Application of tmem119 gene or protein in anti-gastric cancer drug development

Abstract

The application relates to the field of biological medicine, and particularly relates to application of a TMEM119 gene or protein in development of an anti-gastric cancer drug, and particularly to application of a TMEM119 inhibitor in preparation of an anti-gastric cancer drug, wherein the inhibitor can be a small-molecule nucleotide for targeted knockdown of the TMEM119 gene or protein, the small-molecule nucleotide is shRNA or siRNA, the shRNA is a double-stranded oligonucleotide composed of complementary base sequences shown in SEQ ID NO. 1-2 or SEQ ID NO. 3-4; and the siRNA has a guide strand with a base sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8. Experiments prove that when the expression of the TMEM119 gene of human gastric cancer cells is knocked down, the viability and the colony formation ability of the human gastric cancer cells are significantly reduced, thereby inhibiting the proliferation and survival of the gastric cancer cells, and further achieving the effect of restraining the occurrence, development, recurrence or metastasis of the gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the TMEM119 gene or protein in the development of anti-gastric cancer drugs. Background Technology

[0002] Gastric cancer is one of the most serious malignant tumors threatening human health worldwide. In terms of clinical prognosis, gastric cancer is characterized by high metastasis and recurrence rates, leading to a significantly shortened overall survival time for patients; the 5-year survival rate for patients with advanced gastric cancer remains below 20%. Currently, chemotherapy remains the primary treatment for advanced gastric cancer, but the development of chemotherapy resistance and treatment-related toxicities greatly limit its effectiveness, ultimately resulting in poor patient prognosis. Therefore, screening for biomarkers that can be used for early diagnosis or targeted therapy of gastric cancer is of significant clinical importance for improving the current state of disease diagnosis and treatment.

[0003] TMEM119 is a member of a family of transmembrane proteins widely distributed in biological membranes, also known as osteoblast-inducing factor. Studies have confirmed that TMEM119 can regulate osteoblast differentiation through the BMP2-RUNX2 and ATF4 / RUNX2 / Osterix signaling pathways. In recent years, the role of TMEM119 in osteosarcoma has gradually attracted attention. TMEM119 is highly expressed in osteosarcoma and is associated with poor patient survival; mechanistically, it can promote the migration and invasion of osteosarcoma cells through the TGF-β / BMP signaling pathway. However, research on TMEM119 in gastric cancer is still relatively limited, and its specific mechanism of action in the development and progression of gastric cancer remains unclear. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an application of the TMEM119 gene or protein in the development of anti-gastric cancer drugs, and designs shRNA that targets and inhibits TMEM119, thereby knocking down the expression of TMEM119, reducing the viability and clonogenic ability of human gastric cancer cells, inhibiting the proliferation and survival of gastric cancer cells, and thus curbing the occurrence, development, recurrence or metastasis of gastric cancer, providing a new target for anti-gastric cancer treatment.

[0006] (II) Technical Solution

[0007] In a first aspect, the present invention provides the application of a TMEM119 inhibitor in the preparation of an anti-gastric cancer drug, wherein the inhibitor is a small molecule nucleotide that targets and knocks down the TMEM119 gene or protein, and the small molecule nucleotide is shRNA, which is a double-stranded oligonucleotide TMEM119-shRNA1 composed of complementary base sequences shown in SEQ ID NO. 1-2 or a double-stranded oligonucleotide TMEM119-shRNA2 composed of complementary base sequences shown in SEQ ID NO. 3-4;

[0008] Alternatively, the small nucleotide is siRNA having a guide strand with a base sequence as shown in SEQ ID NO.7 or SEQ ID NO.8.

[0009] Preferably, the drug comprises an active ingredient and a carrier, wherein the active ingredient is a small molecule nucleotide that targets and knocks down the TMEM119 gene or protein, and the carrier is a lentiviral vector.

[0010] Preferably, the lentiviral vector is pHBLVTM.

[0011] Secondly, the present invention provides an anti-gastric cancer drug, characterized in that it comprises an active ingredient and a carrier, wherein the active ingredient is a small molecule nucleotide that targets and knocks down the expression of TMEM119, and the carrier is a lentiviral vector.

[0012] Preferably, the small molecule nucleotide is a double-stranded oligonucleotide composed of complementary base sequences as shown in SEQ ID NO. 1-2 or a double-stranded oligonucleotide composed of complementary base sequences as shown in SEQ ID NO. 3-4.

[0013] Preferably, the lentiviral vector is pHBLVTM.

[0014] Preferably, the drug further comprises an auxiliary carrier, the auxiliary carrier including pSPAX2 and pMD2G.

[0015] Preferably, the drug is an injectable preparation, which further comprises pharmaceutically acceptable excipients, such as cryoprotectants, surfactants, ion stabilizers, activity maintainers, or penetration enhancers.

[0016] (III) Beneficial Effects

[0017] This invention uses single-cell sequencing technology to compare and analyze the expression profiles of primary gastric cancer lesions and peritoneal metastases, discovering that TMEM119 is abnormally highly expressed in peritoneal metastases. Furthermore, this invention designs small nucleotide shRNAs to target and knock down TMEM119 expression, thereby reducing TMEM119 expression in human gastric cancer cells. Experiments have confirmed that when TMEM119 gene expression is knocked down in human gastric cancer cells, the viability and colony-forming ability of the cancer cells are significantly reduced. This means that knocking down the expression of the TMEM119 gene or protein can inhibit the proliferation and survival of gastric cancer cells, thereby achieving the effect of inhibiting the occurrence, development, recurrence, or metastasis of gastric cancer. Attached Figure Description

[0018] Figure 1 To compare the expression differences of TMEM119 in gastric cancer tissue samples and control tissue samples.

[0019] Figure 2 The mRNA transcription level of TMEM119 in each group of cells was detected by qRT-PC method. The shCtrl group was the transfection control group; the shRNA-1 group was the first TMEM119 interference lentiviral vector transfection group; and the shRNA-2 group was the second TMEM119 interference lentiviral vector transfection group.

[0020] Figure 3 Western blot analysis was used to detect the expression level of TMEM119 protein in lentivirally infected MKN1 cells. "-" represents the transfection control group; shRNA-1 group was the first group transfected with TMEM119 interfering lentiviral vector; shRNA-2 group was the second group transfected with TMEM119 interfering lentiviral vector. *p<0.05,**p<0.01.vs.Control; #p<0.05,##p<0.01.vs.NC.

[0021] Figure 4 The changes in clonogenic ability of human gastric cancer cells MKN1 (Figure A) and HGC27 (Figure B) after knockdown of TMEM119 expression in gastric cancer cells.

[0022] Figure 5 The image shows the decrease in the viability of human gastric cancer cells during culture after TMEM119 was knocked down in human gastric cancer cells HGC27 (Figure A) and MKN1 (Figure B). Detailed Implementation

[0023] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1

[0025] The expression of TMEM119 in gastric cancer tissue samples and healthy control tissue samples was compared using single-cell sequencing technology. The experimental methods are as follows:

[0026] I. Preparation of Experimental Samples

[0027] 1. Gastric cancer tissue samples: Patients diagnosed with primary gastric cancer by pathology (clinical stage I-IV, excluding those who underwent preoperative radiotherapy and chemotherapy, had other malignant tumors or serious underlying diseases) were selected. Cancerous lesion tissue was obtained during surgery (avoiding necrotic areas, and taking mixed tissue from the core and peripheral areas of the tumor, about 100mg / case). A total of biological duplicate samples n≥15 cases were included.

[0028] Healthy control tissue samples: Patients who underwent surgery for benign diseases (such as gastric ulcers or gastric polyps) during the same period were selected, and normal gastric mucosal tissue (with pathological verification of no epithelial dysplasia or cancer) was obtained from the lesion site at a distance of ≥5cm. A total of biological duplicate samples n≥10 cases were included.

[0029] All samples were collected with the patient's informed consent. Immediately after collection, the samples were placed in ice-based tissue preservation solution containing 1% penicillin-streptomycin and transported to the laboratory for processing within 30 minutes.

[0030] 2. Matching of sample clinical information

[0031] The clinical characteristics of the patients in the two groups of samples were recorded, including age (mainly 50-70 years old), gender (balanced male-female ratio), and location of gastric mucosal lesions (antrum, body, etc.). Propensity score matching was used to reduce the influence of confounding factors on the results.

[0032] II. Preparation of Single-Cell Suspension

[0033] 1. Tissue dissociation (to avoid cell damage and cross-contamination)

[0034] Fresh tissue was rinsed three times with pre-cooled PBS (containing 1% penicillin and antibiotics) to remove blood, mucus, and other impurities. The tissue was then minced using a sterile scalpel to a homogenate size of less than 1 mm³. 5 mL of tissue dissociation buffer (containing 0.2% collagenase I, 0.1% hyaluronidase, and 0.05% DNase I, in serum-free DMEM / F12 medium) was added, and the mixture was incubated at 37°C with 5% CO2 and shaken for 40 min, gently pipetting every 10 min. Cell dispersion was observed under a microscope. After dissociation, an equal volume of DMEM / F12 medium containing 10% fetal bovine serum (FBS) was added. Terminate the reaction with 12 culture medium, then filter sequentially through 70μm and 40μm cell sieves to remove undissociated tissue fragments and cell clusters; next, centrifuge at 1200 rpm and 4℃ for 5 min, discard the supernatant, resuspend the cells in pre-chilled PBS, add trypan blue staining solution (final concentration 0.4%), and count the percentage of viable cells under an inverted microscope, ensuring a viable cell rate ≥85% (viable cells appear transparent, dead cells appear blue), and adjust the cell concentration to 1×10⁻⁶. 6 -1.5×10 6 cells / mL, for later use.

[0035] III. Single-cell sequencing library construction and sequencing

[0036] 1. Single-cell capture and cDNA synthesis

[0037] The 10x Genomics Chromium X single-cell sequencing platform was used, following the kit instructions (e.g., ChromiumSingle Cell 3' Reagent Kits v3.1): 10 μL of single-cell suspension, after concentration adjustment, was used to bind individual cells to reverse transcription primers containing unique cell barcodes and molecular identifiers (UMIs) via a microfluidic chip, forming water-in-oil droplets (GEMs). These GEMs were then placed in a PCR instrument for reverse transcription (reaction conditions: 50℃ for 90 min, 85℃ for 5 min, and incubation at 4℃) to convert intracellular mRNA into cDNA. After demulsification, all cDNA was collected and amplified by PCR (amplification conditions: 98℃ for 30 s, 15 cycles: 98℃ for 10 s, 63℃ for 30 s, 72℃ for 30 s, and a final 72℃ for 5 min) to obtain sufficient cDNA product.

[0038] 2. Library Construction and Quality Inspection

[0039] The amplified cDNA was fragmented, end-repaired, A-tailed, and ligated with adapters. Library fragments containing TMEM119 gene-related sequences were enriched by PCR (primer design referenced the TMEM119 gene NM_001130822.2 sequence, upstream primer: 5'-ggcagctggctggatgg-3', downstream primer: 5'-atgttgaccaggtctcggaa-3'). The size of the library fragments (target fragment range 200-600bp) was detected using an Agilent 2100 bioanalyzer, and the library concentration (≥2nM) was detected using a Qubit 4.0 quantitative PCR instrument to ensure that the library quality met sequencing requirements.

[0040] 3. High-throughput sequencing

[0041] The qualified library was loaded into the Illumina NovaSeq 6000 sequencing platform, using the PE150 (paired-end 150bp) sequencing mode and a sequencing depth of ≥50,000 reads per cell (to ensure coverage of the TMEM119 gene transcript). Sequencing quality was monitored in real time during the sequencing process, requiring a Q30 (error rate ≤0.1%) base ratio of ≥90% to avoid adapter contamination or low-quality data interference.

[0042] IV. Bioinformatics Analysis (Focusing on TMEM119 Expression Differences)

[0043] 1. Raw data preprocessing

[0044] Cell Ranger (10x Genomics software, v6.1.2) was used to perform quality control on the raw sequencing data (FASTQ file): reads containing adapter sequences and low-quality bases (Phred score <20) were removed; different cells were distinguished by cell barcode; PCR amplification bias was corrected using UMI; and a gene expression matrix for each cell was obtained (rows: genes, columns: cells, values: UMI count). Cell-level quality control was performed by filtering out the following cells to reduce noise: ① cells with <200 or >6000 gene detections (excluding dead cells, doublet cells, or low-quality cells); ② cells with >15% mitochondrial gene content (excluding stress-damaged cells); ③ cells with a TMEM119 gene UMI count of 0 (not included in subsequent gene expression analysis).

[0045] 2. Cell Clustering and Cell Type Annotation

[0046] Seurat software (v4.0.5, R package) was used to standardize the quality-controlled cell expression matrix (using the Normalize Data function with Log Normalization), select features (using the Find Variable Features function to screen for hypervariable genes), and perform principal component analysis (PCA). Based on the PCA results, dimensionality reduction was performed using t-SNE (t-distributed Stochastic Neighbor Embedding) or UMAP (Uniform Manifold Approximation and Projection), and cell clustering was performed using the Find Clusters function (resolution set to 0.8) to obtain different cell subpopulations. Referring to known gastric tissue cell type markers (e.g., epithelial cells: CDH1, KRT19; immune cells: CD45, CD3D; fibroblasts: COL1A1, FAP), the marker genes of each cluster were identified using the Find Markers function to complete cell type annotation (with a focus on gastric epithelial cell subpopulations, as TMEM119 is mainly expressed in epithelial cells).

[0047] 3. Differential expression analysis of TMEM119

[0048] UMI counts of the TMEM119 gene were extracted from each cell type in the gastric cancer group and the healthy control group. The count was standardized using the UMI count per 10,000 transcripts (TP10K) to eliminate the influence of cell sequencing depth differences on expression levels. The expression levels of TMEM119 in the two groups were compared by cell type. The significance of differences was calculated using the Wilcoxon rank-sum test (one-sided test). The screening criteria were |log2(TP10K in gastric cancer group / TP10K in healthy control group)|>1 and the corrected p-value (padj)<0.05, which were considered to be significantly different expressions of TMEM119. The expression distribution of TMEM119 in each cell type in the two groups was displayed by violin plot.

[0049] Experimental results are as follows Figure 1 As shown, the expression of TMEM119 in gastric cancer tissue samples was significantly higher than that in healthy control tissue samples.

[0050] Example 2

[0051] This embodiment designs siRNA targeting the TMEM119 gene and synthesizes the corresponding shRNA sequence based on the target. The siRNA and shRNA sequences are shown in Table 1-2. The specific steps are as follows:

[0052] 1. Enzymatic digestion of lentiviral vectors

[0053] The lentiviral vector plasmid pHBLVTM was subjected to double digestion with restriction endonucleases BamHI and EcoRI (the reaction system contained the vector plasmid, BamHI, EcoRI, and digestion buffer, etc.), and incubated at 37°C for 4 hours. After digestion, the products were separated by agarose gel electrophoresis, and the linearized pHBLVTM vector fragment was recovered (undigested circular plasmids were removed).

[0054] 2. Preparation of shRNA insert fragments

[0055] Two complementary single-stranded DNA oligonucleotides were artificially synthesized: both single strands carried shRNA sequences targeting the target gene (TMEM119), and their ends were designed with BamHI and EcoRI restriction endonuclease recognition sites and sticky end complementary sequences that matched the vector.

[0056] Two single-stranded DNAs were mixed in an equimolar ratio and subjected to an annealing reaction (the procedure was: heating at 95°C for 5 minutes, followed by slow cooling to room temperature to allow the single strands to fully complement each other and form a double strand), ultimately yielding a double-stranded shRNA insert fragment with sticky ends.

[0057] 3. Ligation and Transformation Identification of Recombinant Vectors

[0058] The annealed shRNA double-stranded insert fragment was ligated with the pHBLV™ vector recovered by double digestion with BamHI / EcoRI using T4 DNA ligase overnight at 16°C to construct a recombinant lentiviral vector.

[0059] The ligation product was transformed into DH5α competent cells using a heat shock method (the competent cells were thawed on ice, the ligation product was added, and the cells were incubated on ice for 30 min; then heat-shocked at 42°C for 90 seconds, followed by an immediate ice incubation for 2 min; after recovery in antibiotic-free medium, the cells were inoculated onto LB agar plates containing ampicillin). After single colonies grew, positive colonies were picked and amplified, plasmids were extracted, and sequenced to verify the accuracy and orientation of the inserted fragment (using universal or specific primers).

[0060] 4. Packaging, concentration, and titer determination of lentiviruses

[0061] Plasmid extraction: A large number of recombinant pHBLV™ vectors, which have been verified by sequencing, are extracted, along with helper plasmids pSPAX2 (providing viral structural proteins and regulatory factors) and pMD2G (providing the envelope protein VSVG).

[0062] Cell transfection: The recombinant pHBLVTM, pSPAX2, and pMD2G were co-transfected into 293T packaging cells using liposome transfection (Lipofectamine 3000) (ensuring that the cell confluence was 70%-80% and the cells were in good condition before transfection).

[0063] Virus collection: Cell culture supernatant was collected at 24h, 48h and 72h after transfection, and all supernatants were combined.

[0064] Virus concentration: The virus particles were concentrated by removing some of the culture medium components using ultracentrifugation (25,000 rpm, 4℃ for 2 h) or PEG precipitation.

[0065] Titer determination: The concentrated virus solution was serially diluted using either quantitative PCR (using the fluorescent reporter gene GFP carried by the vector) or limiting dilution method to infect target cells (e.g., 293T cells). After culture, the proportion of fluorescently positive cells was detected by flow cytometry, and the viral titer (unit: TU / mL, transduction units per milliliter) was calculated based on the dilution factor, ensuring a titer ≥ 1 × 10⁻⁶. 8 TU / mL, meeting the requirements for subsequent experiments.

[0066] Through the above steps, the construction, packaging, and activity verification of recombinant lentiviral vectors targeting the target gene can be completed, providing tool viruses for subsequent gene interference experiments.

[0067] Table 1: siRNA sequences

[0068]

[0069] Table 2: shRNA sequences

[0070]

[0071] The TMEM119-shRNA in Table 2 is cleaved by the Dicer enzyme in cells to generate corresponding double-stranded siRNA. siRNA is the "end product" of shRNA after enzymatic cleavage, thereby exerting its function of silencing the target gene TMEM119. The siRNA is a double-stranded structure composed of a guide strand (see SEQ ID NO. 7-8 in Table 1) and a transit strand. The guide strand binds complementary to the TMEM119 gene mRNA, thereby knocking down the expression of the TMEM119 gene in cells.

[0072] Example 3

[0073] In this embodiment, the lentivirus constructed in Example 2 was used to infect gastric cancer cells to investigate the inhibitory effect of the lentivirus on TMEM119 in human gastric cancer cells HGC27 and MKN1. The experimental steps are as follows:

[0074] 1. Culture and inoculation of gastric cancer cells

[0075] Human gastric cancer cells in logarithmic growth phase (such as HGC27 and MKN1; metastatic gastric cancer cell models and primary gastric adenocarcinoma tissues, respectively) were selected and resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS), and the cell density was adjusted to 2 × 10⁻⁶ cells / year. 5 ) cells / mL; add 2mL of cell suspension to each well and seed in a 6-well plate, incubate at 37℃ and 5% CO2 for 24h. When the cells adhere and reach 70% confluence, perform lentiviral infection. The lentiviral concentration in the transfection system is 1μg / mL.

[0076] 2. Lentiviral infection and grouping experiment: 3 groups were set up:

[0077] Control group (shCtrl): Complete culture medium containing lentivirus carrying TMEM119-shNC; TMEM119-shNC is composed of complementary SEQ ID NO.5 and SEQ ID NO.6.

[0078] shRNA1 group: complete culture medium containing lentivirus carrying TMEM119-shRNA1;

[0079] shRNA2 group: Complete culture medium containing lentivirus carrying TMEM119-shRNA2;

[0080] All of the above lentiviruses carry a GFP fluorescent marker.

[0081] The multiplicity of infection (MOI) for each group was determined to be 10 based on preliminary experiments. After gentle mixing, the mixture was cultured for a longer period.

[0082] 3. qRT-PCR detection of TMEM119 mRNA transcription level

[0083] (1) Total RNA extraction and genomic DNA removal: Human gastric cancer cells MKN1 were infected with three groups of lentiviruses for 48 h, and the cells were collected from each group. Total RNA was extracted using Trizol reagent. The RNA concentration and purity were measured (OD260 / OD280 should be between 1.8 and 2.0). 1 μg of total RNA was taken and genomic DNA was removed using DNase I (incubated at 37℃ for 30 min, then heated at 65℃ for 10 min to inactivate the enzyme).

[0084] (2) cDNA synthesis: Using total RNA after removing genomic DNA as a template, cDNA was synthesized using a reverse transcription kit (such as PrimeScript RT Master Mix). The reaction conditions were: 37℃ for 15 min, 85℃ for 5 s, and stored at 4℃.

[0085] (3) The qPCR reaction was performed using the SYBR Green real-time PCR kit. The reaction system was 20 μL (containing 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL each of upstream and downstream primers, 2 μL of cDNA template, and 7 μL of RNase-free H2O).

[0086] Reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, for a total of 40 cycles; finally, melting curve analysis was performed (95℃, 15s, 60℃, 1min, 95℃, 15s).

[0087] Each sample was tested in triplicate. Actin was used as an internal control. The relative expression level of TMEM119 mRNA was calculated using method 2-ΔΔCt (see experimental results). Figure 2 ).

[0088] The primer sequences used for qRT-PCR are as follows:

[0089] The primer set for TMEM119:

[0090] Upstream primer (5'-CGGCCTATTACCCATCGTCC-3'), downstream primer (5'-CTGGGCTAACAAGAGAGACCC-3').

[0091] Internal control Actin primers: forward primer (5'-CATGTACGTTGCTATCCAGGC-3'), reverse primer (5'-CTCCTTAATGTCACGCACGAT-3').

[0092] like Figure 2 As shown, compared to the shCtrl group, both groups of lentivirus transfection carrying TMEM119-shRNA effectively inhibited the mRNA transcription level of TMEM119 in gastric cancer cells MKN1. Among them, shRNA-1 reduced the mRNA transcription level of TMEM119 by 40%, while shRNA-2 reduced the mRNA transcription level of TMEM119 by about 60%, indicating that shRNA-2 has a higher knockdown efficiency for TMEM119.

[0093] 4. Western blot analysis to detect the expression level of TMEM119 protein.

[0094] Human gastric cancer cells MKN1 and HGC27 were infected with the aforementioned three groups of lentiviruses for 48 h. Cells from each group were collected, and total protein was extracted using RIPA lysis buffer containing a protease inhibitor. Protein concentration was determined using a BCA protein quantification kit, and an equal amount of protein (approximately 30 μg) was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h. Primary antibodies (rabbit anti-human TMEM119 polyclonal antibody, 1:1000 dilution; mouse anti-human Actin monoclonal antibody, 1:5000 dilution) were added, and the membrane was incubated overnight at 4°C. After washing, HRP-labeled secondary antibody (goat anti-rabbit IgG, 1:5000 dilution) was added, and the membrane was incubated at room temperature for 1 h. Finally, the membrane was developed using an ECL chemiluminescence assay kit, and the band grayscale values ​​were quantitatively analyzed using ImageJ software. The relative expression level of TMEM119 protein was calculated using Actin as an internal control (see [link to ECL chemiluminescence assay]). Figure 3 ).

[0095] Figure 3 The results showed that, compared with the negative group, both groups of lentiviruses carrying TMEM119-shRNA could effectively knock down the expression level of TMEM119 protein in gastric cancer cells MKN1 and HGC27.

[0096] like Figure 4 The image shows changes in gastric cancer cells MKN1 and HGC27 after infection with lentiviruses carrying TMEM119-shRNA1 or TMEM119-shRNA2, as observed under SEM. Compared to the shCtrl group, the number and density of viable MKN1 and HGC27 cells were significantly reduced, indicating that knocking down TMEM119 expression levels in gastric cancer cells can reduce the migration ability of human gastric cancer cells (e.g., ...). Figure 4 (Figure A) and clone-forming ability (e.g.) Figure 4 (Figure B in the middle).

[0097] The CCK-8 assay was used to detect the decrease in cell viability after infection of gastric cancer cells MKN1 and HGC27 with lentiviruses carrying TMEM119-shRNA1 and TMEM119-shRNA2 (with initial viability as 1). Figure 5 As shown, after MKN1 cells were treated with TMEM119-shRNA1 for 96 hours, cell viability decreased by approximately 60% compared to the control group (e.g., Figure 5 (Figure B); After treating HGC27 cells with TMEM119-shRNA2 for 96 h, cell viability decreased by 70% compared to the control group (e.g., Figure B). Figure 5 (See Figure (A) in the text). The experiments in this embodiment demonstrate that by designing shRNA or siRNA that targets and knocks down TMEM119, the motility, invasiveness, and migration ability of gastric cancer cells can be reduced.

[0098] These experimental results indicate that inhibiting the expression level of the TMEM119 gene or protein in cells can suppress the proliferation and survival of gastric cancer cells. The shRNA and siRNA designed in this invention have a high knockdown function of TMEM119 and are expected to be developed into effective biological agents that can then be used to inhibit the occurrence, development, recurrence or metastasis of gastric cancer.

[0099] 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. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of TMEM119 inhibitor in the preparation of anti-gastric cancer drugs, wherein the inhibitor is a small molecule nucleotide that targets and knocks down the TMEM119 gene or protein, wherein the small molecule nucleotide is shRNA, which is a double-stranded oligonucleotide composed of complementary base sequences shown in SEQ ID NO.1-2 or a double-stranded oligonucleotide composed of complementary base sequences shown in SEQ ID NO.3-4; Alternatively, the small nucleotide is siRNA having a guide strand with a base sequence as shown in SEQ ID NO.7 or SEQ ID NO.

8.

2. The application according to claim 1, characterized in that, The drug comprises an active ingredient and a carrier, wherein the active ingredient is a small molecule nucleotide that targets and knocks down the TMEM119 gene or protein, and the carrier is a lentiviral vector.

3. The application according to claim 2, characterized in that, The lentiviral vector is pHBLVTM.

4. An anti-gastric cancer drug, characterized in that, It comprises an active ingredient and a carrier, wherein the active ingredient is a small molecule nucleotide that targets and knocks down TMEM119 expression, and the carrier is a lentiviral vector; the small molecule nucleotide is shRNA, which is a double-stranded oligonucleotide composed of complementary base sequences shown in SEQ ID NO.1-2 or a double-stranded oligonucleotide composed of complementary base sequences shown in SEQ ID NO.3-4; Alternatively, the small nucleotide is siRNA having a guide strand with a base sequence as shown in SEQ ID NO.7 or SEQ ID NO.

8.

5. The anti-gastric cancer drug according to claim 4, characterized in that, Its features are, The lentiviral vector is pHBLVTM.

6. The anti-gastric cancer drug according to claim 5, characterized in that, The drug also includes an auxiliary carrier, which includes pSPAX2 and pMD2G.

7. The anti-gastric cancer drug according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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