GC tumor and para-cancerous tissue samples were collected from 60 patients treated in the Department of Pathology, The Affiliated Hospital of Qingdao University (Qingdao, China) between 2013 and 2015. Both primary tumors and metastatic lymph nodes were used to prepare tissue microarrays from formalin-fixed paraffin-embedded tissues. Patient follow-up data were obtained from the hospital's medical records and through direct communication with patients. The histopathological diagnosis and grading were performed by two experienced pathologists, and clinicopathological characteristics were recorded. The clinicopathological characteristics included age, sex, tumor differentiation degree, Lauren type, T stage, N stage, M stage and TNM stage (21). A total of 60 pairs of GC tissues and para-cancerous tissue samples were collected from patients who underwent surgery at the Affiliated Hospital of Qingdao University (Qingdao, China), and the present study cohort included 45 male and 15 female patients, with a median age of 64.42 years (mean ± SD, 64.42±10.11 years; range, 46–84 years). Survival data, such as overall survival (OS) and disease-free survival (DFS) were also collected to perform survival analysis via the Kaplan-Meier method and values were compared using the log-rank test. The studies involving human participants were reviewed and approved by The Ethics Committee at the Affiliated Hospital of Qingdao University (approval no. AHQDGC2019022; Qingdao, China). The patients provided their written informed consent to participate in the present study.

The expression profile of LINC00467 was examined using 45 pairs of GC tissues and their corresponding normal adjacent tissues sourced from the GSE63089 database. The GSE63089 dataset (22) can be accessed through the Gene Expression Omnibus (GEO) on the NCBI website. The specific URL is https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63089. This dataset contains expression data from 45 paired gastric cancer tissues and gastric normal tissues, which were used in the present study to examine the expression profile of LINC00467. The Gene Expression Profiling Interactive Analysis (GEPIA) online database (http://gepia2.cancer-pku.cn/#index) includes RNA-seq data on 9,736 tumor and 8,587 control tissue samples derived from The Cancer Genome Atlas Program (TCGA) and Genotype-Tissue Expression (GTEx) databases, which provide tools to enable gene expression profiling as a function of disease type or stage and to conduct corresponding analyses (23). The GEPIA database was utilized to assess LINC00467 expression profiles in 33 cancer types including GC, with P<0.05 as the significance threshold. In the bioinformatics analysis section, the MERAV database (http://merav.wi.mit.edu/) was also utilized for exploration analysis to assess LINC00467 expression in gastric cancer tissues. LINC00467-targeted miRNAs were predicted using miTarget (https://www.mitarget.org/), TargetScan (https://www.targetscan.org/vert_80/) and StarBase (https://rnasysu.com/encori/).

The subcellular localization of LINC00467 was predicted using two online software tools: Genecards (https://www.genecards.org/, version accessed: 2025, database updated regularly) is a comprehensive genomic database providing gene annotation and functional prediction, including subcellular localization insights. RNALocate (http://www.rnalocate.org/, version 2.0, updated in 2023) is a specialized database for RNA subcellular localization, integrating experimental data and computational predictions. These tools were used to inform the design of subsequent FISH and nuclear/cytoplasmic fractionation experiments.

The AGS, HGC27 and MKN45 GC cell lines and the control GES-1 human cell line were from bnbio and were grown in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO₂ incubator. Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transient transfection of cells with siRNAs specific for LINC00467 (si-LINC00467), miR-141-3p mimics or inhibitors, or corresponding controls (si-NC, miR-NC and miR-NC inhibitor) from Shanghai GenePharma Co., Ltd. Detailed information regarding transfection sequences is provided in Table SI. siRNAs and mimics/inhibitors were transfected at a final concentration of 50 nM, unless otherwise specified in experiments. Transient transfections were performed using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, cells were transfected at 70–80% confluence in serum-free medium for 6 h at 37°C, after which the medium was replaced with fresh complete medium. For proliferation assays (RTCA, EdU and colony formation), cells were harvested at 24–72 h post-transfection. For migration/invasion assays (Transwell and wound healing), cells were analyzed at 48 h post-transfection. For qPCR and western blotting, samples were collected at 48 h post-transfection to assess gene/protein expression. For luciferase reporter assays, luciferase activity was measured 48 h after co-transfection of reporter vectors and oligonucleotides. These parameters were optimized to ensure efficient transfection and minimal cytotoxicity, with all experiments performed in triplicate unless stated otherwise.

The methods employed for qPCR have been previously described (24). An RNAiso kit (catalog no. 9109; Takara Bio, Inc.) was used to extract total RNA based on the manufacturer's protocol, after which cDNA was prepared from 1 µg of RNA with a TaqMan miRNA Reverse Transcription Kit (catalog no. 4366596; Applied Biosystems; Thermo Fisher Scientific, Inc.) for miRNA quantification assays, after which a TaqMan miRNA kit (catalog no. 15187092; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR analyses, with U6 used as a normalization control. For mRNA expression analyses, 2 µg of total RNA was reverse-transcribed using a PrimeScript first-strand cDNA synthesis kit (catalog no. K1622; Applied Biosystems; Thermo Fisher Scientific, Inc.), after which SYBR^® Premix Ex Taq™ II (catalog no. RR0820; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct qPCR, based on the manufacturer's protocol. The primers used are provided in Table SI. For the miRNA quantification assays, initial denaturation was performed at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. For the mRNA expression analyses, initial denaturation was performed at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 30 sec. All analyses were conducted using a LightCycler 480 (Roche Diagnostics GmbH). GAPDH was used to normalize mRNA expression levels and the 2^−ΔΔCq method used to calculate relative gene expression (25).

An xCELLigence system E-Plate RTCA (Roche Diagnostics GmbH) was used to monitor proliferation (26) by assessing impedance values in each well and these results were used to calculate cell index (CI) values. Briefly, cells were plated at 1×10⁴ cells/well and grown for 24 h, after which they were treated with si-LINC00467, si-NC, miR-141-3p mimics, miR-141-3p inhibitors or corresponding control oligonucleotides using Lipofectamine 3000 and the CI was monitored for 15 h. CI values were used for normalization of the proliferation data.

For HGC27 cells, colony formation assays were performed as follows. Human gastric HGC27 cells were seeded into 6-well plates at a density of 4,000 cells per well in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h of incubation at 37°C in a humidified 5% CO₂ atmosphere to allow cell attachment, the medium was replaced with fresh complete medium. Cells were then cultured for 10 days under the same conditions (37°C, 5% CO₂), with treatments including transfection of si-LINC00467, si-NC, miR-141-3p mimics, miR-141-3p inhibitors or corresponding controls as described in the experimental design. Following incubation, colonies were fixed with 70% ethanol at room temperature for 5 min to preserve cell morphology. Fixed colonies were stained with 0.05% crystal violet at room temperature for 10 min, allowing visualization of cell clusters. Colonies were defined as aggregates containing ≥50 cells. Images were captured using a brightfield microscope, and colonies were manually counted to quantify proliferation capacity, with colony diameters measured to assess growth efficiency. Data are presented as the mean ± SD from triplicate wells.

Plates were not coated with any substrate prior to cell seeding. Experiments were performed using standard tissue culture-treated 24-well plates. Human gastric cancer AGS and HGC27 cell lines were used. siRNA oligonucleotides (si-LINC00467 and si-NC) and miRNA mimics/inhibitors (miR-141-3p mimics, inhibitors and miR-NC) were transfected at a 50 nM final concentration (Shanghai GenePharma Co., Ltd.) All images were captured at ×100 magnification using a Nikon Eclipse Ti fluorescence microscope (Nikon Corporation). A 100-µm scale bar was included in each figure panel to indicate magnification. Images were acquired from 5 random fields per well using NIS-Elements software (Nikon Corporation). Values were averaged across triplicate wells for each condition. Cells were not serum-starved prior to or during the assay. Cells were maintained in DMEM supplemented with 10% FBS throughout the experiment to ensure optimal proliferation. An EdU kit (BeyoClick™ EdU Cell Proliferation Kit; Beyotime Institute of Biotechnology) was used to assess GC cell proliferative activity. Briefly, human gastric cancer AGS or HGC27 cells were added to 24-well plates (2×10⁴/well) and were treated as appropriate for 72 h, after which EdU was added for 3 h at 37°C. Cells were then fixed for 15 min with 4% paraformaldehyde at room temperature, permeabilized with 0.3% Triton X-100 for 15 min, combined for 30 min with click reaction mixture and stained for 10 min with Hoechst 33342. The cells were imaged using fluorescence microscopy and the number of proliferating cells was averaged to calculate the labeling index. Labeling Index was calculated as: Labeling Index=(total number of Hoechst-stained nuclei/number of EdU-positive cells) ×100. Values were averaged across triplicate wells for each condition. Statistical analysis was performed using GraphPad Prism 6 (Dotmatics).

Appropriately treated cells were added to 6-well plates and grown to confluence, at which time a sterile pipette tip was used to generate a scratch wound in the monolayer. Loose cells were washed away with PBS and fresh medium was then added. After 24 and 48 h post-wounding, the monolayer was imaged with a light microscope (ECLIPSE Ts2 microscope; Nikon Corporation).

GC cell migration and invasion were assessed using Transwell assays. Briefly, complete DMEM was added to the lower chambers of wells in a 24-well plate, with GC cells (2×10⁴ in 200 µl) added to the upper chamber of a Transwell insert. For invasion assays, Transwell inserts (8-µm pores) were precoated with Matrigel (BD Biosciences, San Jose, CA) diluted 1:8 with cold serum-free DMEM and incubated at 37°C for 1 h to allow the Matrigel to solidify into a gel; for migration assays, inserts were uncoated. The plates were then incubated at 37°C in a humidified 5% CO₂ incubator for 24 or 48 h to assess migration or invasion, after which cells on the upper surface were removed with a cotton swab and remaining cells were fixed with 4% paraformaldehyde prior to being stained for 15–20 min with 0.5% crystal violet (w/v) at room temperature for 20 min, rinsed with PBS, and imaged using a brightfield light microscope (Nikon Eclipse Ts2) at ×100 magnification.

GC cells were added to 24-well plates overnight. Cells were then co-transfected with pGL6-miR containing wild-type (WT) or mutated (Mut) sequences of the DPYSL3 3′-UTR or LINC00467 binding region, along with miR-141-3p mimics, miR-141-3p inhibitors, or corresponding miR-NC controls using Lipofectamine^® 3000 Transfection Reagent (catalog no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 48 h post-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (catalog no. E1910; Promega Corp.), with firefly luciferase activity normalized to Renilla luciferase activity to account for transfection efficiency. Data were expressed as the ratio of firefly to Renilla luciferase activity.

GC cells were first treated under non-denaturing conditions and were then hybridized with LINC00467 probes (Shanghai GenePharma Co., Ltd.). The procedures were performed in accordance with the manufacturer's instructions. DAPI (1:1,000) was then used to stain nuclei for 5 min, after which a confocal microscope (Leica Biosystems) was used to image the cells. ImageJ (National Institutes of Health) was used to analyze the images to assess the subcellular localization of LINC00467.

Protein samples were isolated from GC cell lines (AGS and HGC27) at 48 h after transfection. Cells were lysed in RIPA Lysis Buffer (catalog no. P0013B; Beyotime Institute of Biotechnology) containing 1X Protease Inhibitor Cocktail (catalog no. P2417; MilliporeSigma) on ice for 30 min, followed by centrifugation at 12,000 × g for 15 min at 4°C. Supernatant protein concentrations were quantified using the BCA Protein Assay Kit (catalog no. 23225; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg/sample) were separated on 10% gels using SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). Blots were blocked with 5% non-fat milk for 1 h in TBST at room temperature for 1 h, after which they were stained with anti-DPYSL3 (catalog no. ab126787; Abcam) and anti-GAPDH (catalog no. #2118; Cell Signaling Technology, Inc.). After washing, membranes were incubated with HRP-linked goat anti-mouse IgG (1:2,000; catalog no. 7076; Cell Signaling Technology, Inc.) or HRP-linked goat anti-rabbit IgG (1:2,000 dilution; catalog no. 7074; Cell Signaling Technology, Inc.) at room temperature for 1 h each. An enhanced chemiluminescence kit (catalog no. BB-3501; Cytiva) was used to detect protein bands, with Image Lab 2.0 (Bio-Rad Laboratories, Inc.) used for data analysis. GAPDH served as a loading control.

Cells were transfected with biotinylated miR-141-3p mimics or miR-141-3p mutants using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, the cells were collected, washed with PBS and lysed on ice for 15 min. Following centrifugation, 100 µl aliquots of the lysates were reserved as input samples. The remaining samples were incubated with M-280 streptavidin-coupled Dynabeads™ (Invitrogen; Thermo Fisher Scientific, Inc.) at 4°C for ~3.5 h. Subsequently, the bound RNA was eluted using wash buffer and TRIzol^® (Beyotime Institute of Biotechnology) was used to purify the RNA for subsequent analysis. The biotinylated miR-141-3p mimics and mutants were synthesized by Guangzhou RiboBio Co., Ltd.

Cells were transfected with miR-141-3p mimics and the miR-141-3p mutant using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, argonaute 2 (AGO2) immunoprecipitation was performed using an RNA immunoprecipitation kit (catalog no. P0101; Guangzhou Geneseed Biotech. Co., Ltd.) according to the manufacturer's protocol. Antibodies against IgG (catalog no. ab172730; Abcam) and anti-AGO2 (catalog no. ab32381; Abcam) were obtained. Lysates (500 µg total protein) were incubated overnight at 4°C with 30 µl Protein A/G magnetic beads pre-bound to either 5 µg anti-AGO2 antibody (catalog no. ab186733; Abcam) or 5 µg IgG control (catalog no. ab172730; Abcam). Beads were washed sequentially with Wash Buffers 1–3. Bound RNA-protein complexes were eluted by incubation. Input (10% of total lysate) served as the positive control. The input was utilized as the positive control and IgG served as the negative control. To confirm the efficiency of immunoprecipitation, Western blotting was conducted using the anti-AGO2 antibody and the harvested RNAs were subjected to qPCR for further analysis.

GC cells were seeded at 1×10⁴ cells per well in 96-well plates and analyzed using a colorimetric glucose uptake assay kit (catalog no. 36503; AAT Bioquest, Inc.) and a colorimetric L-lactate assay kit (catalog no. 13815; AAT Bioquest, Inc.) following the manufacturer's instructions. For the ATP assay, 3×10⁵ cells cultured in 6-well plates were lysed in 200 µl/well lysis buffer on ice and the lysates were subsequently centrifuged at 4°C. The ATP content in the supernatant was determined using an enhanced ATP assay kit (catalog no. S0027; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The content was normalized to the cell number. Each experiment was performed in quintuplicate.

The ECAR and cellular OCR were assessed using the Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience; Agilent Technologies, Inc.) according to the manufacturer's protocol. ECAR and OCR were determined using Seahorse XFe Glycolysis Stress Test Kit (cat no. 103020-100; Agilent Technologies) and Seahorse XF Cell Mito Stress Test Kit (cat no. 103015-100; Agilent Technologies), respectively. Briefly, 1×10⁴ cells per well were seeded into a Seahorse XFe 96 cell culture microplate (Seahorse Bioscience; Agilent Technologies, Inc.). After baseline measurements, for ECAR, glucose, the oxidative phosphorylation inhibitor oligomycin and the glycolytic inhibitor 2-deoxyglucose (2-DG) were sequentially injected into each well at indicated time points; and for OCR, oligomycin, the reversible inhibitor of oxidative phosphorylation p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rot/AA) were sequentially injected.

In the measurements of glycolysis, a stepwise injection protocol was employed to examine the metabolic behavior of cells. Glucose was administered, at a final concentration of 10 mM per well, to initiate the glycolytic process. Subsequently, oligomycin was introduced, with a final concentration of 2 µM per well, to inhibit ATP synthase and redirect energy production towards glycolysis. Finally, 2-DG was injected at a final concentration of 50 mM per well. Prior to these injections, cells were cultured in XF Glycolysis stress test assay medium devoid of glucose to maintain appropriate cellular conditions. During the first injection, glucose underwent catabolism via the glycolytic pathway, which resulted in the generation of pyruvate along with ATP, NADH, water and protons. The subsequent injection of oligomycin specifically hindered mitochondrial ATP synthesis, thereby promoting a shift in energy production towards glycolysis. This shift enabled assessment of the cellular maximum glycolytic capacity. By comparing the measurements of glycolysis with the glycolytic capacity, the extent of the glycolytic reserve can be determined.

The initial introduction of oligomycin (2 µM final concentration per well), an ATP synthase inhibitor, led to a reduction in oxygen consumption rate associated with mitochondrial respiration and ATP production. Subsequently, the administration of FCCP (1 µM final concentration per well), an uncoupling agent, caused the collapse of the proton gradient and mitochondrial membrane potential. This disruption allowed for unimpeded electron flow through the electron transport chain, which maximized oxygen utilization by complex IV and yielded peak respiration. The disparity between the basal respiration and the maximal respiration levels determined the spare respiratory capacity, which represented the respiratory capability available to meet increased energy demands. In the last injection stage, Rot/AA (0.5 µM final concentration per well), was introduced. This combination completely halted mitochondrial respiration and facilitated the calculation of non-mitochondrial respiration attributed to processes occurring outside the mitochondria. Data were analyzed by Seahorse XFe 96 Wave software (version 2.6.1; Agilent Technologies, Inc.). ECAR was presented in mpH/min and OCR was presented in pmols/min.

HGC27 cells that had been transduced with sh-LINC00467 or sh-NC were subcutaneously implanted into the right flanks of 4-week-old male BALB/c nude mice (18–20 g body weight; Viltalriver Inc.), with each mouse receiving 5×10⁶ cells in 150 µl of FBS-free culture medium. A total of 12 mice (6 per experimental group) were used. Mice were housed in a climate-controlled facility (22±2°C; 55±5% humidity; 12 h light/dark cycle) with free food and water access. Tumors were measured twice per week, with tumor volume being defined as tumor volume=(width × width × length)/2. Each experimental group included 6 mice. On day 22 post-implantation, mice were anesthetized using 1% sodium pentobarbital (50 mg/kg, Sigma-Adrich; Merck KGaA), a method widely accepted for rodent anesthesia (27). Following anesthesia, cervical dislocation was performed to ensure complete cessation of life. The largest volume measured was 808.195 mm³ and the largest diameter measured was 11.93 mm. Subsequently, tumors were excised and weighed for further analysis. The Ethics Committee of the Affiliated Hospital of Qingdao University approved the present animal study (approval no. AHQU-MAL20190810; Qingdao, China).

SPSS (version 19.0; IBM Corp.) and GraphPad Prism 6 (version 6; Dotmatics) were used for all statistical analyses. Correlations between patient clinicopathological features (age, sex, differentiation grade, Lauren type, T stage, N stage, M stage and TNM stage) and LINC00467 expression were assessed via χ² tests, while other differences between groups (e.g., si-NC vs. si-LINC00467, miR-NC vs. miR-141-3p mimics/inhibitors), clinical stage groups (e.g., TNM stage I/II vs. III/IV), cell line groups (e.g., GC cells vs. GES-1) and in vivo groups (e.g., sh-NC vs. sh-LINC00467 mice) were compared using unpaired Student's t-test and one-way ANOVA followed by Tukey's post hoc test. Paired t-tests were used to compare gene expression between tumor and adjacent normal tissues from the same patients (LINC00467 expression in 60 GC tumor/paracancerous tissue pairs; miR-141-3p expression in the same 60 paired samples). For non-paired datasets (e.g., MERAV), unpaired t-tests were performed to assess group differences. Spearman's correlation analyses were used to evaluate the association between LINC00467 and miR-141-3p or DPYSL3 in tumors. P<0.05 was considered to indicate a statistically significant difference.

In pursuit of identifying key lncRNAs implicated in the advancement of GC, a comprehensive analysis was conducted utilizing microarray data on gene expression encompassing 45 pairs of GC tissues and their corresponding normal adjacent tissues (GSE63089). Notably, LINC00467 exhibited upregulation (logFC=0.2982; P=0.0025) within GC tissues (Fig. S1A-B). Subsequent exploration within the GEPIA and MERAV databases demonstrated an increased expression of this lncRNA within GC tumor tissues compared with that of normal tissue (Figs. 1A and S1C). Furthermore, expression pattern analyses of tumor and normal tissues sourced from the TCGA and GTEx databases demonstrated a consistent upregulation of LINC00467 in comparison to adjacent normal tissues across 16 cancer types: Breast invasive carcinoma, cervical and endocervical cancer types, colorectal adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, liver HCC, lung adenocarcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, rectum adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, thymoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma (Fig. S1D).

To elucidate the regulatory mechanisms governing the LINC00467 molecule in GC, a comprehensive examination of LINC00467 expression levels was conducted utilizing qPCR on a cohort of 60 freshly frozen GC tissues. This cohort consisted of 20 cases from clinical stages I and II, alongside 40 cases from clinical stages III and IV (as detailed in Table I). Subsequently, the association between LINC00467 and the clinicopathological characteristics of GC were explored. The analysis (Table I) demonstrated significant associations between LINC00467 expression and the N stage (P=0.015) as well as the TNM stage (P=0.028). Nevertheless, the expression pattern of LINC00467 was not significantly associated with age (P=0.791), sex (P=0.371), differentiation (P=0.184), Lauren type (P=0.688), T stage (P=0.278), or M stage (P=0.085).

Consistent with these results, qPCR results indicated significant upregulation of LINC00467 in 60 GC tumor tissues compared with para-cancerous normal samples (Fig. 1B; P<0.0001). Patients were then stratified into LINC00467-low (n=30) and -high (n=30) groups based on median LINC00467 expression. Survival analysis using Kaplan-Meier curves suggested that high LINC00467 expression in patients was associated with significantly shorter OS and DFS (P<0.05; Fig. 1C and D). The expression levels of LINC00467 were further analyzed across different T stages (T1-T4) and compared with normal tissues in the TCGA database. The results demonstrated a significant upregulation of LINC00467 in tumor tissues compared with normal tissues (P<0.001). However, no significant differences were observed between different T stages within the tumor group, which suggested that the LINC00467 is primarily upregulated in the presence of tumors rather than varying significantly across tumor stages (Fig. 1E). LINC00467 was similarly upregulated in three GC cell lines compared with that of control GES-1 gastric cells, with the highest expression levels in HGC27 cells (Fig. 1F). These findings indicated that LINC00467 upregulation in GC was associated with the progression of GC.

To explore the functional role of LINC00467 in GC cells, the LINC00467 expression was silenced using siRNA (Fig. 2A). The siRNA with the greatest knockdown efficiency (si-R1) was used in subsequent assays. Silencing of LINC00467 was found to suppress HGC27 cell proliferation in RTCA (Fig. 2B) and colony formation (Fig. 2C) assays. EdU assays demonstrated that the knockdown of LINC00467 also reduced EdU uptake compared with transfection with the control si-NC (Fig. 2D). LINC00467 silencing suppressed the proliferation of MKN45 cells (Fig. S2). In order to explore the mechanism underlying the modulation of GC tumor growth by LINC00467, the effects of si-LINC00467 transfection on invasive and migratory activity were examined with wound healing assays, which demonstrated that knockdown decreased the migration of HGC27 cells (Fig. 2E). Transwell assays further demonstrated that the knockdown of LINC00467 suppressed the migratory and invasive activity of HGC27 cells (Fig. 2F). The functional impact of LINC00467 knockdown on tumor growth was then assessed in vivo by transducing HGC27 cells with LINC00467-specific or control short hairpin (sh) RNAs and implanted them subcutaneously into the nude mice. Tumors derived from LINC00467-silenced cells grew more slowly and were smaller in size compared with sh-NC control tumors (Fig. 2G and H). The number of liver metastases were also significantly decreased in mice implanted with shLINC00467-expressing tumors compared with those implanted with control tumors (Fig. 2I and J; P=0.0376), which suggested that the loss of shLINC00467 was sufficient to interfere with the metastatic ability of HGC27 cells. Together, the findings suggested that knockdown of LINC00467 suppresses the growth of GC cells.

The findings from the analysis of GSE63089 demonstrated that LINC00467 serves a key role in metabolic pathways (Fig. S1E). The results demonstrated that silencing LINC00467 led to a significant decrease in glucose consumption, ATP production (Fig. 3A and B) and lactate production (Fig. 3C) in both MKN45 and HGC27 cell lines. Additionally, assessments of the ECAR and OCR further confirmed the impact of LINC00467 on aerobic glycolysis in GC cells (Fig. 3A). Specifically, LINC00467 knockdown resulted in a decrease in ECAR, which were associated with a decrease in glycolytic flux and an increase in OCR, and suggested enhanced mitochondrial respiration (Fig. 3D-G). In summary, these findings suggested that the downregulation of LINC00467 decreases aerobic glycolysis in GC cells.

Predictive analyses suggested that LINC00467 is primarily localized in the cytoplasm of human cells (Figs. S3A and 3B). To evaluate the location, nuclear and cytoplasmic cell fractions were analyzed using qPCR, which confirmed the presence of LINC00467 in both the cytoplasm (>50%) and the nucleus (<50%) (Fig. 4A), and FISH analyses of human tissue samples, which yielded similar results (Fig. 4B). A prediction analysis on potential target genes of LINC00467 was conducted using bioinformatics tools including miTarget, TargetScan and StarBase, which indicated a putative binding site for miR-141-3p within LINC00467, consistent with the downregulation in GC cells. Subsequent investigations included the assessment of miR-141-3p expression in GC cells (Fig. 4C). Biotinylated miR-141-3p mimics and corresponding mutant plasmids were designed and transfected into MKN45 and HGC27 cells for a biotin pulldown assay. Notably, LINC00467 exhibited higher enrichment in the precipitate of WT biotin-coupled miR-141-3p compared with the corresponding mutant plasmids (Fig. 4D), which indicated a potential interaction through base pairing. Given the reported role of lncRNAs in sequestering miRNAs in an AGO2-dependent manner (28), the enrichment of LINC00467 following AGO2 immunoprecipitation in GC cells transfected with WT or mutated miR-141-3p mimics was evaluated. Enhanced enrichment of LINC00467 in WT miR-141-3p-transfected cells suggested the involvement of AGO2 in facilitating the interaction between LINC00467 and miR-141-3p (Fig. 4E and F). Subsequent FISH assays confirmed the co-localization of LINC00467 and miR-141-3p in the cytoplasm of MKN45 and HGC27 cells (Fig. 4G). Furthermore, bioinformatic analyses and luciferase reporter assays corroborated the association between LINC00467 and miR-141-3p, which underscored the downregulation of miR-141-3p in GC tissues compared with normal tissues (Fig. 4H and I). Spearman's correlation analyses demonstrated a negative correlation between LINC00467 and miR-141-3p (r=−0.2951; P=0.0221; Fig. 4J).

The possibility of mutual regulation between LINC00467 and miR-141-3p was then evaluated. The transfection of HGC27 and MKN45 cells with miR-141-3p inhibitors and mimics significantly decreased and increased expression levels, respectively (Fig. 5A and B; both P<0.0001). In order to investigate interactions between LINC00467 and miR-141-3p, luciferase reporter constructs containing WT or mutant (Mut LINC00467) versions of the putative miR-141-3p binding site in LINC00467 were prepared, which demonstrated that transfection with the miR-141-3p mimic was sufficient to suppress the activity of the WT but not Mut LINC00467 reporter vectors (Fig. 5C) in MKN45 cells. LINC00467 knockdown also significantly increased the expression of miR-141-3p (Fig. 5D; P<0.0001), whereas miR-141-3p silencing or overexpression led to increased or decreased LINC00467 expression, respectively (Fig. 5E and F). Thus, the results suggested that LINC00467 may function as a ceRNA that sequesters miR-141-3p in GC cells.

LINC00467 bonded to miR-141-3p, which suggested that LINC00467 may serve as a ceRNA for binding and sequestration of the miRNA. The RTCA and colony formation assay results indicated that transfection with the miR-141-3p inhibitor reversed the effects of LINC00467 knockdown on GC cell proliferation, demonstrated by RTCA assays (Fig. 6A) and colony assays (Fig. 6B). Transwell assays demonstrated that transfection with the miR-141-3p inhibitor reversed the LINC00467 knockdown-dependent reductions in migration and invasion by GC cells (Fig. 6C). Together, these results suggested that LINC00467 knockdown suppressed GC oncogenesis via a mechanism dependent upon miR-141-3p sequestration.

miR-141-3p was identified as a direct regulator of DPYSL3 in GC, with computational analyses indicating a putative binding site within the 3′-UTR of DPYSL3 (Fig. 7A). Validation through luciferase reporter assays in MKN45 cells confirmed the binding interaction (Fig. 7B). Subsequent investigations demonstrated downregulation of DPYSL3 at both mRNA (Fig. 7C) and protein levels (Fig. 7D) in HGC27 cells post-transfection with si-LINC00467, while transfection with miR-141-3p mimics led to significantly decreased DPYSL3 expression levels (Fig. 7E and F). Notably, analysis of the AKT pathway involvement in GC cell metabolism demonstrated that DPYSL3 knockdown in MKN45 cells significantly attenuated GLUT1 expression without affecting HK2 and lactate dehydrogenase A/C expression (Fig. 7G; P<0.0001) (29,30). These findings suggest a mechanism wherein DPYSL3 facilitates glucose influx via increased GLUT1 expression, thereby potentially activating the AKT signaling pathway.

Validation experiments were conducted to confirm the mechanism by which LINC00467 induced GC cell proliferation, migration and invasion via the LINC00467/miR-141-3p/DPYSL3 axis. Rescue experiments employing a miR-141-3p inhibitor and mimics were designed to investigate LINC00467′s role in tumorigenesis. Analysis via qPCR and western blotting demonstrated that LINC00467 knockdown resulted in decreased mRNA and expression levels of DPYSL3 in MKN45 cells (Fig. 8A), while upregulation of LINC00467 significantly enhanced DPYSL3 expression levels in AGS cells (Fig. 8B). Additionally, the effects observed upon silencing or overexpressing LINC00467 were reversed by the miR-141-3p inhibitor or mimics, respectively (Fig. 8A and B). Further analyses sought to determine if the biological functions of LINC00467 in GC cells could be modulated by the miR-141-3p inhibitor or mimics. Results indicated that the miR-141-3p inhibitor counteracted the suppression of proliferation, migration and invasion caused by LINC00467 knockdown in MKN45 cells, while miR-141-3p mimics offset the promotion of these activities induced by upregulation of LINC00467 in AGS cells, as evidenced by colony formation and Transwell assays (Fig. 8C-E). Comprehensively, these findings underscore functionality of LINC00467 as a ceRNA for miR-141-3p, orchestrating DPYSL3 expression and in turn, contributing to the progression of GC.