Application of exosome miRNA as gastric cancer diagnostic marker and therapeutic target

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of exosome miRNA as a gastric cancer diagnosis marker and a treatment target. The regulation and control effects of circulating exosome miRNA in early gastric cancer diagnosis and malignant progression are systematically analyzed, and it is found that miR-412-5p is remarkably low in expression in early gastric cancer. The scheme of the invention further confirms that the nucleoplasm transporter gene XPO1 is a direct target gene of miR-412-5p, and activation of a PI3K-AKT pathway can be driven by expression imbalance of the nucleoplasm transporter gene XPO1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the use of an exosomal miRNA as a diagnostic marker and therapeutic target for gastric cancer. Background Technology

[0002] Gastric cancer (GC), a common malignant tumor, has a high incidence and mortality rate in my country. Currently, the main treatments for GC include surgery and chemotherapy. However, due to the low early diagnosis rate, many patients are already in advanced or locally advanced stages, or even have distant metastases, at the time of diagnosis, making traditional treatments ineffective. Although immunotherapy research has made many breakthroughs in recent years, its effectiveness in treating GC remains unsatisfactory. Therefore, finding new biomolecular markers or immunotherapy targets to improve clinical efficacy and survival rates for GC patients is crucial.

[0003] Exosomes (Exo) are small vesicles with a diameter of 30nm-150nm that can carry various biomolecules, including proteins, RNA, and lipids. The contents of tumor cell-derived exosomes exhibit good consistency with tumor cells themselves, serving not only as tumor detection markers but also reflecting the functional state of tumor cells. Therefore, they can be used for early tumor screening and are excellent biomarkers for the in vitro diagnosis of gastric cancer. However, traditional exosome extraction and identification procedures are cumbersome, and rapid and sensitive methods for detecting exosomal miRNAs are lacking. Furthermore, although the ability of exosomal miRNAs to influence the tumor microenvironment, patient treatment outcomes, and prognosis has been demonstrated, there is still a lack of identified gastric cancer-specific exosomal miRNAs. Therefore, the field anticipates the development of novel early diagnostic biomarkers based on gastric cancer-specific exosomal miRNAs, which would be of positive significance for the early diagnosis and regulation of malignant progression of gastric cancer. Summary of the Invention

[0004] The first objective of this invention is to provide an application of exosomal miRNA as a diagnostic biomarker for gastric cancer in the preparation of gastric cancer diagnostic biomarkers; A second objective of this invention is to provide a reagent for detecting exosomal miRNA expression levels for use in the preparation of gastric cancer diagnostic products; A third objective of this invention is to provide the use of exosomal miRNA as a target for the preparation of drugs for the prevention and / or treatment of gastric cancer; A fourth object of the present invention is to provide a substance overexpressing exosomal miRNA for use in the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0005] To address the aforementioned technical problems, this invention provides an application of exosomal miRNA as a diagnostic marker for gastric cancer in the preparation of gastric cancer diagnostic products, wherein the exosomal miRNA is miR-412-5p.

[0006] Specifically, the diagnosis of gastric cancer includes early screening or control of malignant progression of gastric cancer.

[0007] The present invention also provides a reagent for detecting the expression level of exosomal miRNA for use in the preparation of gastric cancer diagnostic products, wherein the exosomal miRNA is miR-412-5p.

[0008] Specifically, the products include reagent kits or reagent strips.

[0009] The present invention also provides the use of exosomal miRNA as a therapeutic target for the preparation of drugs for the prevention and / or treatment of gastric cancer, wherein the exosomal miRNA is miR-412-5p.

[0010] The present invention also provides the use of a substance overexpressing exosomal miRNA for the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0011] Specifically, the effects of the drug include at least one of the following: 1) Inhibits the proliferation of gastric cancer cells; 2) Inhibits the migration of gastric cancer cells; 3) Inhibits the invasion of gastric cancer cells.

[0012] The present invention also provides the use of a synthetic miR-412-5p mimic for the preparation of a drug for treating gastric cancer, wherein the synthetic miR-412-5p mimic can increase the expression level of miR-412-5p.

[0013] This invention systematically analyzes the regulatory role of circulating exosomal miRNAs in the early diagnosis and malignant progression of gastric cancer, focusing on the molecular mechanism by which miR-412-5p mediates signaling pathway reprogramming through targeting the nucleocytoplasmic transporter XPO1, providing novel liquid biopsy biomarkers and potential therapeutic targets for the early diagnosis of gastric cancer.

[0014] This invention systematically analyzes the regulatory role of circulating exosomal miRNAs in the diagnosis and malignant progression of early gastric cancer, and discovers 30 differentially expressed miRNAs, of which 12 miRNAs are upregulated and 18 miRNAs are downregulated. In particular, miR-412-5p is significantly downexpressed in early gastric cancer and has the potential to serve as a non-invasive diagnostic biomarker.

[0015] The present invention further confirms that the nucleoplasmic transporter gene XPO1 is a direct target gene of miR-412-5p, and its dysregulation can drive the activation of the PI3K-AKT pathway.

[0016] The present invention further confirms that overexpression of miR-412-5p can inhibit the migration of gastric cancer cells, while XPO1 rescue can reverse this effect.

[0017] This invention reveals for the first time the core role of the exosomal miR-412-5p-XPO1-p38 dual signal axis in the metabolic reprogramming of gastric cancer, and further confirms that exosomal miRNA can serve as a highly sensitive biomarker for early diagnosis, providing a new strategy for liquid biopsy and targeted intervention in gastric cancer.

[0018] This invention demonstrates that miR-412-5p exerts its anti-cancer effect by targeting XPO1 to inhibit the AKT-MAPK signaling pathway. Experiments confirm that miR-412-5p directly binds to and inhibits the expression of the nuclear export protein XPO1, thereby downregulating the activity of key phosphorylated proteins such as p-p38, inhibiting the proliferation, migration, and invasion of gastric cancer cells. A nude mouse tumorigenesis model further validates its in vivo tumor-suppressive function.

[0019] The present invention validates that the exosomal miRNA-mRNA regulatory network reveals that a dual signaling axis synergistically drives the progression of gastric cancer. The constructed miRNA-mRNA interaction network shows that the miR-412-5p-XPO1-p38 dual signaling axis is synergistically activated in early gastric cancer, jointly promoting tumor cell proliferation, migration and chemotherapy resistance, providing a theoretical basis for multi-target combination therapy. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 These are the exosome identification results from Example 1; where A: transmission electron microscopy image of exosomes; B: particle size analysis; C: nanoflow cytometry identification; Figure 2 This is the basic information on sample sequencing in Example 2; where A: box plot of sample expression level; B: PCA distribution of sample expression level; Figure 3 This is a volcano diagram of differential miRNA expression in Example 2; Figure 4This is the Venn diagram predicting the target genes of differentially expressed miRNAs in Example 2; Figure 5 This is a miRNA-mRNA regulatory network diagram from Example 3; where arrows represent miRNAs, circles represent mRNAs, red indicates an increase, and blue indicates a decrease; Figure 6 This refers to the STRING protein interaction network in Example 2; Figure 7 The results of differential expression and functional intervention of miR-412-5p in gastric cancer cell lines in Example 4 are as follows: (A) miR-412-5p expression analysis, (B) miR-412-5p mimics effect analysis, and (C) miR-412-5p pinhibitor effect analysis. Figure 8 Example 4 illustrates the regulatory effect of miR-412-5p expression intervention on the proliferation of gastric cancer cells; where (A) is the proliferation curve of MGC-803 and (B) is the proliferation curve of HGC-27. Figure 9 The results of the cell migration experiment in Example 4 are shown in the figure. The scale bar in the figure represents a size of 100 μm. Figure 10 The results of the cell invasion experiment in Example 4 are shown in the figure. The scale bar represents a size of 100 μm. Figure 11 The graph shows the results of the scratch assay on MGC-803 cells in Example 4. The scale bar represents a size of 100 μm. Figure 12 The results of the HGC-27 cell scratch assay in Example 4 are shown in the figure. The scale bar represents a size of 100 μm. Figure 13 The results of miR-412-5p targeting and regulating XPO1 gene expression in Example 4 are shown below; (A) XPO1 protein expression level detection; (B) WB data statistics; (C) RT-PCR data analysis; (D) binding site prediction; (E) luciferase reporter gene analysis results. Figure 14 (A) shows the results of XPO1 protein expression level detection; (B) shows the statistical results of WB data; and (C) shows the results of RT-PCR data analysis. Figure 15 (A) shows the results of cell migration and invasion detection; (B) shows the statistical and analytical results of cell migration ability data; (C) shows the statistical and analytical results of cell invasion ability data; the scale bar in the figure represents a size of 100μm. Figure 16(A) shows the results of the cell scratch assay, (B) shows the results of the data statistics and analysis, and (C) shows the results of the cell proliferation assay. The scale bar in the figure represents a size of 100 μm. Figure 17 (A) shows the results of XPO1 protein expression level detection, (B) shows the statistical results of WB data, and (C) shows the results of RT-PCR data analysis. Figure 18 (A) shows the results of cell migration and invasion detection; (B) shows the statistical and analytical results of cell migration ability data; (C) shows the statistical and analytical results of cell invasion ability data; the scale bar in the figure represents a size of 100μm. Figure 19 (A) shows the results of the cell scratch assay, (B) shows the results of the data statistics and analysis, and (C) shows the results of the cell proliferation assay. The scale bar in the figure represents a size of 100 μm. Figure 20 (A) shows the detection of protein expression levels in the signaling pathway by Western blotting (WB), (B) shows the statistical analysis of WB data, (C) shows the analysis of RT-PCR data, (D) shows the detection of protein expression levels by Western blotting (WB), (E) shows the statistical analysis of WB data, and (F) shows the analysis of RT-PCR data. Figure 21 (A) shows the detection of protein expression levels in the signaling pathway by Western blotting (WB), (B) shows the statistical analysis of WB data, (C) shows the analysis of RT-PCR data, (D) shows the detection of protein expression levels by Western blotting (WB), (E) shows the statistical analysis of WB data, and (F) shows the analysis of RT-PCR data. Figure 22 (A) shows the comparison and data statistics of HGC-27 tumors, and (B) shows the comparison and data statistics of MGC-803 tumors. Figure 23 The results of immunohistochemical detection in Example 4 (40x magnification). Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are part of this invention.

[0023] This invention provides the use of exosomal miRNA as an active biomarker for gastric cancer diagnosis in the preparation of gastric cancer diagnostic products, wherein the exosomal miRNA is miR-412-5p.

[0024] In some specific implementations, the gastric cancer diagnosis includes early screening for gastric cancer or regulation of malignant progression.

[0025] In some specific embodiments, the miR-412-5p drives PI3K-AKT pathway activation by targeting the nucleoplasmic transporter XPO1.

[0026] The present invention also provides a reagent for detecting the expression level of the exosomal miRNA for use in the preparation of gastric cancer diagnostic products, wherein the exosomal miRNA is miR-412-5p.

[0027] The present invention also provides the use of exosomal miRNA as a therapeutic target for the preparation of drugs for the prevention and / or treatment of gastric cancer, wherein the exosomal miRNA is miR-412-5p.

[0028] The present invention also provides the use of a substance overexpressing exosomal miRNA for the preparation of drugs for the prevention and / or treatment of gastric cancer.

[0029] In some specific embodiments, the drug has at least one of the following effects: 1) inhibiting the proliferation of gastric cancer cells; 2) inhibiting the migration of gastric cancer cells; 3) inhibiting the invasion of gastric cancer cells.

[0030] The present invention also provides the use of exosome miR-412-5p for the preparation of nucleocytoplasmic transporter XPO1 inhibitor.

[0031] Example 1 In this embodiment, exosomes were separated from the plasma of gastric cancer patients by ultracentrifugation-density gradient method, and their physical properties and molecular markers were identified by transmission electron microscopy and nanoflow cytometry.

[0032] (1) Exosome extraction In this embodiment, the isolation of plasma exosomes strictly followed the differential centrifugation combined with density gradient purification protocol outlined in the International Society for Extracellular Vesicles (ISEV) MISEV 2018 guidelines. 10 mL of EDTA-anticoagulated whole blood was collected from patients with early-stage gastric cancer and healthy volunteers on an empty stomach in the morning. Blood cells were removed by centrifugation at 2000 ×g for 20 minutes at 4°C within 30 minutes. The supernatant was collected and further centrifuged at 12000 ×g (4°C, 45 minutes) to remove platelets and cell debris. The resulting plasma supernatant was filtered through a 0.22 μm Millipore membrane and then centrifuged at 110000 ×g for 90 minutes at 4°C using an Optima XPN-100 ultracentrifuge (Beckman Coulter) to precipitate crude exosomes. To eliminate co-precipitation contamination, the precipitate was resuspended in 4 mL of 30% iodixanol (OptiPrep). TMA density gradient medium solution was prepared, with layers of 20%, 10%, and 5% iodixanol solution added sequentially. The mixture was then ultracentrifuged at 200,000 × g at 4°C for 18 hours. Exosome enrichment was aspirated from the 30%–20% interfacial layer using a syringe, diluted 10-fold with phosphate-buffered saline (PBS), and repeatedly ultracentrifuged (110,000 × g, 70 min) to remove the density gradient medium. The final precipitate was resuspended in 100 μL of sterile PBS.

[0033] (2) Identification of exosomes by transmission electron microscopy Take the extracted exosome suspension and adjust the concentration to 1×10⁻⁶. 8 Particles / mL: Using a micropipette, 5 μL of sample was dropped onto the surface of a 200-mesh carbon film copper grid (Ted Pella Inc.) treated with glow discharge, and allowed to stand at room temperature. After 10 minutes of adsorption, excess liquid was gently aspirated using the edge of filter paper. Subsequently, 2% phosphotungstic acid solution (pH 7.0) was added for negative staining for 5 minutes. The aspiration process was repeated, and the sample was placed in a clean petri dish to air dry for 30 minutes. After sample preparation, field emission transmission electron microscopy (HT7800, Hitachi) was used for observation at an accelerating voltage of 80 kV. The system magnification was adjusted in a gradient from ×20000 to ×150000, focusing on typical areas of the field of view to capture images.

[0034] (3) Nanoparticle tracking analysis The exosome precipitate separated by ultracentrifugation was resuspended in sterile filtered (0.22 μm) 1×PBS buffer. The optimal detection concentration range (1×10⁻⁶) was determined through preliminary experiments. 7 ~5×10 8 To avoid interference from light-blocking effects, samples were placed on ice for 5 minutes before loading to eliminate air bubbles. After gentle mixing with a micropipette, the samples were injected into the NanoSight NS300 sample cell (Malvern Panalytical) using a precision syringe (Hamilton). Instrument parameters were set as follows: laser wavelength 532nm, camera shutter speed 30ms, detection duration 60 seconds / sample, 3 measurements per sample, and ambient temperature constant at 25±0.5℃. Software analysis (NTA version 3.4) employed a dynamic threshold algorithm to automatically track Brownian motion trajectories and calculate the hydrodynamic diameter.

[0035] (4) Nanoflow cytometry The exosomes were resuspended in 0.22 μm filtered PBS buffer (containing 1% BSA) and the concentration was calibrated to 1 × 10⁻⁶. 9particles / mL. Take 50 μL of the suspension and co-incubate with fluorescently labeled antibodies (CD63-FITC and negative control IgGκ, antibody titer 1:50). After shaking in the dark for 30 minutes, stop the reaction by adding 100 times the volume of cold PBS. Remove free antibodies by ultracentrifugation at 100000×g (4℃, 70 minutes). Resuspend the precipitate in anti-quenching mounting medium (ProLong). TM Diamond microspheres were loaded into the sample flow path of the CytoFLEX S nanoflow cytometer (Beckman Coulter). Instrument parameters were calibrated and optimized using 200nm standard fluorescent microspheres (Apogee Mix): the side-scattered light (SSC) threshold was set to 500nm particle triggering, the gain of the 488nm / 638nm dual laser excitation channel was adjusted to the FITC fluorescence minimum detection limit, and the flow rate was controlled at 20μL / min to ensure single-particle resolution.

[0036] In this embodiment, the ultrastructure and biophysical characteristics of exosomes were verified using a multimodal technology platform system, and the results are shown in the attached figure. Figure 1 As shown.

[0037] Transmission electron microscopy analysis showed that >85% of the vesicles in the negatively stained samples exhibited a typical cup-shaped depression structure, with diameters concentrated in the 40-130 nm range (mean 82.3 ± 16.7 nm). The boundaries of the lipid bilayer were clearly distinguishable (membrane thickness 7.2 ± 0.8 nm), and no protein polymers or cell debris were mixed in, confirming that the ultracentrifugation-density gradient purification system effectively maintained the structural integrity of exosomes (e.g., Figure 1 (A). Nanoparticle tracking analysis further quantified the particle size distribution characteristics: the main peak was located at 68.5 nm (accounting for 63.7% ± 5.2%), the cumulative proportion of vesicles in the 30-150 nm range reached 96.8% ± 2.1%, and the polydispersity index (PDI) remained stable at 0.12 ± 0.03 (healthy group) and 0.15 ± 0.04 (gastric cancer group), which meets the size definition standards for small extracellular vesicles of the International Society for Extracellular Vesicles (ISEV) (e.g., ...). Figure 1 (B). Quantification of surface markers was performed using high-sensitivity nanoflow cytometry (e.g., ...). Figure 1 (C)

[0038] Example 2 In this embodiment, RT-qPCR and high-throughput sequencing were used to screen differentially expressed miRNAs. The miRNA-target gene interaction was predicted and verified using a dual algorithm (RNAhybrid + mirmada) and luciferase reporter assay.

[0039] (1) High-throughput sequencing and data preprocessing methods In this embodiment, high-throughput sequencing of exosomal RNA was performed using the Illumina HiSeq 4000 platform in paired-end sequencing mode (2 × 150 bp). After total RNA was tested with an Agilent 2100 Bioanalyzer and confirmed to have a RIN value > 7.0, sequencing libraries were constructed using the NEBNext® Small RNA Library Prep Kit. The library fragment selection range was narrowed down to 140-160 bp to enrich miRNA and small RNA fragments. Raw data quality control was performed using FastQC v0.11.9, removing invalid sequences with adapter contamination > 5%, low-quality bases (Q20 < 90%), and N base percentage > 1%. Valid data were dynamically trimmed using Trimmomatic v0.39 (4 bp window, average quality threshold Q15), retaining clean reads ≥ 17 nt in length. Sequence alignment was mapped to the human reference genome (GRCh38) using Bowtie2 v2.4.5, and miRNA expression profiles were annotated based on miRBase v22.1. Expression level normalization was performed using the TPM (Transcripts Per Million) algorithm to correct sequencing depth bias, while UMI (Unique Molecular Identifier) ​​counting was introduced to eliminate PCR amplification bias.

[0040] (2) Methods for analyzing differential expression of miRNAs In this embodiment, the differential expression profile is constructed based on a negative binomial generalized linear model, and statistical inference of high-throughput sequencing data is achieved using the DESeq2 v1.30.1 package. The original miRNA expression matrix is ​​normalized by library size (Median of Ratios) to correct for sequencing depth bias, and a local dispersion fit strategy is used to eliminate the correlation between gene expression levels and variance. During the model construction phase, sample grouping variables (healthy group vs. gastric cancer group) are introduced, and the hypothesis test statistic is calculated using the Wald test. The significance threshold is set as follows: false discovery rate (FDR) corrected by the Benjamini-Hochberg method ≤ 0.05, while requiring the fold change in expression level (|log2FoldChange|) ≥ 1 (i.e., linear scale Foldchange ≥ 2).

[0041] (3) Methods for predicting and verifying the target genes of differentially expressed miRNAs Bioinformatics prediction of target gene interactions employs a dual-database cross-validation strategy to improve the reliability of early gastric cancer-related miRNA regulatory networks.

[0042] First, based on the 30 differentially expressed miRNA (DEM) sequences obtained through screening, target gene scanning was performed using the RNAhybrid v2.1.7 and mirnada v3.3a algorithms, respectively. The RNAhybrid algorithm was executed with a strict energy threshold (ΔG ≤ -25 kcal / mol), requiring a perfect complementary match between the miRNA seed region (2-8 nt) and the 3'-UTR region of the target gene, and the minimum free energy binding site was calculated using a dynamic programming algorithm. The mirnada analysis integrated cross-species conservation analysis (vertebrate phyloP score > 1.5) and target site accessibility (opening energy calculated by RNAfold > -10 kcal / mol), excluding targets with low conservation and steric hindrance.

[0043] In this embodiment, the results of the sample expression consistency and inter-group heterogeneity analysis are attached. Figure 2 As shown, the intra-group homogeneity and inter-group heterogeneity of the sample expression profiles were verified using a multi-dimensional quality control system.

[0044] like Figure 2 Box plot analysis shown in Figure A indicates that the median absolute variation (MAD) of expression levels in the healthy group and the gastric cancer group remained at 0.12±0.03 and 0.15±0.04, respectively, significantly lower than the difference in MAD between the groups (0.53±0.11, p<0.001). Figure 2 The principal component analysis results shown in Figure B more intuitively present the tight clustering characteristics within groups: the silhouette coefficient of the healthy group samples projected onto the PC1-PC2 plane reaches 0.91 (Silhouette Width), while that of the gastric cancer group is 0.88, and the Bhattacharyya distance between groups is as high as 1.93 (p=4.2×10). -7 ).

[0045] This high intragroup consistency in expression profiles provides crucial quality control for subsequent differential analysis. It also reveals that the expression patterns of exosomal miRNAs in early gastric cancer patients have molecular characteristics that are significantly different from those in healthy individuals, suggesting the biological basis for their use as diagnostic biomarkers.

[0046] In this embodiment, the results of differentially expressed miRNA screening and statistical validation are attached. Figure 3As can be seen, differential expression analysis based on the negative binomial generalized linear model reveals a significant dysregulation profile of plasma exosomal miRNAs in early gastric cancer. Using the DESeq2 algorithm (v1.30.1), local dispersion fitting and Wald test were performed on the original count matrices of the healthy group and the gastric cancer group. The results showed that among the 1258 miRNAs detected, 30 miRNAs exhibited statistically significant differential expression (FDR < 0.05 after Benjamini-Hochberg correction), and their expression patterns showed bidirectional regulatory characteristics: 12 miRNAs showed significantly increased expression levels (median upregulation magnitude 2.8-fold, range 2.1-5.3-fold), and 18 miRNAs showed significantly decreased expression levels (median downregulation magnitude 3.2-fold, range 2.2-7.1-fold).

[0047] In this embodiment, the results of differential miRNA target gene prediction and intersection validation are attached. Figure 4 As shown. In this embodiment, the targeting regulatory network of differentially expressed miRNAs (DEMs) was analyzed using a dual-algorithm cross-validation system to improve the reliability of the early gastric cancer-related miRNA regulatory network. Figure 4 The results shown are based on the mature sequences of 30 differentially expressed miRNAs (DEMs) obtained through screening (miRBase v22.1). Target gene scanning was performed using RNAhybrid v2.1.7 and mirnada v3.3a algorithms, respectively. The RNAhybrid algorithm was executed with a free energy threshold ΔG ≤ -25 kcal / mol, requiring complete complementarity between the miRNA seed region (2-8 nt) and the 3'-UTR region of the target gene, and a 3'-UTR binding site vacancy ≤ 2 bp. The mirnada analysis integrated multi-species conservation in vertebrates (phyloP score > 1.5) and target site accessibility (opening energy calculated by RNAfold > -10 kcal / mol), excluding low-conservation and sterically hindered targets. Therefore, the number of RNAhybrid target genes and mirnada target genes can be preliminarily predicted.

[0048] Example 3 This embodiment utilizes the construction of miRNA-mRNA regulatory networks and protein interaction networks to analyze the activity of the PI3K-AKT / MAPK signaling pathway.

[0049] (1) Construction of miRNA-mRNA regulatory network Visualization analysis of the miRNA-mRNA interaction network was performed using Cytoscape software (v3.9.1). A directed graph model was constructed based on the regulatory relationship matrix of differentially expressed miRNAs (DEMs) and target differentially expressed mRNAs (DEGs). Lists of DEMs and target DEGs were imported as network nodes, where DEMs were defined as source nodes and target DEGs as target nodes. Node attribute tables labeled gene ID, log2FoldChange value, and FDR significance level. The establishment of interaction edges strictly followed the cross-linking target relationships of the two databases (intersection prediction results of RNAhybrid and mirmada), retaining only regulatory pairs with free energy binding sites (ΔG ≤ -20 kcal / mol) verified by TarPmiR.

[0050] (2) Methods for constructing protein interaction networks The interaction network of proteins encoded by differentially expressed genes was systematically analyzed using the STRING database (v11.5), with a moderate confidence threshold (interaction score > 0.7) to ensure the biological reliability of the interaction relationships. First, the Ensembl IDs of the differentially expressed genes (DEGs) were entered into the database. The "multiple proteins" analysis mode was selected, activating four sources of interaction evidence: experimental validation (Experimental Data), database curated, co-expression, and conserved co-localization (Textmining).

[0051] In this embodiment, the visualization results of the miRNA-mRNA regulatory network and the functional analysis results of the core hub genes are attached. Figure 5 As shown.

[0052] As attached Figure 5 The results show that the directed miRNA-mRNA interaction network constructed based on Cytoscape v3.9.1 systematically presents the regulatory topology of differentially expressed miRNAs (DEMs) and their targets (DEGs). Specifically, the nuclear export protein XPO1 (log2FC=-2.8) is targeted and inhibited by miR-412-5p, which activates the AKT-MAPK cascade (phosphorylation of p-MAPKThr202 / Tyr204 increases by 3.1-fold, p=5.7×10⁻⁶). -5 ).

[0053] In this embodiment, the experimental verification and functional analysis results of the key protein interaction network are attached. Figure 6 As shown.

[0054] STRING protein interaction network analysis (confidence threshold > 0.7) combined with experimental validation system confirmed that there is a direct physical interaction between the core proteins of the KRT5-PI3K-AKT and XPO1-AKT-MAPK signaling axis in early gastric cancer.

[0055] Example 4 This embodiment is based on the study of the mechanism by which miR-412-5p mediates the AKT-MAPK signaling pathway to regulate gastric cancer.

[0056] In this embodiment, the mechanism by which miR-412-5p mediates the AKT-MAPK signaling pathway to regulate gastric cancer is investigated.

[0057] This embodiment further constructs a stable transfection cell model of miRNA mimics / inhibitor, and evaluates the cell proliferation, migration and invasion capabilities through experiments such as Transwell assay, scratch assay, MTT assay and nude mouse tumor model. The experimental methods involved are as follows.

[0058] Detection of miR-412-5p expression in cells using RT-PCR In this embodiment, total RNA extraction was performed using TRK lysis buffer (LC Science, catalog number LCS-R100) in conjunction with a silica-based adsorption column. Cells grown to 80% confluence were washed with PBS, then lysed at room temperature for 5 minutes with 1 mL of lysis buffer. The lysate was homogenized using a 20-gauge needle (repeated 10 times) and centrifuged at 12,000 × g for 10 minutes (4°C). The supernatant was mixed with an equal volume of 70% ethanol and transferred to an RNA purification column (Zymo Research, catalog number R1016). The cells were washed three times with RNAWash Buffer I / II (containing guanidine isothiocyanate), and finally eluted with 30 μL of RNase-free water. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (RIN values ​​>9.0), and RNA concentration was determined using a NanoDrop ND-1000 (A260 / A280 ratio stable between 1.95 and 2.05). The reverse transcription reaction employed a poly(A) tailing method to enhance miRNA detection sensitivity: 1 μg total RNA was reacted with 2 μL ATP (10 mM) and 1 μL poly(A) polymerase (NEB, catalog number M0276S) at 37°C for 30 minutes. Then, 5 μL of Oligo(dT) universal primers (sequence: 5'-CAGGTCCAGTTTTTTTTTTTTTTTVN-3′) and 200 U M-MLV reverse transcriptase (Thermo, catalog number 28025013) were added, followed by extension at 42°C for 60 minutes and inactivation at 70°C for 15 minutes. The resulting cDNA was stored at -80°C for later use. Quantitative PCR was performed using the SYBR Green method on a CFX96 Touch system (Bio-Rad). The reaction system contained 10 μL of 2×SYBR Premix (Takara, catalog number RR820A), 1 μL of cDNA template, 0.4 μL of forward specific primers (miR-412-5p: 5′-TAGGTGAGGTGAGGTGAGTAG-3′; U6 snRNA: 5′-CTCGCTTCGGCAGCACA-3′) and 0.4 μL of universal reverse primers (5′-CAGGTCCAGTTTTTTTTTTTTTTT-3′). The amplification program was: 95℃ pre-denaturation for 30 seconds; followed by 95℃ for 5 seconds and 62℃ for 30 seconds (40 cycles in total); finally, a melting curve was generated (65℃ to 95℃, increments of 0.5℃ / 5 seconds). Each sample was tested in triplicate. The amplification efficiency was verified by standard curve analysis to be 98.3%-102.6% (R²>0.995). Expression levels were calculated using 2... -△△Ct The method used U6 snRNA as an internal reference, and outliers were removed by Grubbs test (α=0.05).

[0059] Design and Synthesis Methods of miR-412-5p mimics and inhibitors In this embodiment, the mimics were synthesized using a double-stranded RNA mimicry strategy: the sense strand (5'-UAGGUGAAGUUGGUCCCACUGC-3') and the antisense strand (5-GCAGUGGGACCAACUUCACCUA-3') were synthesized in a solid-phase manner by Integrated DNA Technologies (IDT). Key modifications included: (1) Two phosphate thioate bonds are introduced at the 3′ end of the positive chain to enhance nuclease resistance; (2) The 5′ end of the antisense strand is labeled with a FAM fluorescent group (Ex / Em: 492 / 517nm) for transfection tracking; (3) 2′-O-methyl modified locked nucleic acid (LNA) is inserted at the central position of the double strand, locking the nucleotides at positions 9-12 (5′-GUUG-3′) to form a rigid structure.

[0060] After synthesis, the fraction was purified by HPLC (Agilent 1260 Infinity II, C18 reversed-phase column) with a mobile phase of 0.1 MTEAA buffer (pH 7.0) / acetonitrile gradient elution. The fraction with the main peak retention time of 12.3 ± 0.2 min was collected and lyophilized at -80℃.

[0061] In this embodiment, the inhibitor is designed as a single-chain antagonistic molecule with a sequence that is inversely complementary to the mature miR-412-5p (5'-GCAGUGGGACCAACUUCACCUA-3'), and enhances cell membrane permeability through full-chain phosphorylation (Phosphorothioate backbone) and 3' end cholesterol modification (Cholesterol-TEG). Locked nucleic acid (LNA) modification covers positions 2-8 (5'-CAGUGGG-3') and positions 15-21 (5'-UUCACCU-3'), significantly increasing the dissociation temperature (Tm=78.5℃, an increase of 21.3℃ compared to the unmodified sequence). Synthesis was performed using the ÄKTA oligopilot plus system (Cytiva), with the 5' end protected in DMT-on mode. After deprotection with trichloroacetic acid, the product was cleaved by ammonolysis. The crude product was desalted using a Sephadex G-25 column to remove small molecule impurities. Finally, the molecular weight (measured value: 7,489.2 Da, theoretical value: 7,488.6 Da, error <0.01%) and HPLC purity >98% were verified by mass spectrometry (MALDI-TOF MS).

[0062] Cell migration and invasion experiments In this embodiment, cell migration ability was assessed using a matrix-free Transwell system (Corning, catalog number 3422), while invasion ability was detected using a matrix-simulated in vivo microenvironment (BD BioCoat™, catalog number 354480). Cells stably transfected with miR-412-5p mimics / inhibitor were grown to the logarithmic growth phase, digested with 0.25% trypsin-EDTA, and resuspended in serum-free RPMI-1640 medium (Gibco, catalog number 31800022) to adjust the density to 5 × 10⁶ cells / year. 4 Cells / mL. In the migration assay, 200 μL of cell suspension was added to the upper chamber of the Transwell membrane (polycarbonate membrane with 8 μm pore size), and the lower chamber was filled with 600 μL of complete culture medium containing 10% fetal bovine serum as a chemokine source. For the invasion assay, 30 μL of Matrigel (1.8 mg / mL, prepolymerized at 4°C for 30 minutes) was pre-coated onto the Transwell membrane, and the cell seeding density was increased to 1 × 10⁻⁶ cells / mL. 5 Cells / mL were used to overcome the matrix barrier. Cells were incubated at 37°C and 5% CO2 for specific time periods (migration assay: 16 hours; invasion assay: 24 hours). Afterward, the chambers were removed, and unmigrated cells on the upper surface of the membrane were gently wiped away with cotton swabs. Cells that migrated / invaded to the lower membrane surface were fixed with 4% paraformaldehyde for 20 minutes, stained with 0.1% crystal violet for 15 minutes, washed three times with PBS, and air-dried. The membrane was cut and placed on a glass slide, and photographed and counted using a randomly selected 5 fields (×200) under an upright microscope (Nikon Eclipse Ci-L). Each sample was replicated in 3 independent wells.

[0063] Cell scratch assay Cell migration ability was assessed using a standardized scratch-healing model. Cells were cultured in six-well plates to near-complete confluence. A 200 μL sterile pipette tip was used to apply constant pressure perpendicular to the bottom of the plate, creating scratches of uniform width at a constant speed. After scratching, the cells were gently washed three times with pre-warmed phosphate-buffered saline (PBS) to remove detached cell debris. The medium was then replaced with a low-serum medium containing 1% fetal bovine serum to reduce interference from cell proliferation on migration ability assessment. The culture plates were continuously incubated at 37°C and 5% CO2, and images were observed and acquired under an inverted microscope at 0 h and 24 h. Image analysis was performed using ImageJ software. Cell migration rate was calculated by measuring the change in pixel width of the scratched area. Migration distance was obtained by comparing the difference between the scratch width and the initial width at each time point. The final data are expressed as the mean ± standard deviation of three independent experiments, and statistical analysis was performed using a t-test.

[0064] Luciferase reporter gene analysis In this embodiment, a fragment containing the predicted binding site in the 3'-UTR of the human XPO1 gene was obtained by PCR amplification and cloned downstream of the firefly luciferase reporter gene in the psiCHECK-2 vector to construct a wild-type recombinant plasmid (WT-XPO1-3'UTR). Site-directed mutagenesis was used to substitute bases in the miR-412-5p seed sequence binding region to obtain a mutant plasmid (MUT-XPO1-3UTR). HEK293T cells were cultured at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 24-well plates. The recombinant plasmid and miR-412-5p mimic (miR-412-5pmimic) or its negative control (NC mimic) were co-transfected using a Lipofectamine 3000 transfection system. Forty-eight hours after transfection, cells were collected, and luciferase activity was measured using a dual-luciferase reporter gene assay system. Using Renida luciferase activity as an internal control, the ratio of firefly luciferase to Renida luciferase activity was calculated and normalized to the negative control group. The final values ​​are expressed as the mean ± standard deviation of three independent experiments. Student's t-test was used for statistical analysis of intergroup comparisons.

[0065] Western blot analysis of protein expression In this embodiment, cell protein samples were prepared using a modified RIPA lysis buffer (Beyotime, catalog number P0013B), supplemented with 1× phosphatase inhibitor (Roche, catalog number 4906837001) and 1× protease inhibitor (Sigma, catalog number S8830) to maintain the stability of phosphorylated proteins. Gastric cancer cells were washed with pre-cooled PBS and then added with lysis buffer (200 μL / 100000 ppm). 6Cells were lysed on ice for 30 minutes. The lysate was sonicated (Branson S450D, 20% power, 3-second pulses per cycle, 5 times total), then centrifuged at 16,000×g for 20 minutes (4℃), and the supernatant was transferred to a new tube. Protein concentration was determined using the BCA method (Pierce™, catalog number 23225), and the final concentration was adjusted to 4 μg / μL with 0.1% SDS. The cells were then boiled in 5×Laemmli buffer (containing 100 mM DTT) for 10 minutes to denature. The electrophoresis system used a 10% separating gel and a 5% stacking gel, with 40 μg of protein loaded per well, using a pre-stained protein marker (Thermo, catalog number 26616) as a reference. Electrophoresis parameters were set to a constant voltage of 80V (stacking gel) to 110V (separating gel), and stopped when bromophenol blue migrated to the bottom of the gel. The proteins were then transferred to a 0.22 μm PVDF membrane (Millipore, catalog number IPVH00010) using a wet transfer method (Bio-RadTrans-Blot® system). Transfer conditions were optimized for different molecular weights: XPO1 (214 kDa) was transferred at a constant current of 200 mA for 120 minutes; phosphorylated MAPK proteins (p-p38 / p38, 43 kDa; p-ERK1 / 2, 42 / 44 kDa; p-JNK, 46 / 54 kDa) were transferred at a constant current of 250 mA for 90 minutes, maintained at 4°C throughout. After transfer, the membranes were washed with TBST (Tris 25 mM, NaCl 150 mM, pH 7.4) containing 0.05% Tween-20 and blocked with 5% skim milk-TBST blocking buffer at room temperature with shaking for 1 hour. Primary antibodies were incubated overnight at 4°C (oscillation speed 200 rpm). Working concentrations of target protein antibodies were strictly in accordance with the Cell Signaling Technology guidelines: XPO1 (rabbit antibody, 1:1000, #46249), p-p38 (T180 / Y182, rabbit antibody, 1:2000, #4511), p38 (rabbit antibody, 1:1000, #8690), p-ERK1 / 2 (T202 / Y204, rabbit antibody, 1:5000, #4370), ERK1 / 2 (rabbit antibody, 1:5000, #4695), p-JNK (T183 / Y185, rabbit antibody, 1:1000, #4668), JNK (rabbit antibody, 1:1000, #9252), β-actin (mouse antibody, 1:8000, #3700). After washing with TBST, the membrane was incubated with fluorescent secondary antibody (IRDye® 800CW anti-rabbit / anti-mouse, LI-COR, 1:15000) at a temperature away from light for 1 hour. Signal acquisition was performed using an Odyssey CLx dual-color infrared imaging system (channel 1: 800nm ​​for target protein detection; channel 2: 680nm for β-actin internal reference detection), with a resolution set to 84μm to ensure weak signal capture capability.

[0066] Design and synthesis of XP01 knockdown siRNA In this embodiment, the stability of the secondary structure (ΔG ≤ -2.5 kcal / mol) was predicted using the Sfold algorithm, and three candidate sequences were finally screened: siXPO1-1:5′-GCACGAACUUCUGGACCAATT-3′ (target site: 1124-1142), siXPO1-2:5′-GGACAAAGCCUAUGAAUAUTT-3′ (target site: 1589-1607), and siXPO1-3:5′-CUGGAAUUGUGAAGAGCAATT-3′ (target site: 2317-2335). The chemical synthesis was performed by Integrated DNA Technologies (IDT) using a double-stranded RNA symmetry modification strategy: phosphorylation modification (5′-P-) was added to the 5′ end of the sense strand, and a dithiophosphate linkage (PS linkage) was introduced to the 3′ end of the antisense strand to enhance nuclease resistance. A 2'-fluorouracil (2'-FU) modification was inserted at the central position (positions 9-11) of the double strand, improving the loading efficiency of the ribozyme complex (RISC) (increasing the melting temperature Tm by 8.3 °C). Synthesis was performed using a solid-phase phosphoramidite method with DMT-on mode to protect the 5' hydroxyl group. After deprotection with concentrated ammonia at 55 °C for 16 hours, purification was achieved by HPLC (Agilent 1260 Infinity II, XBridge C18 column) using a 0.1 M triethylamine acetate (TEAA, pH 7.0) / acetonitrile gradient elution (5%-95%). The main peak (purity >98%) with a retention time of 14.2 ± 0.3 min was collected and lyophilized at -80 °C. Functional validation was performed using a dual reporter gene system: HEK293T cells were transfected with the pmirGLO vector (Promega) containing the XPO13'UTR wild-type or mutant binding site, with a co-transfection of 50 nM siRNA. Dual-luciferase activity was measured 48 hours later using the Promega Dual-Luciferase Kit. The selection criteria were: 1) wild-type group luciferase activity inhibition rate >70%; 2) mutant group inhibition rate <15%; 3) non-target gene (e.g., GAPDH) expression fluctuation <10%. Results showed that siXPO1-2 exhibited the best inhibitory efficacy (inhibition rate 82.4±3.7%, p=1.2×10⁻⁶). 6 Furthermore, it did not induce a non-specific immune response (TLR3 / 7 / 8 activation detection △Ct>5).

[0067] Construction of XP01 overexpression system in cells In this embodiment, gastric cancer tissue cDNA was used as a template, and the XPO1 open reading frame was amplified using a high-fidelity enzyme (PrimeSTAR Max, Takara#R045Q) (primers: F-5′-CTAGCTAGCGCCACCATGGCCGAGACCGAGGAG-3′ containing the NheI site and Kozak sequence; R-5′-CCGCTCGAGTCAGTCCTCCGGGATGTTC-3′ containing the Xhol site). The PCR conditions were 98℃ for 10 seconds → 68℃ for 2 minutes (30 cycles). The product was verified by 1.2% agarose gel electrophoresis as a 214 kDa band. The amplified fragment was digested with NheI / Xhol (NEB #R3131S / #R0146S) and then ligated with the linearized vector using In-Fusion HD enzyme (Takara #638909) at 50°C for 15 minutes at a ligation ratio of 3:1 (insert fragment: vector). The ligation was then transformed into Stbl3 competent cells (Thermo #C737303) and plated. Positive clones were initially screened by colony PCR (vector primers CMV-F / R), and those positive were verified by Sanger sequencing (100% coverage, Q30>99%). Recombinant plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G were co-transfected into HEK293T cells at a mass ratio of 4:3:1 (PEI transfection reagent, Polysciences#24765, N / P=7). Viral supernatant was collected 48 hours post-transfection, filtered through a 0.45 μm PVDF membrane, and concentrated to a titer of 2 × 10⁻⁶ using a 100 kDa ultrafiltration tube (Merck Millipore#UFC910024). 9 TU / mL (Lenti-X qRT-PCR Titration Kit, Takara#631235). Target cells were infected with MOI=40, and 8 μg / mL polybrene (Sigma#H9268) was added to enhance infection efficiency. After 72 hours, 1.5 μg / mL puromycin (Invivogen #ant-pr-1) was added for continuous selection for 14 days. Single clones were isolated using limiting dilution and overexpression validation was performed.

[0068] KPT-330 inhibits XP01 function in cells. The stock solution (MedChemExpress, catalog number HY-70042) was prepared with DMSO to a storage concentration of 10 mM (stored at -80℃ protected from light). The working solution was serially diluted with serum-free medium before the experiment (final DMSO concentration ≤0.1%, preliminary experiments confirmed that this concentration had no effect on cell viability). Gastric cancer cells were seeded in 6-well plates (density 2 × 10⁶ m³ / well). 5When the cells / well reached 70% confluence, the medium was replaced with KPT-330 (concentration gradient: 0 nM, 25 nM, 50 nM, 100 nM, 200 nM), and incubated at 37℃ and 5% CO2 for 12 h, 24 h, and 48 h, respectively. To eliminate solvent interference, an equal volume of DMSO solvent control group (0.1%) was set up, and the positive control group used the classic XPO1 inhibitor Leptomycin B (LMB, 20 nM, Sigma#L2913).

[0069] Nude mouse tumor-bearing experiment The tumor-bearing animal model was established using BALB / c-nu / nu immunodeficient female mice (4-6 weeks old, 18 ± 2 g), strictly following AAALAC internationally certified animal ethics guidelines. Prior to the experiment, the mice were acclimatized in an SPF-grade barrier environment for 7 days (temperature 22 ± 1℃, humidity 55 ± 5%, 12h light cycle), and their feed and bedding were sterilized by 60Co-Y irradiation. Cells were digested with 0.25% trypsin-EDTA, resuspended in pre-chilled PBS, centrifuged to remove the supernatant, and mixed with matrix gel (Corning #356231) at a volume ratio of 7:3 (final concentration 2 × 10⁻⁶). 7 Cells / mL), maintained fluidity on ice to prevent gelation. After disinfecting the abdominal skin of nude mice with povidone-iodine, 100 μL of a cell-Matrix gel suspension (containing 2 × 10⁻⁶ cells / mL) was subcutaneously injected into the groin area. 6 (Cells), injection angle maintained at 30° to avoid penetrating the muscle layer. To verify the accuracy of tumor localization, a 10% Evans Blue dye (Sigma #E2129) tracer group was added, and microsurgical observation confirmed no vascular damage at the injection site (bleeding <5μL). Tumor-bearing animals were housed individually, and weight changes and wound healing were monitored daily. Analgesia was administered with butorphanol (1mg / kg, subcutaneous injection, every 12 hours). Tumor growth dynamics were monitored using a three-dimensional digital vernier caliper (Mitutoyo #500-196-30) to measure the major axis (L) and minor axis (W) every 72 hours. The volume was calculated using the formula: V=L*W 2 / 2. The experimental endpoint is based on the following criteria: 1. Tumor volume > 1500 mm 3 2. Weight loss >20% of initial value; 3. Impaired mobility or ulceration. Euthanasia was performed using the CO2 progressive asphyxiation method (filling rate 30% cavity volume / min). After the tumor tissue was fixed in 4% paraformaldehyde for 24 hours, it was embedded in paraffin to prepare 4μm serial sections.

[0070] Immunohistochemical detection of XP01 and p-p38 expression in mouse tumor tissues Tumor tissue obtained from nude mice after the tumor-bearing experiment was terminated was fixed in 4% paraformaldehyde (Sigma#P6148) for 12 hours, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare 4μm serial sections. After dewaxing at 60℃ for 1 hour, the sections were sequentially rehydrated with xylene I / II (10 minutes each), graded ethanol, and rinsed with PBS (pH 7.4) 3×5 minutes. Antigen retrieval was performed using the heat-induced epitope retrieval (HIER) method: sections were immersed in 10mM sodium citrate buffer (pH 6.0, containing 0.05% Tween-20), placed in a microwave retrieval system (BioGenex#HK866) at 95℃ for 20 minutes (800 W), and then allowed to cool naturally to room temperature. Sections were blocked with 3% BSA-TBST (containing 0.1% Triton X-100) at room temperature for 30 minutes, and then incubated overnight at 4°C with primary antibody working solution (XPO1: rabbit anti-CST#46249, 1:200; p-p38 (T180 / Y182): rabbit anti-CST#4511, 1:500; dilution buffer: SignalStain® Antibody Diluent #8112). The negative control used homologous IgG instead of the primary antibody (concentration matched), and the positive control used a human gastric cancer tissue microarray (US Biomax#ST801c). The next day, sections were washed with TBST for 3×5 minutes, and then incubated with HRP-labeled secondary antibody (EnVision+System-HRP Labeled Polymer Anti-Rabbit, DAKO#K4003) at room temperature for 1 hour. Signal amplification was performed using TYRAMIDE signal amplification technology (TSA Plus Cyanine 3 / 5 Kit, PerkinElmer#NEL753001KT): sections were reacted with TSA working solution (1:50 dilution) in the dark for 10 minutes, followed by DAB staining (Vector Laboratories#SK-4105) for 30 seconds (monitored dynamically under a microscope), and counterstaining with hematoxylin (Sigma#GHS316) for 45 seconds. After dehydration and clearing with graded ethanol, the sections were mounted with neutral resin (Thermo#SP15-500).

[0071] In this embodiment, the results based on the above experimental method are as follows.

[0072] Differential expression and functional intervention validation of miR-412-5p in gastric cancer cell lines In this embodiment, to systematically evaluate the expression characteristics of miR-412-5p in gastric cancer cells, six common gastric cancer cell lines (including HGC-27, MGC-803, GES-1, SGC-7901, SNU-719, and NCI-N87) were selected, and their expression levels were detected using real-time quantitative PCR (RT-PCR). Each experiment was performed in triplicate and repeated three times to ensure the reliability of the results. Fluorescence signal acquisition was performed using the SYBR Green assay, with U6snRNA as an internal reference gene. Expression levels were analyzed by 2... -△△Ct The method is used for relative quantification.

[0073] The results are attached. Figure 7 The results showed that miR-412-5p exhibited significant heterogeneity in expression across different cell lines: its expression was highest in human gastric mucosal epithelial cells HGC-27, while its expression level in the poorly differentiated gastric cancer cell line MGC-803 was significantly lower than that in other groups, with statistically significant differences (p<0.01). This suggests that there may be differences in expression inhibition or transcriptional regulation of this molecule in gastric cancer models with different degrees of malignancy. Figure 7 (A). Based on this, to further explore the functional mechanism of miR-412-5p in gastric cancer development, genetic intervention experiments were conducted in the high-expression model HGC-27 and the low-expression model MGC-803. Chemically synthesized miR-412-5p inhibitor and mimics were introduced via liposome transfection, with a scrambled sequence used as a negative control. Cells were collected 48 hours after transfection, total RNA was extracted and reverse transcribed into cDNA, and the intervention efficiency was detected by RT-PCR. The results showed that transfection with the inhibitor in HGC-27 significantly reduced the expression level of endogenous miR-412-5p, with an inhibition efficiency of over 60%; correspondingly, transfection with mimics in MGC-803 significantly increased its expression, with an overexpression efficiency of more than 4-fold. Both groups showed highly significant differences compared with the blank control group and the negative control group (p<0.001). Figure 7 (B and C). This result confirms that the oligonucleotide sequence used can effectively regulate the expression of the target miRNA, and the intervention system is stable and reliable, providing an effective tool and experimental basis for subsequent phenotypic analysis and molecular mechanism exploration.

[0074] Regulatory Effect of miR-412-5p Expression Intervention on Gastric Cancer Cell Proliferation In this embodiment, to further investigate the regulatory role of miR-412-5p in the proliferation of gastric cancer cells, HGC-27 and MGC-803 cell models were selected, and the effect of this molecule on cell proliferation was systematically evaluated using the MTT assay. HGC-27 cells were transfected with miR-412-5pinhibitor to inhibit its endogenous expression, while MGC-803 cells were transfected with miR-412-5pmimics to achieve its overexpression. Corresponding control groups were also established.

[0075] like Figure 8 The MTT assay results showed that, after 48 hours of intervention, compared with the wild-type group and the negative control group, the HGC-27-inhibitor group exhibited significantly enhanced cell proliferation activity (p<0.01), with an absorbance increase of approximately 40%, indicating that inhibiting miR-412-5p significantly promoted the proliferation of this cell line. Conversely, in the MGC-803-mimics group, overexpression of miR-412-5p significantly inhibited cell proliferation (p<0.001), with an absorbance decrease of approximately 35% compared to the control group, indicating a significant suppression of the proliferation rate. These results consistently suggest that miR-412-5p may act as a tumor suppressor in gastric cancer cells, and its expression level is negatively correlated with cell proliferation activity, providing experimental evidence for further elucidating its function in the development and progression of gastric cancer.

[0076] miR-412-5p inhibits the migration and invasion of gastric cancer cells. In this embodiment, to further investigate the effect of miR-412-5p on the migration and invasion ability of gastric cancer cells, the Transwell migration assay and scratch healing assay were used to systematically evaluate cell motility behavior. The results are as follows: Figure 9-12 As shown.

[0077] like Figure 9 The results showed that, in the migration experiment, compared with the wild-type control group, the number of transmembrane cells in the HGC-27-inhibitor group with interfering miR-412-5p expression was significantly increased (p<0.01), indicating enhanced migration ability; while in the MGC-803-mimics group with overexpression of miR-412-5p, the number of transmembrane cells was significantly reduced (p<0.001), indicating that migration ability was inhibited.

[0078] like Figure 10-12 The results shown were further validated by a scratch test: 24 hours after intervention, the scratch closure rate in the HGC-27-inhibitor group was significantly higher than that in the control group, while the healing rate in the MGC-803-mimics group was significantly delayed. Figure 10Regarding invasive ability, the results were consistent with the migration assay, as demonstrated by the matrix gel-coated Transwell chamber assay: downregulation of miR-412-5p promoted the invasion of HGC-27 cells. Figure 12 Upregulating its expression significantly weakens the invasive ability of MGC-803 cells. Figure 11 The above series of experimental results indicate that miR-412-5p can significantly inhibit the migration and invasion of gastric cancer cells, suggesting that it may play an important role in regulating the metastatic potential of gastric cancer.

[0079] miR-412-5p targets and regulates the gene expression of XP01. Based on the aforementioned research results, which suggest a potential targeting relationship between miR-412-5p and XPO1, this embodiment further conducted verification experiments at the gene transcription and protein expression levels. Total RNA and total protein were extracted from cells in the HGC-27-inhibitor group, MGC-803-mimics group, and corresponding control groups, respectively. Changes in XPO1 expression were detected using real-time quantitative PCR (qRT-PCR) and Western blotting techniques.

[0080] like Figure 13 The experimental results showed that, at the protein level, XPO1 protein expression was significantly upregulated in HGC-27 cells with interfering miR-412-5p expression, while XPO1 protein expression was significantly decreased in MGC-803 cells with overexpression of miR-412-5p, and the differences were statistically significant (p<0.01). Figure 13 (A, B). At the mRNA level, the trend of XPO1 transcript changes was consistent with the protein results, indicating that miR-412-5p can negatively regulate XPO1 expression at the post-transcriptional level. Figure 13 (C). To further confirm the direct targeting relationship between the two, the binding site of miR-412-5p to the 3′-UTR region of the XPO1 gene was predicted using bioinformatics methods. Figure 13 In a study, miR-412-5p was used to construct luciferase reporter gene vectors containing wild-type and mutant target sequences. Luciferase activity analysis showed that miR-412-5p significantly inhibited the luciferase activity of the wild-type XPO1 reporter vector (p<0.001), while having no significant effect on the mutant vector. This confirms that miR-412-5p achieves its targeted regulatory role by specifically binding to the 3′-UTR region of XPO1. Figure 13 (E).

[0081] Mediating role of XP01 in miR-412-5p regulation of malignant phenotype in gastric cancer cells In this embodiment, to clarify whether XPO1 mediates the regulation of the biological behavior of gastric cancer cells by miR-412-5p, an XPO1-overexpressing lentiviral vector and a specific siRNA interference fragment were constructed using genetic manipulation. MGC-803 cell lines with low miR-412-5p expression were selected, and three experimental models were established: a negative control group (NC group), a miR-412-5p mimics group, and a group co-transfected with mimics and XPO1-overexpressing virus (mimics+OE group). Western blot and qRT-PCR results showed that, compared with the NC group, XPO1 was significantly downregulated at both mRNA and protein levels in the mimics group (p<0.01); while in the mimics+OE group, exogenous XPO1 introduction effectively reversed the XPO1 inhibition caused by miR-412-5p overexpression, and its expression level was significantly higher than that in the mimics group (p<0.05), confirming that the expression level of XPO1 is directly regulated by miR-412-5p and can be restored by genetic manipulation (e.g., Figure 14 Based on this, the motility, invasion, and proliferation abilities of the three groups of cells were further evaluated using Transwell migration and invasion assays, cell proliferation and cell scratch assays. Figure 15-16 The results showed that overexpression of miR-412-5p significantly inhibited cell migration, invasion, and proliferation (p<0.01), while co-transfection with the XPO1 overexpression vector partially rescued this inhibitory effect, restoring cell migration, invasion, and proliferation (p<0.05). These results indicate that XPO1, as a key downstream target gene of miR-412-5p, plays an important mediating role in regulating the migration, invasion, and proliferation of gastric cancer cells.

[0082] To further clarify the functional role of XPO1 in the miR-412-5p regulatory pathway, a recovery experiment was conducted in HGC-27 cells that highly express miR-412-5p. This part of the experiment included three treatment groups: a blank control group (NC group), a miR-412-5p inhibitor group, and a group co-transfected with inhibitor and XPO1-specific siRNA (Inhibitor + siRNA group). qRT-PCR and Western Blot analysis revealed that inhibiting miR-412-5p significantly increased XPO1 expression at both transcriptional and translational levels (p < 0.01); however, when XPO1 expression was simultaneously interfered with, both its mRNA and protein levels were significantly lower than in the inhibitor-only treatment group (p < 0.05). Figure 17This indicates that the XPO1 expression state was successfully reversed through genetic means. At the functional level, a comprehensive evaluation of malignant phenotypes using Transwell assays, scratch assays, and MTT proliferation assays revealed that inhibiting miR-412-5p enhanced cell migration, invasion, and proliferation (p<0.01), while combined downregulation of XPO1 effectively attenuated these promoting effects (p<0.05). Figure 18-19 These results consistently demonstrate that XPO1, as a key downstream target of miR-412-5p, mediates the molecule's regulatory role in the malignant behaviors of gastric cancer cells, including migration, invasion, and proliferation.

[0083] miR-412-5p regulates the AKT-MAPK signaling pathway by targeting XP01. In this embodiment, based on previous data suggesting that miR-412-5p may be involved in regulating the AKT-MAPK signaling pathway, the expression levels of key phosphorylated proteins in this pathway were first systematically detected in the MGC-803 cell line. The protein and transcriptional levels of p-p38, p-ERK1 / 2, and p-JNK in the MAPK signaling pathway were analyzed using Western blotting and real-time quantitative PCR, respectively.

[0084] like Figure 18 The results showed that miR-412-5p overexpression significantly inhibited the activity of p-p38 protein, indicating a functional association with this signaling pathway. Figure 20 (AC). To further elucidate its regulatory mechanism, cells were divided into four groups for intervention: blank control group (NC), miR-412-5p mimics group, mimics and XPO1 overexpression vector co-transfection group (mimics+OE), and treatment group treated with XPO1-specific inhibitor KPT-330 after co-transfection (mimics+OE+KPT-330). The experimental results showed that XPO1 overexpression could partially reverse the inhibitory effect of miR-412-5p on key signaling molecules such as p-p38, and the rescue effect was significantly weakened after KPT-330 was applied to inhibit XPO1 function, indicating that XPO1 functionally mediates the regulatory role of miR-412-5p on the AKT-MAPK signaling pathway (AC). Figure 20 These results collectively support the existence of a molecular axis in which miR-412-5p regulates the AKT-MAPK signaling pathway by targeting XPO1, providing new experimental evidence for understanding the tumor-suppressive mechanism of this miRNA in gastric cancer.

[0085] To further verify the regulatory role of the miR-412-5p-XPO1 molecular axis in the AKT-MAPK signaling pathway, a complementary mechanism study was conducted in HGC-27 cells. Western blotting and qPCR were used to detect key phosphorylated proteins in this pathway (including p-p38, p-ERK1 / 2, and p-JNK). The results showed that inhibiting miR-412-5p significantly enhanced the phosphorylation level of p-p38, suggesting its involvement in the negative regulation of this signaling pathway (e.g., ...). Figure 21 To clarify the functional role of XPO1 in this process, cells were divided into four groups: blank control (NC), miR-412-5p inhibitor group, inhibitor and XPO1 siRNA co-transfection group (inhibitor + siRNA), and treatment group with exogenous recombinant XPO1 protein added (inhibitor + siRNA + rXPO1). The results showed that knocking down XPO1 could, to some extent, attenuate the enhanced p-p38 phosphorylation effect caused by miR-412-5p inhibition; and after supplementing rXPO1 protein, this rescue effect was further enhanced, indicating that XPO1 functionally mediates the regulation of the AKT-MAPK signaling pathway by miR-412-5p (e.g., AC). Figure 21 (Df). These data consistently support the following mechanism: miR-412-5p exerts a tumor-suppressive effect in gastric cancer by targeting and inhibiting XPO1, thereby negatively regulating the phosphorylation state of key proteins in the AKT-MAPK signaling pathway.

[0086] Based on a nude mouse tumorigenesis model, this study validated that miR-412-5p inhibits gastric cancer growth via the XP01 / p-p38 signaling axis. In this embodiment, to further verify the regulatory effect and molecular mechanism of miR-412-5p on the malignant behavior of gastric cancer at the in vivo level, a cell line stably intervening in miR-412-5p expression was constructed and a subcutaneous tumorigenesis experiment was conducted in nude mice. Wild-type HGC-27 and MGC-803 cells transfected with an empty vector, HGC-27-inhibitor cells stably inhibiting miR-412-5p, and MGC-803-mimics cells stably overexpressing miR-412-5p were subcutaneously seeded into BALB / c nude mice, and tumor volume and weight were continuously observed and measured. Figure 22 The results showed that, compared with the wild-type control group, the growth rate and final tumor weight of the xenografts in the HGC-27-inhibitor group were significantly increased (p<0.01). Figure 22 In contrast, MGC-803-mimics showed significantly reduced tumorigenicity (p<0.01). Figure 22(B) To further verify the in vivo mechanism of action at the molecular level, immunohistochemical analysis was performed on tumor tissue. The results showed that the expression levels of XPO1 and p-p38 proteins in the HGC-27-inhibitor group were significantly increased compared to the control group, while the expression of both proteins was significantly downregulated in the MGC-803-mimics group. The differences were statistically significant (p<0.05). Figure 23 (40x magnification). This in vivo experiment not only confirmed that miR-412-5p can inhibit the tumorigenicity of gastric cancer cells in vivo, but also showed that XPO1 and its downstream p-p38 signaling molecules play a key role in miR-412-5p-mediated tumor suppression, providing solid in vivo experimental evidence for further clarifying its signal regulation mechanism.

[0087] In summary, this study first detected the expression of miR-412-5p in various gastric cancer cell lines using RT-PCR, revealing significant heterogeneity in its expression. Specifically, the expression level was significantly reduced in the poorly differentiated gastric cancer cell line MGC-803, while it was higher in human gastric mucosal epithelial cells HGC-27. This finding is consistent with previous studies on the dysregulation of various miRNAs in tumors, further confirming the important role of miRNAs in tumorigenesis and development. By constructing miR-412-5p overexpression and knockdown cell models, the inhibitory effect of miR-412-5p on the proliferation, migration, and invasion of gastric cancer cells was demonstrated. MTT assay results showed that overexpression of miR-412-5p significantly inhibited the proliferation of MGC-803 cells, while knockdown promoted the proliferation of HGC-27 cells. Transwell scratch assays further demonstrated that miR-412-5p can inhibit the migration and invasion of gastric cancer cells. These results confirm the tumor-suppressive function of miR-412-5p at the cellular level, laying the foundation for subsequent mechanistic studies. Regarding mechanistic research, bioinformatics analysis and experimental verification revealed that XPO1 is a direct target gene of miR-412-5p. Dual-luciferase reporter gene assays confirmed that miR-412-5p can directly bind to the 3'-UTR region of the XPO1 gene, inhibiting its expression. Western blot and qRT-PCR results further showed that overexpression of miR-412-5p downregulates the mRNA and protein expression levels of XPO1, while knockdown of miR-412-5p upregulates XPO1 expression. These results not only confirm the targeting relationship between miR-412-5p and XPO1 but also reveal the post-transcriptional mechanism by which miR-412-5p regulates XPO1 expression. As a nuclear export protein, XPO1 is upregulated in various tumors, and its high expression is closely related to malignant tumor progression and poor prognosis. Further functional experiments revealed that XPO1 plays a key mediating role in the regulation of malignant phenotypes in gastric cancer cells by miR-412-5p. By constructing XPO1 overexpression and knockdown models, it was found that XPO1 overexpression could partially reverse the inhibitory effect of miR-412-5p on the proliferation, migration, and invasion of gastric cancer cells, while XPO1 knockdown could attenuate the pro-cancer effect induced by miR-412-5p knockdown. These results confirm that XPO1 is a key downstream effector molecule of miR-412-5p, mediating the tumor-suppressive function of miR-412-5p. Furthermore, this study also found that miR-412-5p affects the activity of the AKT-MAPK signaling pathway by regulating XPO1 expression. Western blot results showed that miR-412-5p overexpression could inhibit p-p38 phosphorylation levels, while XPO1 overexpression or treatment with the XPO1 inhibitor KPT-330 could reverse this effect.These results indicate that miR-412-5p regulates the activity of the AKT-MAPK signaling pathway by targeting XPO1, thereby affecting the malignant behavior of gastric cancer cells. At the animal level, the inhibitory effect of miR-412-5p on gastric cancer growth was confirmed through tumorigenesis experiments in nude mice. The results showed that overexpression of miR-412-5p significantly inhibited the tumorigenicity of MGC-803 cells in nude mice, while knockdown of miR-412-5p promoted tumor growth of HGC-27 cells. Immunohistochemical analysis further showed that the expression level of miR-412-5p was negatively correlated with the expression of XPO1 and p-p38. These results validate the anti-tumor function and mechanism of miR-412-5p at the in vivo level, providing experimental evidence for subsequent clinical studies.

[0088] In summary, this study systematically explored the biological function and molecular mechanism of miR-412-5p in the development and progression of gastric cancer through in vitro and in vivo experiments. The results consistently showed that miR-412-5p expression was downregulated in gastric cancer tissues, and its expression level was negatively correlated with the proliferation, migration, and invasion capabilities of gastric cancer cells, suggesting that it may act as a potential tumor suppressor gene involved in the regulation of gastric cancer progression. Mechanistic studies revealed that miR-412-5p directly targets the XPO1 gene, thereby regulating the activity of the AKT-MAPK signaling pathway and ultimately affecting the malignant biological behavior of gastric cancer cells. These findings not only enrich our understanding of the mechanisms of gastric cancer development but also provide new potential targets for the clinical diagnosis and treatment of gastric cancer.

[0089] In summary, this embodiment demonstrates through in vitro and in vivo experiments that miR-412-5p inhibits the proliferation, migration, and invasion of gastric cancer cells by targeting XPO1 to regulate the AKT-MAPK signaling pathway. These findings not only reveal the anti-tumor function and mechanism of miR-412-5p in gastric cancer but also provide new potential targets for the diagnosis and treatment of gastric cancer.

[0090] Example 5 This embodiment discloses a drug for targeted therapy of gastric cancer based on overexpression of exosomal miR-412-5p. The drug has the following specific effects: 1) inhibiting the proliferation of gastric cancer cells; 2) inhibiting the migration of gastric cancer cells; 3) inhibiting the invasion of gastric cancer cells.

[0091] In this embodiment, the pharmaceutical preparation comprises a therapeutically effective amount of an active ingredient capable of overexpressing miR-412-5p. It should be noted that the term "therapeutically effective amount" refers to the dosage of the drug required to produce an effective effect. This "therapeutically effective amount" can be adjusted and varied according to actual circumstances and is ultimately determined by medical personnel, taking into account factors such as the route of administration and the nature of the preparation, the recipient's weight, age, and other general characteristics, as well as the nature and severity of the disease being treated.

[0092] In this embodiment, the pharmaceutical formulation further includes other pharmaceutically acceptable carriers and / or excipients compatible with the active ingredient. It should be noted that "other pharmaceuticals" here refers to drugs that are compatible with the active ingredient, do not cause inactivation, reduced activity, or hydrolysis of the active ingredient, and that, after synergistic action with the active ingredient, can improve the therapeutic effect of the active ingredient. The aforementioned carriers can be commonly used drug carriers in the art, such as chitosan, liposomes, alginate, agar, fibrin, collagen, and synthetic polymer carriers. The aforementioned excipients are physiologically inactive, do not affect the efficacy, content determination, or stability of the active drug in the pharmaceutical formulation, and their primary purpose is to facilitate the preparation and clinical application of the formulation.

[0093] In this embodiment, the pharmaceutical preparation is a pharmaceutically acceptable oral dosage form. Examples include powders, granules, pills, capsules, tablets, ointments, liquid preparations, gels, etc. Accordingly, depending on the actual dosage form, the administration method can be selected from inhalation, inhalation, nasal administration, sublingual administration, parenteral administration, etc.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The use of an exosomal miRNA as a diagnostic marker for gastric cancer in the preparation of gastric cancer diagnostic products, characterized in that, The exosomal miRNA is miR-412-5p.

2. The use according to claim 1, characterized in that, The diagnosis of gastric cancer includes early screening or control of malignant progression.

3. The use of a reagent for detecting exosomal miRNA expression levels in the preparation of gastric cancer diagnostic products, characterized in that, The exosomal miRNA is miR-412-5p.

4. The use according to claim 3, characterized in that, The products include reagent kits or reagent strips.

5. The use of an exosomal miRNA as a therapeutic target in the preparation of drugs for the prevention and / or treatment of gastric cancer, characterized in that, The exosomal miRNA is miR-412-5p.

6. The use of a substance overexpressing exosomal miRNA for the preparation of drugs for the prevention and / or treatment of gastric cancer, characterized in that, The exosomal miRNA is miR-412-5p.

7. The use according to claim 6, characterized in that, The effects of the drug include at least one of the following: 1) Inhibits the proliferation of gastric cancer cells; 2) Inhibits the migration of gastric cancer cells; 3) Inhibits the invasion of gastric cancer cells.

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