gene knockout lentiviral plasmid lentiCRISPRv2-Puro-sg-ADAMTS2 (sg-ADAMTS2), ADAMTS2 overexpression plasmid PLVX-Puro-ADAMTS2 (oe-ADAMTS2), COL3A1 overexpression plasmid PLVX-Puro-COL3A1 (oe-COL3A1), blank plasmid OE-NC, ADAMTS2 overexpression mutant plasmids PLVX-Puro-ADAMTS2-A41-A48 and PLVX-Puro-ADAMTS2-A1062-A1073 (ADAMTS2-MutA41-A48 and ADAMTS2-MutA1062-A1073) were all purchased from Qingke Biotechnology Co., Ltd., and their sequence information is shown in Table 2 below.

[0044] Table 2 Guide RNA Sequence Information Table

[0045] Escherichia coli DH5α competent cells were purchased from Thermo Fisher Scientific.

[0046] The wild-type BALB / c-Nude mice used in this invention were all obtained from Wuhan Shubaili Biotechnology Co., Ltd. The BALB / c-Nude mice were 6 weeks old and housed in an SPF-grade environment. All animal experiments have been approved by the Animal Ethics Committee of Wuhan University of Science and Technology.

[0047] Table 3 Reagent Information Table

[0048] Table 3 (continued)

[0049] Table 4 RT-qPCR Primer Sequence List

[0050] Example 1: Upregulation of ADAMTS2 expression in gastric cancer promotes gastric cancer progression Three datasets related to gastric cancer (GSE122796, GSE172159, and GSE111556) were downloaded from the GEO database. GSE122796 is a gastric cancer gene RNA sequencing dataset, GSE172159 is a peritoneal metastasis mRNA expression profile dataset, and GSE111556 focuses on the mutual reprogramming sequencing data between cancer cells and related mesenchymal stem cells in gastric cancer. First, differentially expressed gene sets in gastric cancer were obtained through differential analysis and plotted using a volcano plot (…). Figure 1 A) and heat map ( Figure 1 B) is presented, followed by an intersection analysis of the differential gene sets of the three datasets. Figure 1 C), ultimately identifying 446 common genes that were simultaneously associated with significantly differential expression in gastric cancer, peritoneal metastasis, and tumor stemness.

[0051] GO pathway analysis of 446 common genes showed that this common gene set was mainly enriched in extracellular matrix-related pathways. Figure 1 D). Further screening of five extracellular matrix-related pathways with significant differences was performed for intersection analysis. Figure 1 (E), ultimately identifying ADAMTS2 as the co-screening result. This result suggests that the ADAMTS2 gene may play a crucial role in the development and progression of gastric cancer, peritoneal metastasis, and maintenance of tumor stemness.

[0052] The expression characteristics and clinical relevance of the ADAMTS2 gene were analyzed using the GEPIA2 online platform. Pan-cancer analysis showed that ADAMTS2 expression was significantly upregulated in gastric cancer and most other cancer tissues. Figure 2 A). Analysis of gastric cancer tissue and adjacent normal tissue showed that ADAMTS2 expression was significantly higher in gastric cancer tissue than in adjacent normal tissue. Figure 2 B, P<0.001). Further pathological staging analysis revealed that ADAMTS2 expression levels gradually increased with the progression of gastric cancer pathological stages (B, P<0.001). Figure 2 C, P<0.001).

[0053] Survival analysis showed that high ADAMTS2 expression was significantly associated with poorer clinical prognosis in gastric cancer patients: compared with patients with low ADAMTS2 expression, patients with upregulated ADAMTS2 expression had poorer overall survival. Figure 2 D, P<0.05, suggesting that this gene may serve as a potential biomarker for gastric cancer prognosis. Next, the endogenous expression of ADAMTS2 was detected in gastric cancer cell models, and suitable cell models were selected to verify the effect of ADAMTS2 on gastric cancer progression. Using human gastric mucosal epithelial cells (GES-1) as a reference, RT-qPCR and Western blot experiments were used to detect the endogenous expression of ADAMTS2 in four gastric cancer cell models (SGC-7901, MGC-803, AGS, and HGC-27). Quantitative analysis showed that the mRNA level of ADAMTS2 in the SGC-7901 and AGS cell lines (…) was significantly lower than that in the HGC-7901 and AGS cell lines. Figure 2 E) and protein levels ( Figure 2 Both F and G were significantly overexpressed, therefore SGC-7901 and AGS cell lines were selected for subsequent validation experiments.

[0054] Example 2: Downregulation of ADAMTS2 expression inhibits tumor stemness, proliferation, migration, and invasion of gastric cancer cells. To clarify the functional role of ADAMTS2 in gastric cancer cells, this embodiment was validated through the following systematic experiments: First, wild-type SGC-7901 cells and wild-type AGS cells were transfected with the CRISPR-Cas9 plasmid carrying ADAMTS2-targeting sgRNA—sg-ADAMTS2. After selection for puromycin resistance, single clones were picked and analyzed by RT-qPCR. Figure 3 A) and Western blot ( Figure 3 B) Verify the knockout efficiency at both the mRNA and protein levels. Figure 3 C represents the quantitative analysis by Western blot. The results showed that ADAMTS2 expression was significantly inhibited in both cell lines, and the SGC-7901 and AGS cell lines with stable ADAMTS2 knockout were successfully obtained.

[0055] The regulatory effect of ADAMTS2 on the stemness of gastric cancer cells was evaluated using a tumor spheroid formation assay: the control group consisted of wild-type SGC-7901 cells and wild-type AGS cells, while the knockout group consisted of the aforementioned ADAMTS2 stably knocked-out SGC-7901 and AGS cell lines. The experimental results are as follows: Figure 4 As shown in Figure A, the quantitative analysis by ImageJ software shows ( Figure 4 B), compared with the control group, the tumor spheres formed by the knockout group cells in the suspension culture system were significantly smaller, suggesting that ADAMTS2 deficiency can effectively inhibit the tumor stemness of gastric cancer cells.

[0056] To further investigate the effects of ADAMTS2 on the proliferation, migration, and invasion of gastric cancer cells, three functional experiments were systematically conducted using stably knocked-out ADAMTS2 SGC-7901 and AGS cell lines: colony formation assay, wound healing assay, and Transwell invasion assay. The control group used wild-type SGC-7901 and wild-type AGS cells, while the knockout group used the aforementioned stably knocked-out ADAMTS2 SGC-7901 and AGS cell lines. The results showed that ADAMTS2 gene knockout significantly inhibited the malignant phenotype of gastric cancer cells. In the colony formation assay (… Figure 5 A), as shown by ImageJ software quantitative analysis ( Figure 5 (B) Compared with the control group, the knockout group showed a significant reduction in the number of clones formed by both SGC-7901 and AGS cells. Regarding cell migration ability, the wound healing assay results were as follows: Figure 6 A, as shown by ImageJ software quantitative analysis ( Figure 6 B), the scratch closure rate of ADAMTS2 knockout cells was significantly lower than that of the control group. Further analysis using Transwell assays (…) Figure 7 A) Assessing cell invasion ability, quantitative analysis using ImageJ software revealed ( Figure 7 (B) The number of transmembrane cells in the knockout group was significantly reduced compared to the control group, and the difference was statistically significant. These results preliminarily indicate that ADAMTS2 knockout inhibits tumor stemness, proliferation, migration, and invasion of gastric cancer cells.

[0057] Example 3: Knockdown of ADAMTS2 inhibited in vivo tumorigenesis and peritoneal metastasis in gastric cancer. To determine the effect of ADAMTS2 on tumor growth in gastric cancer cells, the knockout group used stably knocked-out SGC-7901 cells, and the control group used wild-type SGC-7901 cells. Cell suspensions in logarithmic growth phase (1×10⁻⁶ cells) were used. 7 Mix ( / mouse) with matrix gel at a volume ratio of 1.5:1 and inject into the groin area of ​​nude mice to construct a xenograft model. Figure 8 A). The results of subcutaneous transplant tumor morphology comparison showed that the knockout group had a significantly lower tumor mass compared to the control group ( Figure 8 B). During tumor formation in the nude mouse subcutaneous xenograft model, the size of the subcutaneous xenograft was measured and recorded using vernier calipers, and finally, a growth curve of the subcutaneous xenograft was plotted. Figure 8 (C) The results showed that the growth rate of subcutaneous xenografts in the knockout group was significantly lower than that in the control group, suggesting that ADAMTS2 downregulation inhibited the growth of gastric tumors. ADAMTS2 immunohistochemical staining showed ( Figure 8(D) Compared with the control group, the expression level of ADAMTS2 protein in the tumor tissue of the knockout group was significantly decreased, confirming the effectiveness of the in vivo model. Sirius red staining results showed that ( Figure 8 (E) Compared with the control group, the area of ​​collagen fiber deposition in tumor tissue was significantly reduced in the ADAMTS2 knockout group, indicating that ADAMTS2 affects the tumor microenvironment by regulating collagen synthesis. Knocking out the ADAMTS2 gene inhibits the progression of gastric cancer in two ways: firstly, it directly reduces the proliferative capacity of tumor cells, manifested as a decrease in tumor mass / volume, which is consistent with the experimental results of knocking down ADAMTS2 to inhibit the proliferation of gastric cancer cells; secondly, downregulation of ADAMTS2 leads to a decrease in collagen content in the collagen network of gastric tumor matrix, thereby inhibiting the growth of subcutaneous xenografts of gastric cancer.

[0058] Meanwhile, to verify the effect of ADAMTS2 on peritoneal metastasis of gastric cancer, the knockout group used SGC-7901 cells stably knocked out by ADAMTS2, while the control group used SGC-7901 cells that were not knocked out by ADAMTS2. Cells in the logarithmic growth phase were planted at a rate of 5 × 10⁻⁶ cells per cell line. 6 Injected subserosally into the stomach of nude mice. The nude mice were then dissected. Figure 9 A), statistical analysis revealed ( Figure 9 B), the knockout group not only had a significantly lower incidence of intraperitoneal metastasis compared to the control group, but also a significantly smaller spread range. This indicates that the ability of gastric cancer cells to proliferate and spread in the peritoneum was significantly inhibited after ADAMTS2 downregulation, suggesting that ADAMTS2 may be a potential target for the treatment of gastric cancer.

[0059] Example 4: High expression of COL3A1 in gastric cancer cells promotes the occurrence and development of gastric cancer. Transcriptome sequencing was performed on the SGC-7901 cell line with stable ADAMTS2 knockout, and differentially expressed genes such as... Figure 10 As shown in A (screening criteria: |log2FC|>2, p<0.05), the set of genes with significant downregulation was obtained through screening. Figure 10 B), GO / KEGG analysis showed that COL3A1 is a core hub gene (B), Figure 10 C). ADAMTS2 deficiency leads to synergistic downregulation of extracellular matrix-related pathways (C). Figure 11 A). GSEA analysis showed that four key pathways were inhibited, including extracellular matrix tissue (NES=-1.3, P<0.05). Figure 11 B) Extracellular matrix degradation (NES=-1.66, P<0.001, Figure 11 C) Cell-to-cell adhesion via plasma membrane adhesion molecules (NES = -1.29, P < 0.05) Figure 11 D) Membrane adhesion mediated by molecules (NES = -1.55, P < 0.001) Figure 11E).

[0060] Based on the potential functional link between ADAMTS2 and COL3A1, ADAMTS2 was immunoprecipitated using a FLAG-tagged mass spectrometer coupled with Co-IP mass spectrometry. The PPI network was identified by mass spectrometry. Figure 12 A, Transcriptome-mass spectrometry intersection display (Venn Figure 12 B), COL3A1 not only directly interacts with ADAMTS2, but the expression level of COL3A1 is also positively correlated with ADAMTS2. Western blot analysis of IP-ADAMTS2 and IP-COL3A1 showed that ADAMTS2 and COL3A1 physically bind to each other (B). Figure 12 C). Survival curves for COL3A1 were analyzed using the GEPIA2 database. The results showed that patients with high COL3A1 expression had a significantly lower overall survival compared to patients with low COL3A1 expression (HR=1.9, p<0.01). Figure 12 D).

[0061] Spatial localization analysis of COL3A1 was performed using the publicly available gastric cancer spatial transcriptome sequencing database GSM6177607. The mapping of the gastric cancer spatial transcriptome sequencing database showed that COL3A1 is specifically highly expressed at the tumor invasion front. Figure 13 A represents HE-stained sections from spatial transcriptome sequencing, with different colors used to display different parts of the tumor. Simultaneously, the major expression sites of genes in the low-expression groups selected from transcriptome sequencing are mapped onto the gastric cancer spatial transcriptome. Quantification of the mapped sites of each gene in the low-expression groups from transcriptome sequencing is shown below. Figure 13 As shown in Figure B, the mapping results of COL3A1 in the spatial transcriptome sequencing of gastric cancer show that the expression sites of COL3A1 are mainly concentrated in the fibroblasts of the outer layer of gastric cancer lesions. Figure 13 C).

[0062] Example 5: Knockdown of ADAMTS2 inhibits ECM remodeling in gastric cancer In immunofluorescence experiments, the knockout group used ADAMTS2 stably knocked-out SGC-7901 and AGS cells, while the control group used wild-type SGC-7901 and wild-type AGS cells. Three key biomarkers related to extracellular matrix (ECM) remodeling were detected. The results showed that, compared with the control group, the knockout group had significantly higher levels of type III collagen (Collagen III). Figure 14 A, Figure 14 B) Type I collagen (Collagen I; Figure 14 C, Figure 14 D) and fibronectin; Figure 14 E, Figure 14The expression levels of F) showed a significant decreasing trend. This result suggests that ADAMTS2 gene knockout can effectively inhibit ECM remodeling in gastric cancer cells. The loss of ADAMTS2 directly alters the composition of the tumor microenvironment, providing important experimental evidence for elucidating the regulatory mechanism of ADAMTS2 in gastric cancer development. Combined transcriptome data ( Figure 10 C) and protein interaction verification ( Figure 12 C) confirmed that ADAMTS2 deficiency leads to a decrease in the content of major ECM components (Collagen III, Collagen I, and Fibronectin), which in turn reduces matrix stiffness and disrupts the extracellular matrix of gastric tumor cells, inhibiting invasion and metastasis. Figure 13 The results of spatial transcriptome sequencing were consistent. Preliminary findings indicate that knockdown of ADAMTS2 inhibits extracellular matrix remodeling by directly regulating COL3A1 expression, thereby suppressing gastric cancer cell adhesion and matrix invasion, and ultimately inhibiting gastric cancer progression and peritoneal metastasis.

[0063] Example 6: Overexpression of COL3A1 can relieve the inhibitory effect of ADAMTS2 knockdown on tumor stemness, proliferation, migration, and invasion of gastric cancer cells. To further verify that ADAMTS2 participates in the process of gastric cell carcinoma by targeting COL3A1, stable transgenic cells overexpressing COL3A1 were constructed again in the previously constructed stable ADAMTS2 knockout SGC-7901 and AGS cells: The COL3A1 overexpression plasmid —oe-COL3A1 and the blank plasmid OE-NC were transfected into the stable ADAMTS2 knockout SGC-7901 and AGS cells, respectively. After selection by puromycin resistance, single clones were picked and analyzed by RT-qPCR. Figure 15 A) and Western blot ( Figure 15 B) Experimental verification, as shown by ImageJ software quantitative analysis, indicates that ( Figure 15 (C) Compared with SGC-7901 and AGS cells that stably knocked out ADAMTS2, the mRNA and protein levels of SGC-7901 and AGS cells that overexpressed COL3A1 and stably knocked out ADAMTS2 were significantly increased, indicating that the construction of stable cells overexpressing COL3A1 was successful.

[0064] To further verify the effects of the ADAMTS2 / COL3A1 regulatory axis on the in vitro tumor stemness, proliferation, migration, and invasion of gastric cancer cells, stable transfected cells were subjected to tumor spheroid formation assays, colony formation assays, wound healing assays, and Transwell assays. The sg ADAMTS2+oe nc group used SGC-7901 and AGS cells stably knocked out ADAMTS2 by transfection with the blank plasmid OE-NC, while the sg ADAMTS2+oe COL3A1 group used SGC-7901 and AGS cells stably knocked out ADAMTS2 by transfection with the oe-COL3A1 plasmid.

[0065] Results of tumor spheroid formation experiment as follows Figure 16 As shown in Figure A, the quantitative analysis by ImageJ software shows ( Figure 16 (B) Compared with the sgADAMTS2+oe nc group, the tumor spheres formed by cells in the sg ADAMTS2+oe COL3A1 group in the suspension culture system were significantly larger, suggesting that overexpression of COL3A1 in gastric cancer cells leading to excessive accumulation of type III collagen can relieve the inhibitory effect of knockdown of ADAMTS2 on tumor stemness in gastric cancer cells.

[0066] Results of the colony formation experiment are as follows Figure 17 As shown in Figure A, the quantitative analysis by ImageJ software shows ( Figure 17 (B) Compared with the sgADAMTS2+oe nc group, the number of clones formed in the sgADAMTS2+oe COL3A1 group was significantly increased, suggesting that overexpression of COL3A1 can relieve the inhibition of ADAMTS2 knockdown on the proliferation of gastric cancer cells. Wound healing experiment results are as follows: Figure 18 As shown in Figure A, the quantitative analysis by ImageJ software shows ( Figure 18 (B) Compared with the sg ADAMTS2+oe nc group, the migration rate of cells in the sg ADAMTS2+oeCOL3A1 group was significantly increased, suggesting that overexpression of COL3A1 can relieve the inhibition of ADAMTS2 knockdown on the migration ability of gastric cancer cells. Transwell assay results are as follows: Figure 19 As shown in Figure A, the quantitative analysis by ImageJ software shows ( Figure 19 (B) Compared with the sg ADAMTS2+oe nc group, the sg ADAMTS2+oe COL3A1 group showed a significant increase in the number of transmembrane cells, suggesting that COL3A1 overexpression can relieve the inhibition of ADAMTS2 knockdown on the invasive ability of gastric cancer cells. In conclusion, high expression of COL3A1 leads to excessive accumulation of type III collagen, promoting tumor stemness, proliferation, migration, and invasion of gastric cancer cells.

[0067] Example 7: Mutation of the A1062-A1073 domain inhibited the catalytic activity of ADAMTS2 on COL3A1. This embodiment uses AlphaFold protein structure prediction to resolve the three-dimensional spatial conformation of ADAMTS2. Figure 20 A, and the first molecular docking simulation of ADAMTS2 and COL3A1 was performed using GARMM (https: / / gramm.compbio.ku.edu / ), revealing the potential interaction mode between ADAMTS2 and COL3A1 proteins. Figure 20 B). Predictive analysis suggests that the A41-A48 and A1062-A1073 domains may be related to the catalytic activity of ADAMTS2.

[0068] To verify this hypothesis, the following experiment was conducted in this embodiment: The control group used the wild-type SGC-7901 cell line; Wild-type ADAMTS2-WT group: wild-type ADAMTS2 overexpression plasmid —oe-ADAMTS2 was transfected into wild-type SGC-7901 cell line; The mutant ADAMTS2-MutA41-A48 group: The ADAMTS2 overexpression plasmid with the A41-A48 domain mutant was transfected into the wild-type SGC-7901 cell line. Mutant ADAMTS2-MutA1062-A1073 group: ADAMTS2 overexpression plasmid with A1062-A1073 domain mutant in wild-type SGC-7901 cell line—ADAMTS2-MutA1062-A1073.

[0069] Transfection efficiency was verified at the mRNA and protein levels using RT-qPCR and Western blot, respectively, after transfection. Western blot results are shown below. Figure 20 As shown in C, the quantitative analysis by ImageJ software shows ( Figure 20(D) The overexpression efficiency of ADAMTS2 in the three stable expression cell lines was comparable and significantly higher than that of the wild-type SGC-7901 cell line, proving the successful construction of the three stable overexpression cell lines. However, the expression level of COL3A1 showed significant differences. Compared with the wild-type ADAMTS2-WT group, the COL3A1 protein level in the mutant ADAMTS2-MutA1062-A1073 group was significantly lower than that in the wild-type ADAMTS2-WT group, but there was no significant difference compared with the control group. The COL3A1 protein level in the mutant ADAMTS2-MutA41-A48 group was close to that in the wild-type ADAMTS2-WT group and significantly higher than that in the control group. This finding directly confirms that the A1062-A1073 domain is the key catalytic active site for ADAMTS2 to regulate COL3A1 protein metabolism, providing important experimental evidence for further analysis of the enzymatic activity mechanism of ADAMTS2.

[0070] Example 8: Mutations in the A1062-A1073 domains caused ADAMTS2 to lose its promoting effect on the stemness, proliferation, migration, and invasion of gastric cancer tumors. To verify the effects of A1062-A1073 domain mutations on the stemness, proliferation, migration, and invasion of gastric cancer tumors, stable wild-type and mutant SGC-7901 and AGS cells were subjected to tumor spheroid formation, colony formation, wound healing, and Transwell assays. Grouping criteria were the same as in Example 7.

[0071] The results of the tumor spheroid formation experiment showed that ( Figure 21 A) The tumor spheres formed by the mutant ADAMTS2-MutA1062-A1073 group in the suspension culture system were significantly smaller than those in the wild-type ADAMTS2-WT group, but there was no significant difference compared with the control group. Figure 21 B). The results of the colony formation experiment show that ( Figure 22 A), the number of colonies formed in the mutant ADAMTS2-MutA1062-A1073 group was significantly less than that in the wild-type ADAMTS2-WT group, but there was no significant difference in the number of colonies formed compared with the control group. Figure 22 B). The results of the wound healing experiment showed that ( Figure 23 A), the migration rate of the mutant ADAMTS2-MutA1062-A1073 group was significantly lower than that of the wild-type ADAMTS2-WT group, and there was no significant difference in migration rate between the two groups. Figure 23 B). Transwell experimental results show that ( Figure 24 A), the number of transmembrane cells in the mutant ADAMTS2-MutA1062-A1073 group was significantly lower than that in the wild-type ADAMTS2-WT group, but there was no significant difference in the number of transmembrane cells between the mutant and control groups. Figure 24(B) By comparing the functional differences of SGC-7901 and AGS cells in the wild-type ADAMTS2-WT group and the mutant ADAMTS2-MutA1062-A1073 group, this study reveals for the first time that the A1062-A1073 domain is the region affecting the catalytic activity of ADAMTS2, and its structural integrity directly affects the protein's oncogenic function. When this domain is mutated, the biological effects of ADAMTS2 in promoting tumor stemness maintenance, cell proliferation, migration, and invasion are specifically eliminated, indicating that this site may be a key molecular target for intervening in the malignant progression of gastric cancer.

[0072] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of ADAMTS2 inhibitors in the preparation of drugs for the treatment of gastric cancer.

2. The use according to claim 1, characterized in that: The ADAMTS2 inhibitor is a CRISPR-Cas9 plasmid carrying ADAMTS2-targeting sgRNA.

3. The use according to claim 2, characterized in that: The sgRNA sequence targeting the ADAMTS2 gene is selected from any one of SEQ ID NO:1~3.

4. The use according to claim 1, characterized in that: The ADAMTS2 inhibitor is a reagent that targets and blocks the activity of the ADAMTS2 enzyme. It works by specifically binding to the A1062-A1073 domains of ADAMTS2.

5. The use according to claim 1, characterized in that: The treatment of gastric cancer includes at least one of (1) to (4): (1) Reduces the proliferative capacity of gastric cancer cells; (2) Reduce the collagen content of the gastric tumor matrix; (3) Reduce the incidence of peritoneal metastasis in gastric cancer; (4) Reduce the extent of gastric cancer metastases.

6. The use according to claim 1, characterized in that: The drug also contains a COL3A1 expression inhibitor.

7. The use of reagents for detecting ADAMTS2 and / or COL3A1 in the preparation of kits or systems for predicting the prognosis of gastric cancer.

8. The application according to claim 7, characterized in that: The reagents used to detect ADAMTS2 and / or COL3A1 include at least one of the following four reagents: (1) Reagents for detecting the endogenous expression level of ADAMTS2 mRNA in gastric cancer cell lines by RT-qPCR; (2) Reagents for detecting the protein expression level of ADAMTS2 in gastric cancer cell lines by Western blot; (3) Reagents for RT-qPCR detection of endogenous expression levels of COL3A1 mRNA in gastric cancer cell lines; (4) Reagents for Western blot detection of COL3A1 protein expression level in gastric cancer cell lines.

9. A gastric cancer risk assessment or prognostic system, characterized in that, include: The acquisition module is configured to acquire the expression level of the ADAMTS2 gene in the subject's biological sample; An assessment module is configured to assess the risk or prognosis of a subject having gastric cancer based on the acquisition results obtained by the acquisition module. The acquisition module and the evaluation module are connected wirelessly and / or via wired means.

10. A gastric cancer risk assessment or prognostic instrument, characterized in that, Includes the gastric cancer risk assessment or prognosis system as described in claim 9.

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