Application of IGFBP7 in preparation of medicine for treating gastric adenocarcinoma

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to application of IGFBP7 in preparation of a medicine for treating gastric adenocarcinoma. IGFBP7 closely related to gastric adenocarcinoma is screened out as a molecular marker through multiple bioinformatics technologies, expression of IGFBP7 in gastric adenocarcinoma is detected through immunohistochemistry, and prognosis of a patient is evaluated by establishing a scoring system combining dyeing intensity and positive cell proportion. Experimental results show that IGFBP7 knock-down can effectively inhibit proliferation and migration of gastric adenocarcinoma cells, induce apoptosis and trigger S-phase arrest. Furthermore, a co-culture experiment of gastric adenocarcinoma cells and mononuclear cells reveals that IGFBP7 can promote polarization of M2 type macrophages in a gastric adenocarcinoma tumor immune microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, and in particular relates to the application of IGFBP7 in the preparation of drugs for the treatment of gastric adenocarcinoma. Background Technology

[0002] Gastric adenocarcinoma is one of the most common malignant tumors of the digestive system, ranking among the leading causes of death from malignant tumors, with persistently high incidence and mortality rates. Despite continuous advancements in surgical, radiotherapy, and chemotherapy treatments, the overall prognosis for patients with gastric adenocarcinoma remains unsatisfactory, with high rates of recurrence and metastasis. Therefore, the search for novel molecular biomarkers that can be used to assess patient prognosis and guide treatment is of significant clinical importance.

[0003] IGFBP7 is a low-affinity insulin-like growth factor (IGF) conjugate expressed in both epithelial and mesenchymal tumors. Previous studies have shown that IGFBP7 plays an important role in the development and progression of tumors such as malignant melanoma, glioma, and thyroid cancer. However, research on its expression patterns and prognostic impact in gastric adenocarcinoma is still in its early stages, especially regarding its mechanism of action in gastric adenocarcinoma, which has not yet been systematically elucidated. Summary of the Invention

[0004] In view of the problems and shortcomings of the existing technology, the present invention aims to provide the application of IGFBP7 in the preparation of drugs for the treatment of gastric adenocarcinoma. The IGFBP7 inhibition strategy provided by the present invention has good effect in the treatment of gastric adenocarcinoma, and provides a new direction for the prognostic assessment of gastric adenocarcinoma and the development of immune-related targeted drugs.

[0005] This invention is achieved through the following technical solution: The application of siRNA sequences specifically targeting the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3, in the preparation of drugs for the treatment of gastric adenocarcinoma.

[0006] The amino acid sequence of IGFBP7 is as follows: MERPSLRALLLGAAGLLLLLLPLSSSSDTCGPCEPASCPPLPPLGCLLGETRDACGCCPMCARGEGEPCGGGGAGRGYCAPGMECVKSRKRRKGKAGAAAGGPGVSGVCVCKSRYPVCGSDGTTYPSGCQLRAASQRAESR GEKAITQVSKGTCEQGPSIVTPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKRGHYGVQRTELLPGDRDNLAIQTRGGPEKHEVTGWVLVSPLSKEDAGEYECHASNSQGQASASAKITVVDALHEIPVKKGEGAEL (SEQ ID No.1).

[0007] siIGFBP7-1: (5′-3′)UGGUAUCUCCUCUAAGUAATT (SEQ ID No. 2); siIGFBP7-2: (5′-3′)CGAGCAAGGUCCUUCCAUATT (SEQ ID No. 3).

[0008] Furthermore, the drug inhibits tumor growth by suppressing IGFBP7 expression.

[0009] Furthermore, the drug inhibits the proliferation and migration of gastric adenocarcinoma tumors by inhibiting IGFBP7 expression.

[0010] Furthermore, the drug induces S-phase arrest in tumor cells by inhibiting IGFBP7 expression.

[0011] Furthermore, the drug inhibits tumor-associated M2 macrophage polarization by suppressing IGFBP7 expression.

[0012] The present invention also provides the use of siRNA sequences specifically targeting the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3, in the preparation of gastric adenocarcinoma detection kits.

[0013] Furthermore, the kit also includes rabbit polyclonal anti-IGFBP7 antibody, HRP-labeled goat anti-rabbit secondary antibody, and DAB chromogenic reagent.

[0014] The present invention also provides a medicament for treating gastric adenocarcinoma, wherein the medicament contains a sequence of siRNA specifically targeting the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3.

[0015] Beneficial effects:

[0016] (1) This invention uses a combination of bioinformatics methods to screen for the protein IGFBP7, which is closely related to the prognosis of gastric adenocarcinoma. Immunohistochemical staining was used to detect its expression in tumor tissues of 93 patients with gastric adenocarcinoma. The results showed that IGFBP7 was highly expressed in gastric adenocarcinoma tissues, and its expression level was significantly correlated with patient prognosis. Patients with high IGFBP7 expression had significantly worse prognoses than those with low expression, indicating that IGFBP7 has potential prognostic value. A scoring standard can be established using immunohistochemical IHC, allowing IGFBP7 to be used as a new prognostic biomarker.

[0017] (2) This invention demonstrates through in vitro cell experiments that knocking down IGFBP7 expression can effectively inhibit the proliferation and migration of gastric adenocarcinoma cells, induce apoptosis, and arrest the cell cycle in the S phase. This reveals the potential of IGFBP7 as a therapeutic target, and inhibiting its expression can directly produce an anti-tumor effect.

[0018] (3) The co-culture experiment further revealed that IGFBP7 can promote the polarization of tumor-associated macrophages to the M2 type, indicating that it participates in the regulation of the tumor immune microenvironment. Therefore, inhibiting IGFBP7 expression can not only directly inhibit tumor cells, but may also exert a synergistic anti-tumor effect by remodeling the immune microenvironment (inhibiting M2 polarization).

[0019] This invention is the first to discover that IGFBP7 protein is highly expressed in gastric adenocarcinoma tissues and is associated with patient clinical prognosis. It proposes an IGFBP7 typing method based on the immunohistochemical IHC scoring system, filling a gap in the field of gastric adenocarcinoma stratification assessment. This invention utilizes siRNA technology to effectively knock down IGFBP7 expression, significantly inhibiting gastric adenocarcinoma cell proliferation and inducing apoptosis and cell cycle arrest. Furthermore, this invention uses siRNA technology to overexpress IGFBP7 in gastric adenocarcinoma cells and co-cultures them with macrophages, promoting macrophage polarization towards the M2 morphology, indicating a close relationship between IGFBP7 and the tumor immune microenvironment and validating its therapeutic potential.

[0020] The IGFBP7 inhibition strategy provided by this invention has good efficacy and safety in the treatment of gastric adenocarcinoma. IGFBP7 has the dual value of being a prognostic biomarker and a precision therapeutic target, providing a new direction for the prognostic assessment of gastric adenocarcinoma and immuno-targeted therapy, and has broad clinical application prospects. Attached Figure Description

[0021] Figure 1 This is a graph showing the differential expression of the IGFBP7 gene in pan-cancer tissues based on the TIMER2.0 and GEPIA2 databases in an embodiment of the present invention.

[0022] Figure 2This embodiment of the invention uses a volcano plot to perform differential gene analysis based on transcriptome information downloaded from the TCGA database.

[0023] Figure 3 This is a Power value curve of the WGCNA selection threshold in an embodiment of the present invention;

[0024] Figure 4 This is a gene clustering tree diagram of the WGCNA partitioning module in this embodiment of the invention;

[0025] Figure 5 This is a heatmap of inter-module correlations after WGCNA gene clustering in an embodiment of the present invention;

[0026] Figure 6 This is a Venn diagram of TCGA differentially expressed genes, GEO differentially expressed genes, and WGCNA key module genes in an embodiment of the present invention.

[0027] Figure 7 This is a bar chart showing the expression of IGFBP7 in gastric adenocarcinoma tissue and normal tissue based on the TCGA and GEO databases in this embodiment of the invention.

[0028] Figure 8 These are immunohistochemical staining images and scoring statistics of IGFBP7 protein expression in gastric adenocarcinoma tissue in this embodiment of the invention; wherein, A is HE staining image of normal gastric mucosa tissue (hematoxylin-eosin); B is immunohistochemical image of IGFBP7 in normal gastric mucosa; C is HE staining image of gastric adenocarcinoma tissue; D is immunohistochemical image of IGFBP7 in gastric adenocarcinoma.

[0029] Figure 9 This is a survival curve diagram of IGFBP7 protein H-Score grouping based on IHC in an embodiment of the present invention;

[0030] Figure 10 This is a graph showing the mRNA and protein expression levels of gastric adenocarcinoma cells after IGFBP7 knockdown, detected by real-time quantitative polymerase chain reaction and Western blotting in an embodiment of the present invention.

[0031] Figure 11 This is a graph showing the effect of CCK-8 assay on the proliferation ability of gastric adenocarcinoma cells after IGFBP7 knockdown in an embodiment of the present invention.

[0032] Figure 12 This is a graph showing the results of the scratch test in this invention to detect the effect of IGFBP7 knockdown on the migration ability of gastric adenocarcinoma cells;

[0033] Figure 13 This is a flow cytometry diagram showing the effect of IGFBP7 knockdown on the cell cycle distribution of gastric adenocarcinoma cells in an embodiment of the present invention.

[0034] Figure 14 This is a flow cytometry diagram of the effect of IGFBP7 knockdown on apoptosis-inducing effects in gastric adenocarcinoma cells in an embodiment of the present invention.

[0035] Figure 15 This is a graph showing the gene ontology enrichment analysis results of IGFBP7 in gastric adenocarcinoma in an embodiment of the present invention;

[0036] Figure 16 This is a diagram showing the results of the Kyoto Gene and Genome Encyclopedia analysis of IGFBP7 in gastric adenocarcinoma in this invention embodiment;

[0037] Figure 17 This is a correlation diagram of IGFBP7 expression and gastric adenocarcinoma immune microenvironment score analyzed based on the CIBERSORT algorithm in an embodiment of the present invention;

[0038] Figure 18 This is an immunohistochemical image of co-expression of CD163 and IGFBP7 proteins in gastric cancer tissue in an embodiment of the present invention; wherein, A is a region of gastric adenocarcinoma with low expression of IGFBP7; B is the expression of CD163 in M2 macrophages around the tumor in the same field of view; C is a region of gastric adenocarcinoma with high expression of IGFBP7; D is the expression of CD163 in M2 macrophages around the tumor in the same field of view.

[0039] Figure 19 This is a diagram showing the results of THP-1 cells being induced into M0 type macrophages by PMA treatment in an embodiment of the present invention.

[0040] Figure 20 This is a graph showing the results of real-time quantitative polymerase chain reaction detection of CD68 and CD14 mRNA expression levels in an embodiment of the present invention.

[0041] Figure 21 This is a Western blot diagram showing the expression level of IGFBP7 protein after transfection and overexpression of gastric adenocarcinoma cells in an embodiment of the present invention.

[0042] Figure 22 This is a flow cytometry result of the polarization state of monocytes and gastric adenocarcinoma cells after co-culturing, as described in this embodiment of the invention. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0044] Unless otherwise specified, the following examples and comparative examples are parallel experiments, using the same processing steps and parameters.

[0045] Example 1: Evaluation of the value of IGFBP7 in the prognosis of gastric adenocarcinoma based on bioinformatics analysis.

[0046] (1) Data sources: Transcriptome sequencing (RNA-Seq) data of gastric adenocarcinoma patients were downloaded from the Tumor Immunological Assessment Resource Bank Platform (TIMER2.0) and the Gene Expression Profiling Interaction Analysis Platform (GEPIA2). Transcriptome sequencing data and corresponding clinical information of gastric adenocarcinoma patients were downloaded from the TCGA database of the Cancer Genomics Data Analysis Platform (UCSC Xena platform). Transcriptome sequencing data and corresponding clinical information of gastric adenocarcinoma patients in the GSE13911 and GSE29272 datasets were downloaded from the GEO database.

[0047] (2) Data preprocessing, differential expression analysis, and weighted gene co-expression network analysis: The TCGA and GSE13911 expression matrices were read and annotated using R language (Rversion 4.4.3) (annotated according to Ensembl and GPL570 gene IDs), and normalization and differential expression analysis were performed using the DESeq2 package. Weighted co-expression network analysis and rapid hierarchical clustering were performed using the WGCNA and FlashClust packages to identify core genes in the gastric adenocarcinoma-related modules.

[0048] (3) Screening of core genes and validation of expression levels: The intersection of differentially expressed genes in the TCGA dataset, the GEO dataset, and the WGCNA clustered genes was used to screen out the gene with the largest differential expression level, IGFBP7. The expression values ​​of IGFBP7 gene in the tumor group and the normal group were extracted, and the expression differences in the test set TCGA, GSE13911 and the validation set GSE29272 were evaluated using the Wilcoxon Signed-Rank Test.

[0049] Figure 1 To analyze the differential expression of the IGFBP7 gene in pan-cancer tissues based on the TIMER2.0 and GEPIA2 databases; Figure 2 Volcano plot for differential gene analysis of transcriptome information downloaded from the TCGA database; Figure 3 Power value curve for selecting threshold for WGCNA; Figure 4 Gene clustering tree diagram for dividing WGCNA into modules; Figure 5 A heatmap showing the inter-module correlations after WGCNA gene clustering; Figure 6 Venn diagram of differentially expressed genes in TCGA, GEO, and key module genes in WGCNA; Figure 7 A bar chart showing the expression of IGFBP7 in gastric adenocarcinoma and normal tissues based on the TCGA and GEO databases. Figure 1As shown, IGFBP7 is highly expressed in cholangiocarcinoma, colon cancer, esophageal cancer, head and neck cancer, and gastric adenocarcinoma. (Combined with...) Figures 2 to 7 The analysis results showed that IGFBP7 expression was significantly upregulated in gastric adenocarcinoma tissues (P<0.001), indicating that it is a differentially expressed gene associated with gastric adenocarcinoma.

[0050] Table 1 lists the intersection genes (21 in total) of TCGA differentially expressed genes, GEO differentially expressed genes, and WGCNA key module genes. Among them, IGFBP7 has the highest logFC value and is therefore identified as a core gene for further research.

[0051] Table 1: 21 genes identified through combined differential gene selection and WGCNA screening.

[0052]

[0053] Example 2: Evaluation of the prognostic significance of IGFBP7 in clinical DLBCL samples using tissue microarray-based IHC detection.

[0054] (1) Sample source and tissue microarray preparation: Pathological specimens from 93 patients with radical gastric adenocarcinoma who underwent surgical resection and had complete clinical data between January 2015 and December 2022 were collected. The average age of the patients was (65.8±10.4) years. Through review of patient medical records and telephone follow-ups, it was found that none of the patients had received any systemic treatment at the time of diagnosis. After routine dewaxing and graded alcohol dehydration, the paraffin tissues from the above 93 cases were prepared into tissue microarrays according to tissue microarray technology. The diameter of the sampling core was 1.5 mm to ensure that each sample contained at least two duplicate cores.

[0055] (2) IHC staining and marker detection: IGFBP7 protein detection: The expression of IGFBP7 protein in gastric adenocarcinoma tissue samples was detected by immunohistochemistry (IHC) for prognostic assessment of gastric adenocarcinoma. The optimal conditions were used (primary antibody: rabbit polyclonal anti-IGFBP7 antibody from Proteintech, 1:2000 dilution; secondary antibody: HRP-labeled goat anti-rabbit secondary antibody from Proteintech, 1:500 dilution). The staining was performed according to the steps of dewaxing, antigen retrieval, blocking, primary antibody incubation (4℃ overnight), secondary antibody incubation (37℃, 45 min), DAB staining and counterstaining. IGFBP7 staining was mainly located in the cytoplasm of tumor cells.

[0056] The kits used for immunohistochemical testing include:

[0057] (1) Rabbit polyclonal anti-IGFBP7 antibody;

[0058] (2) HRP-labeled goat anti-rabbit secondary antibody;

[0059] (3) DAB colorimetric reagent.

[0060] Immunohistochemical detection methods include the following steps:

[0061] Dewaxing: Immerse the sections sequentially in xylene (10 min), anhydrous ethanol (5 min), 95% ethanol (5 min), and 70% ethanol (5 min); Antigen retrieval: Place the sections in Tris EDTA antigen retrieval solution (pH 9.0) and heat in a 95°C water bath for 15 min; Blocking: Wash three times with phosphate-buffered saline (PBS) (5 min each time), add 1% bovine serum albumin (BSA) and block at room temperature for 2 h; Primary antibody incubation: Add IGFBP7 antibody (1:2000 dilution, 50 μL), incubate overnight at 4°C, and the next day remove the sections and incubate at room temperature for 45 min; Secondary antibody incubation: Wash three times with PBS, add secondary antibody (1:500 dilution, 50 μL)... μL), incubate at 37℃ for 45 min; color development and counterstaining: wash 3 times with PBS, incubate at room temperature for 10 min with 1×DAB color development solution, wash 3 times with PBS, then counterstain with hematoxylin, differentiate with hydrochloric acid and ethanol, blue back, clear, mount, air dry at room temperature overnight, and take pictures the next day.

[0062] (3) Scoring Method and Statistical Analysis: The immunohistochemical staining results were scored using the H-Score method. The criteria for judging staining intensity were: no staining was scored as 0 points, light yellow staining as 1 point, brownish-yellow staining as 2 points, and brownish-red staining as 3 points. For the percentage of positive cells, 5 fields of view were randomly selected under high magnification (400x), and 100 cells were counted in each field of view. The average percentage of positive cells was calculated. Finally, the H-Score was calculated and statistically analyzed according to the formula H-Score = (percentage of light yellow stained cells × 1) + (percentage of brownish-yellow stained cells × 2) + (percentage of brownish-red stained cells × 3). The immunohistochemical staining results were scored by 4 pathologists with associate senior professional titles or above, and the average value was calculated and statistically analyzed. The overall survival (OS) difference between the IGFBP7 high and low expression groups was compared using the Log-rank test (Mantel-Cox test).

[0063] Figure 8 The results of IHC staining of IGFBP7 protein in tissue microarrays from 93 patients with gastric adenocarcinoma and the statistical bar chart based on H-Score are shown. Figure 9 Survival curves after grouping based on IHC-based IGFBP7 protein H-Score scores.

[0064] Figure 8 The results showed that IGFBP7 was mainly strongly positive for cytoplasmic localization; Figure 9The study showed that patients in the high expression group had significantly worse survival than those in the low expression group, leading to the conclusion that IGFBP7 classification based on IHC can effectively differentiate prognoses.

[0065] Table 2 shows the baseline relationship between IGFBP7 expression and clinicopathological parameters in patients with gastric adenocarcinoma.

[0066] Table 2: Relationship between IGFBP7 expression and clinicopathological parameters in patients with gastric adenocarcinoma

[0067]

[0068] As shown in Table 2, high expression of IGFBP7 was associated with higher T stage, N stage, M stage and pathological grade, suggesting that its expression level is positively correlated with the degree of tumor malignancy.

[0069] Table 3 shows the univariate and multivariate Cox regression analysis of IGFBP7 and patient prognosis in the embodiments of the present invention.

[0070] Table 3: Univariate and Multivariate Cox Regression Analysis Tables of IGFBP7 and Patient Prognosis

[0071]

[0072] Table 3 shows that high expression of IGFBP7, T3-4 stage, N2-3 stage, and M1 stage are independent risk factors for 5-year survival after radical resection of gastric adenocarcinoma.

[0073] Example 3: Knockdown of IGFBP7 based on transient siRNA transfection and detection of knockdown efficiency.

[0074] (1) Cell collection and transfection: One day before transfection, MKN-45 cells and human gastric adenocarcinoma cells (AGS) in logarithmic growth phase were seeded in 6-well plates to ensure that the cell density was maintained at 70%~90% at transfection. 250 μL of Opti-MEM was added to a microcentrifuge tube, followed by 10 μL of Lipo2000. The mixture was incubated at room temperature for 5 minutes. 250 μL of Opti-MEM was added to another microcentrifuge tube, followed by 2 μg of siRNA. The mixture was gently mixed and incubated for 5 minutes. Lipo2000 dilution was added to DNA dilution, gently mixed, and incubated at room temperature for 30 minutes to form a DNA-Lipo2000 complex. After 48 hours of culture, the knockdown and overexpression efficiencies were detected by real-time quantitative PCR and Western blotting. The target sequence of the IGFBP7 specific small interfering RNA (siRNA) is:

[0075] siIGFBP7-1: (5′-3′)UGGUAUCUCCUCUAAGUAATT (SEQ ID No. 2);

[0076] siIGFBP7-2: (5′-3′)CGAGCAAGGUCCUUCCAUATT (SEQ ID No. 3).

[0077] (2) Total RNA extraction: 1-5 × 10⁻⁵ RNA was harvested 48 h after transfection. 7 For each cell, add 1 mL of total RNA extraction reagent (TRIzol reagent), lyse at room temperature, and repeatedly pipette to control the volume. Incubate on ice for 5 min. Add 0.2 mL of chloroform, vortex vigorously for 15-30 s, and incubate on ice for 2-3 min. Centrifuge at 12000 g for 15 min at 4 °C, transfer the supernatant to a new tube, add 0.5 mL of isopropanol, and incubate at room temperature for 10 min. Centrifuge at 12000 g for 10 min at 4 °C, discard the supernatant, wash with 1 mL of 75% ice-cold ethanol, and centrifuge at 12000 g for 5 min at 4 °C. After discarding the supernatant, briefly centrifuge at room temperature and discard any remaining liquid. Add 20 µL of DEPC water to dissolve the RNA, incubate at 65 °C for 10-15 min, and then store at -80 °C.

[0078] (3) RNA reverse transcription: Prepare the reverse transcription reaction system on ice. Add 1 µg of RNA sample, 3 µL of 5×gDNA digester mix, and nuclease-free water to a final volume of 15 µL. Incubate at 42 °C for 2 min to remove gDNA. Add reverse transcription premix (containing thermostable reverse transcriptase, reaction buffer, dNTPs, and Mg2+) to the tube, vortex to mix, and centrifuge at low speed. Place the reaction tube in a PCR instrument and set the program as follows: 25 °C for 5 min, 55 °C for 15 min, and 85 °C for 5 min to obtain cDNA.

[0079] (4) Real-time quantitative PCR (qRT-PCR): Prepare a 20 µL reaction system on ice, including 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 1 µL of cDNA template, 10 µL of premix (SYBR Green Mix), and nuclease-free water to a final volume of 20 µL. The program was set as follows: 95℃ for 30 s; 40 cycles: 95℃ for 5 s, 60℃ for 30 s; melting curve stage. Each group was set up in triplicate, and the relative expression was calculated using the 2−ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.

[0080] The primers involved in this application are as follows:

[0081] IGFBP7 F: 5′-CGAGCAAGGTCCTTCCATAGT-3′ (SEQ ID No. 4);

[0082] IGFBP7 R: 5′-GGTGTCGGGATTCCGATGAC-3′ (SEQ ID No. 5);

[0083] GAPDH F: 5′-GGAGCGAGATCCCTCCAAAAT-3′ (SEQ ID No. 6);

[0084] GAPDH R: 5′-GGCTGTTGTCATACTTCTCATGG-3′ (SEQ ID No. 7).

[0085] (5) Protein extraction: 48 h after transfection, collect the cell suspension, centrifuge at 1000 rpm for 3 min at 4 °C, and discard the culture medium. Wash three times with pre-cooled PBS at 4 °C and discard the supernatant. Add 1 mL of RIPA lysis buffer containing 10 µL of protease inhibitor mixture, mix thoroughly, and lyse on ice for 30 min, vortexing gently every 10 min. Centrifuge at 12000 rpm for 10 min at 4 °C, collect the supernatant into a new tube, and store at -80 °C for later use.

[0086] (6) Western Blot Detection: After mixing the protein sample with an equal volume of loading buffer, boil at 95°C for 5 min. Place the prepared gel plate vertically onto the electrophoresis rack, add fresh electrophoresis buffer to the electrophoresis tank, and slowly add the sample using a pipette. Electrophoresis is first performed at a constant voltage of 80V until the protein bands enter the separating gel, then continued at 120V. Immediately after electrophoresis, transfer is performed. Remove the protein gel, place the gel and a 0.45μm polyvinylidene fluoride (PVDF) membrane between filter paper moistened with buffer, forming a transfer sandwich. Place the sandwich in the transfer tank, add transfer buffer, and place the tank on an ice box. Transfer at a constant current of 200mA for 120 min. After transfer, remove the PVDF membrane, add an appropriate amount of 5% skim milk powder, and gently shake at room temperature for 1 h. Subsequently, rabbit polyclonal anti-IGFBP7 antibody is diluted 1:2000 and incubated overnight at 4°C on a shaker. The following day, the membrane was washed three times with TBST (Tris-Borate-Sodium Tween-20 buffer), 5 min each time. The secondary antibody was incubated with shaking at room temperature for 1 h. The membrane was then washed three times with TBST, 5 min each time. ECL chemiluminescence buffer was prepared and uniformly added onto the membrane. After the reaction was carried out in the dark, the membrane was exposed and images were acquired using a chemiluminescence imaging system.

[0087] Figure 10 The image shows a qRT-PCR bar graph of IGFBP7 mRNA levels after transfection with siIGFBP7-1 and siIGFBP7-2, and the results of Western blot analysis of IGFBP7 protein expression. Figure 10As shown, siIGFBP7-1 and siIGFBP7-2 can significantly knock down IGFBP7 mRNA expression (P<0.001); the corresponding protein levels also decreased accordingly, leading to the conclusion that the selected siRNAs can effectively inhibit IGFBP7 expression.

[0088] Example 4: Detection of the effect of IGFBP7 knockdown on gastric adenocarcinoma cell function based on CCK-8 assay, cell scratch assay and flow cytometry.

[0089] (1) CCK-8 assay for cell proliferation activity: Gastric adenocarcinoma cells in logarithmic growth phase and IGFBP7 knockdown cells were thoroughly mixed and centrifuged at 4 ℃ and 1000 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with PBS, resuspended in complete culture medium, and the cell density was adjusted to 1×10⁻⁶. 5 Cells / mL. 100 µL of cell suspension was seeded into 96-well plates; 100 µL of pure culture medium was added to the blank wells. The plates were incubated at 37°C and 5% CO2 for 24, 48, and 72 h. Afterward, the 96-well plates were removed, and 10 µL of CCK-8 reagent was added to each well for 2 h in the dark. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0090] (2) Cell scratch assay to detect cell migration ability: Gastric adenocarcinoma cells and IGFBP7 knockdown cells were seeded in 6-well plates (5×10⁻⁶ cells / wells). 5 Cells / mL were cultured for 24 hours. Using a sterilized pipette tip, horizontal lines were drawn perpendicular to the scratches on the back of a 6-well plate, one line every 3 cm, three lines per well. The culture medium was aspirated, the plate was washed three times with PBS, and the 6-well plate was placed in a 5% CO2 incubator at 37°C for 24 hours. The wound healing rate was observed in five randomly selected fields of view under a 10x magnified microscope.

[0091] (3) Flow cytometry analysis of cell cycle distribution: Cells in the logarithmic growth phase were centrifuged to remove the culture medium, and then washed with pre-chilled PBS. The precipitate was resuspended in 200 μL of PBS, and the cells were added to 1 mL of pre-chilled 70% ethanol (on ice). The cells were gently mixed and fixed overnight at 20°C. The next day, the cells were centrifuged at 400×g for 5 min, the supernatant was discarded, 1 mL of pre-chilled PBS was added, the cells were resuspended, centrifuged at 400×g for 5 min, the supernatant was discarded, and the cells were dispersed to avoid clumping. PI staining solution (50 μg / mL PI + 50 μg / mL RNAase) was added to a 500 μL system for staining, and the cells were incubated at 37°C in the dark for 30 min, mixing once during the staining process. After staining, the cells were stored at 4°C or on ice in the dark. The excitation wavelength of PI was 488 nm, and the maximum emission wavelength was 617 nm.

[0092] (4) Flow cytometry detection of apoptosis: Cells in the logarithmic growth phase were collected, centrifuged to remove the culture medium, and then washed with pre-cooled PBS. An appropriate amount of 1× binding buffer was added to adjust the cell density to 1× 10⁻⁶. 6 / mL, take 200μL of cell suspension (2× 10 5 (Number of cells) were placed in flow cytometry tubes, and appropriate amounts of annexin V labeled with fluorescent dye and 7-AAD were added. The mixture was gently stirred and incubated at room temperature in the dark for 15–20 min. Cells were resuspended in 300 μL of PBS and analyzed by flow cytometry within 1 hour. The excitation wavelengths for FITC were 488 nm (maximum emission wavelength 525 nm), PI, APC, and 7-AAD were 488 nm (maximum emission wavelength 617 nm), APC, and 7-AAD, respectively.

[0093] Figure 11 The graph shows the proliferative activity of IGFBP7 knockdown cells as detected by the CCK-8 assay. Figure 11 As shown, the cell survival rate after 24 h of treatment with siIGFBP7-1 and siIGFBP7-2 was significantly lower than that of the control group (P<0.001). Figure 12 The figure shows the results of a scratch assay to detect the effect of IGFBP7 knockdown on the migration ability of gastric adenocarcinoma cells, as shown in the figure. Figure 12 As shown, the cell migration ability after treatment with siIGFBP7-1 and siIGFBP7-2 was significantly lower than that of the control group; Figure 13 Flow cytometry images showing the effect of knocking down IGFBP7 on cell cycle distribution in gastric adenocarcinoma cells, such as... Figure 13 The proportion of tumor cells in the S phase was significantly increased, leading to the conclusion that IGFBP7 inhibition can induce cell cycle S phase arrest. Figure 14 Image of apoptotic cells detected by flow cytometry using annexin V / 7-AAD. Figure 14 The results showed that the apoptosis rate of cells in both groups was significantly increased after treatment with siIGFBP7-1 and siIGFBP7-2, leading to the conclusion that IGFBP7 knockdown can effectively inhibit the proliferation of gastric adenocarcinoma cells and induce apoptosis.

[0094] Example 5: Detection of the association between IGFBP7 and the immune microenvironment of gastric adenocarcinoma based on GO, KEGG enrichment analysis and immune infiltration analysis.

[0095] (1) Gene Ontology Analysis (GO Analysis): GO analysis was performed using the clusterProfiler package in R (R version 4.4.3). First, the bitr function was used to convert gene IDs. Then, the enrichGO function was called to perform hypergeometric distribution tests on the gene list. Enrichment analysis was performed on the three major ontology categories of biological processes, cellular components, and molecular functions. The p-values ​​were corrected using the BH method, and significantly enriched items (p-value < 0.05) were screened. Finally, the ggplot2 function was used to visualize the results.

[0096] (2) Kyoto Encyclopedia of Genes and Genomes Encyclopedia (KEGG analysis): KEGG analysis was performed using the clusterProfiler package in R (R version 4.4.3). First, the bitr function was used to convert gene IDs, and then the enrichKEGG function was called to perform metabolic pathway tests on the gene list. The genes were compared with the pathways in the KEGG database to identify significant gene enrichment. Finally, the ggplot2 function was used to visualize the results.

[0097] (3) CIBERSORT immune infiltration analysis: The expression matrix in TPM format was read using R language (R version 4.4.3), and then the CIBERSORT package was used for analysis. The cor function was then called to calculate the relationship between immune cells, and finally ggplot2 and pheatmap were used for visualization.

[0098] (4) IHC detection of the correlation between IGFBP7 and M2 macrophages: Immunohistochemical staining was used to detect the correlation between IGFBP7 expression and CD163, a marker of M2 macrophages, in gastric adenocarcinoma tissue. The optimal conditions were used (primary antibody: rabbit polyclonal anti-CD163 antibody from Dako, 1:2000 dilution; secondary antibody: goat anti-rabbit secondary antibody labeled with HRP from Proteintech, 1:500 dilution), and the staining was performed according to the steps of dewaxing, antigen retrieval, blocking, primary antibody incubation (4℃ overnight), secondary antibody incubation (37℃, 45 min), DAB staining and counterstaining.

[0099] Figure 15 The image shows the GO analysis results of IGFBP7 in gastric adenocarcinoma, as follows. Figure 15 As shown, the results indicate that IGFBP7 is significantly associated with extracellular matrix synthesis, spindle microtubule and centromere attachment, and mitochondrial gene expression pathways in gastric adenocarcinoma.

[0100] Figure 16 The image shows the KEGG analysis results of IGFBP7 in gastric adenocarcinoma, as follows. Figure 16As shown, the results indicate that IGFBP7 is associated with the muscle cytoskeleton, neuroactive ligand-receptor interactions, and the cAMP signaling pathway in gastric adenocarcinoma.

[0101] Figure 17 This is a correlation map of IGFBP7 with M2 macrophages in the immune microenvironment of gastric adenocarcinoma analyzed using the CIBERSORT algorithm in this invention. Figure 17 As shown, the results indicate that IGFBP7 is positively correlated with M2 tumor-associated macrophage infiltration.

[0102] Figure 18 This is an immunohistochemical image of co-expression of CD163 and IGFBP7 proteins in gastric cancer tissue in an embodiment of the present invention; wherein, A represents a gastric adenocarcinoma region with low IGFBP7 expression; B represents CD163 expression in M2 macrophages surrounding the tumor in the same field of view; C represents a gastric adenocarcinoma region with high IGFBP7 expression; D represents CD163 expression in M2 macrophages surrounding the tumor in the same field of view; CD163 is a marker of M2 tumor-associated macrophages, such as... Figure 18 As shown, the results indicated that CD163 immunohistochemistry of gastric adenocarcinoma patient samples showed a positive correlation between the level of M2 tumor-associated macrophage infiltration around the tumor and IGFBP7 expression.

[0103] Example 6:

[0104] (1) Human mononuclear cell line THP-1 was cultured in RPMI 1640 complete medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% penicillin-streptomycin antibiotics and 50 μM 2-mercaptoethanol, and incubated at 37°C in a 5% CO2 incubator. When the cell density reached about 80%, differentiation was induced. THP-1 cells were collected and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 100 ng / mL in 6-well plates. Prömböl ester (PMA) was added to a final concentration of 100 ng / mL, and the cells were treated for 48 h to induce differentiation into M0 macrophages. After induction, cell morphology changes were observed under a microscope, and cells were collected for qRT-PCR to detect the mRNA expression levels of macrophage markers CD68 and CD14 to verify the induction efficiency.

[0105] (2) Establishment of overexpression system by transient plasmid transfection: AGS cells in logarithmic growth phase were seeded in 6-well plates 24 h before transfection, so that the cell confluence reached 70%-80% on the day of transfection. Transfection was performed according to the instructions of the liposome transfection reagent: Take a 1.5 mL centrifuge tube and add 125 μL of Opti-MEM medium to dilute 2.5 μg of IGFBP7 overexpression plasmid (or empty vector control plasmid); take another tube and add 125 μL of Opti-MEM to dilute 5 μL of transfection reagent, and let stand at room temperature for 5 min. Add the diluted transfection reagent to the plasmid dilution solution, gently mix, and let stand at room temperature for 15-20 min to form a transfection complex. Aspirate the old medium from the 6-well plate and add 1.75 mL of fresh complete medium without antibiotics to each well. Add the transfection complex dropwise to the well and gently shake to mix. After culturing for 6 h, discard the medium containing the transfection reagent and replace it with 2 mL of fresh complete medium. After culturing for another 72 h, cell lysates were collected, and the expression level of IGFBP7 protein was detected by Western Blot to verify the overexpression efficiency.

[0106] (3) Detection of polarization status in co-culture of gastric adenocarcinoma and macrophages: A co-culture system was established using Transwell chambers. Induced M0 macrophages were seeded into the upper chamber of a Transwell chamber (1×10⁻⁶ cells / mL). 5 Cells / well). AGS cells (including the IGFBP7 overexpression group and the empty vector control group) transfected for 72 h were seeded in the lower chamber of a 6-well plate (2 × 10⁶ cells / well). 5 (cells / well). The upper chamber was placed above the lower chamber to form a non-contact co-culture system. After 48 h of co-culture, macrophages in the upper chamber were collected. After washing the cells with flow cytometry staining buffer, fluorescently labeled antibodies (CD206, CD163) were added, and the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed, resuspended in buffer, and analyzed by flow cytometry within 1 h to analyze the proportion of CD206 and CD163 positive cells and assess the degree of macrophage polarization towards the M2 type.

[0107] Figure 19 This is a diagram showing the results of inducing THP-1 cells into M0 type macrophages through PMA treatment in the invention. Figure 19 As shown, the cells changed from a suspended state to adherent growth, and their shape changed from round to flat and irregular. Granular substances appeared in the cytoplasm, and some cells extended pseudopodia, indicating that they began to differentiate into macrophages.

[0108] Figure 20 This is a graph showing the results of real-time quantitative polymerase chain reaction (qPCR) detection of CD68 and CD14 mRNA expression levels in this invention. Figure 20As shown, the expression levels of CD68 and CD14 mRNA in PMA cells increased after PMA treatment, indicating that the cells had differentiated into M0 type macrophages.

[0109] Figure 21 To detect the expression level of IGFBP7 protein after transfection and overexpression of gastric adenocarcinoma cells using Western blotting in this invention, such as... Figure 21 As shown, the expression level of IGFBP7 protein increased after AGS cells were transfected with plasmids.

[0110] Figure 22 This is a graph showing the results of flow cytometry analysis of macrophage polarization after co-culturing monocytes and gastric adenocarcinoma cells in this invention. Figure 22 The study showed that elevated IGFBP7 expression in gastric adenocarcinoma cells can promote M2 polarization of tumor-associated macrophages.

[0111] In summary, this invention provides a treatment method for gastric adenocarcinoma based on the IGFBP7 protein and its application. Through bioinformatics analysis and tissue microarray (IHC), IGFBP7 was identified for the first time as significantly overexpressed in gastric adenocarcinoma tissues, and its expression level was significantly correlated with overall patient survival, establishing a prognostic assessment method for IGFBP7-based gastric adenocarcinoma. Furthermore, this invention provides siRNA targeting IGFBP7 and its vector. Stable knockdown of IGFBP7 via in vitro siRNA transfection effectively inhibits gastric adenocarcinoma cell proliferation, induces apoptosis, and triggers S-phase arrest. In an in vitro co-culture model, elevated IGFBP7 levels in gastric adenocarcinoma cells promote M2 polarization in macrophages. Compared with existing technologies, the IGFBP7 in this invention possesses the dual value of being both a prognostic biomarker and a precise therapeutic target, providing a new tool for clinical stratification and personalized treatment of gastric adenocarcinoma, and opening new avenues for developing targeted intervention strategies based on tumor-associated macrophages in the tumor microenvironment.

[0112] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of siRNA sequences specifically targeting the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3, in the preparation of drugs for the treatment of gastric adenocarcinoma.

2. The application according to claim 1, characterized in that, The drug inhibits tumor growth by suppressing IGFBP7 expression.

3. The application according to claim 1, characterized in that, The drug inhibits the proliferation of gastric adenocarcinoma cells and induces apoptosis by suppressing IGFBP7 expression.

4. The application according to claim 1, characterized in that, The drug induces S-phase arrest in tumor cells by inhibiting IGFBP7 expression.

5. The application according to claim 1, characterized in that, The drug inhibits tumor-associated M2 macrophage polarization by suppressing IGFBP7 expression.

6. The application of siRNA sequences specifically targeting the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3, in the preparation of gastric adenocarcinoma detection kits.

7. The application according to claim 6, characterized in that, The kit also includes rabbit polyclonal anti-IGFBP7 antibody, HRP-labeled goat anti-rabbit secondary antibody, and DAB chromogenic reagent.

8. A drug for treating gastric adenocarcinoma, characterized in that, The drug contains a siRNA sequence that specifically targets the IGFBP7 gene, as shown in SEQ ID No. 2 or SEQ ID No. 3.

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