Specimen collection

We brought 20 paired GC tissues and matched neighboring non-cancerous tissues, along with corresponding serum samples from Qilu Hospital of Shandong University. All diagnoses were independently confirmed by two experienced pathologists. No patient had experienced chemotherapy or radiotherapy pre-operation. Serum samples from twenty age- and sex-matched healthy individuals were also included as controls. Informed consent was acquired from each participant. The Medical Ethics Committee of Qilu Hospital, Shandong University, gave its approval for this investigation (No. KYLL-202011–223-1).

Cells and cultures

GC cell lines (AGS and HGC-27) and the normal gastric epithelial cell line (GES-1) were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell lines were validated by short tandem repeat (STR) profiling to be mycoplasma contamination-free. Cells were cultivated in RPMI-1640 medium (Gibco, USA) enriched with 10% heat-inactivated FBS (Invitrogen, USA) and 1% penicillin–streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin). Cultures were maintained at 37 °C in a humidified 5% CO₂ condition, with medium changes every 2–3 days. Cells were passaged at 70%–80% confluence via 0.25% trypsin–EDTA (Gibco, USA) for subsequent trials.

Lentiviral transfection and plasmid transfection

Lentiviral vectors encoding Linc-ROR overexpression or short hairpin RNA (shRNA) for gene silencing, both carrying puromycin resistance, were purchased from GeneCopoeia (Guangzhou, China). To determine the optimal infection conditions, AGS and HGC-27 cells were first subjected to lentiviral transduction at a range of MOIs. Based on preliminary optimization experiments aimed at achieving high transduction efficiency while minimizing cytotoxicity, the MOI yielding the highest transduction efficiency with the lowest cytotoxicity was selected for subsequent experiments (MOI 20 for AGS cells and MOI 10 for HGC-27 cells). Cells were infected with the corresponding lentiviruses for 24 h and then selected with 5 μg/mL puromycin for approximately two weeks to create stable cell lines.

Transient transfection of cells was conducted with pcDNA3.1-CARMIL1 or the corresponding empty vector via Lipofectamine 3000 (Invitrogen, USA) as per the manufacturer’s guidelines. Briefly, cells were plated in plates with six wells and grown to 60%–70% confluence at the time of transfection. For each well, 2.5 μg of plasmid DNA was diluted in Opti-MEM (Gibco) and combined with P3000 reagent, followed by incubation with Lipofectamine 3000. The DNA–lipid complexes were applied to the cells, and incubation was conducted at 37 °C. Following 6–8 h, the medium was substituted with new complete medium, and cell collection was conducted 48 h post-transfection for subsequent experiments. For miRNA modulation, GC cells plated in plates with six wells were transfected with miR-145-5p mimics or inhibitors (GenePharma, China) via Lipofectamine™ 3000 (Invitrogen, USA), with scrambled oligonucleotides used as negative controls (NCs). Table S1 lists nucleotide sequences.

Quantitative reverse transcription PCR (RT-qPCR)

Total RNA isolation from tissues, serum samples, and cultured cells was conducted via the RNA Isolation and Extraction Kit (Fastagen Bio, China) as per the manufacturer’s guidelines. RNA purity and level were measured via a NanoDrop 2000 spectrophotometer, and only samples with A260/280 ratios of 1.8–2.1 were included for analysis. cDNA was generated via the PrimeScript™ RT Reagent Kit (GeneCopoeia, China), and RT-qPCR was conducted via SYBR Green Master Mix (GeneCopoeia) on a QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, USA). Using GAPDH for mRNA and U6 for miRNA normalization. Relative levels were assessed via the 2−ΔΔCt technique. Table S2 lists the primer sequences.

Western blot

GC cell lysis was conducted via RIPA buffer (Beyotime, China) enriched with a protease inhibitor cocktail (1:100). Tissue samples were first homogenized using a magnetic bead grinder and further disrupted by brief ultrasonication on ice to ensure complete lysis. Protein levels were measured via a BCA Protein Assay Kit. The separation of equal protein quantities (25 μg) was conducted by 10% SDS-PAGE (80 V for stacking, 120 V for resolving) and then transferred onto 0.22 μm PVDF membranes (Millipore, MA, USA) at 300 mA for 90 min. A 1-h blockage of membranes was conducted with 5% skim milk at room temperature, and incubation was conducted overnight at 4 °C with primary antibodies. After rinsing, a 1-h incubation of membranes was conducted with HRP-conjugated goat anti-rabbit IgG secondary antibodies at room temperature. Enhanced chemiluminescence (ECL) reagents were utilized to detect protein signals, which were then photographed with an Amersham Imager 600 RGB system (GE Healthcare, USA). ImageJ (NIH, USA) was utilized to quantify band intensities, which were then normalized to the corresponding loading controls to calculate relative protein expression levels. Table S3 presents the detailed antibody information.

CCK-8 assay

For the cell viability assay, 2 × 103 GC cells/well were grown on plates with 96 wells and maintained overnight under numerous treatment conditions. At 24 and 48 h post-seeding, 10 μL of CCK-8 reagent (Abbkine, China) was applied to each well, and a 2-h incubation was conducted at 37 °C. Absorbance at 450 nm was read via a BioTek Gen5 microplate reader (BioTek, USA). Each condition was experimented with at least three independent experiments with triplicate wells per condition. Relative cell viability was estimated by the normalization of the absorbance of treated wells to that of the control.

Colony formation assay

AGS and HGC-27 cells (1 × 103 cells/well) were cultured in plates with 96 wells and cultivated for 15 days, with medium replaced every four days, until visible colonies formed. A 4% paraformaldehyde solution was utilized for fixation of the colonies for 20 min, 0.1% crystal violet was utilized for staining for 30 min, and PBS was utilized for rinsing. After air drying, plates were photographed, and colonies with > 50 cells were assessed via an Olympus BX51 microscope (Olympus, Japan). Trials were conducted at least three times independently.

Wound healing assay

Log-phase AGS and HGC-27 cells were plated in plates with six wells (5 × 105 cells/well) and cultivated at 37 °C until reaching ~ 95% confluence. A uniform scratch was formed via a sterile pipette tip, and PBS was utilized to gently wash the cells to eliminate debris before adding 2 mL of serum-free medium per well. Photographs of the wound area were taken at 0 and 24 h post-scratch via an inverted microscope (Olympus, Japan), and the migration rate was quantified by measuring the reduction in wound width. Trials were conducted in triplicate.

Transwell assay

For the migration assay, GC cells were plated into the top chambers of 8-μm pore Transwell inserts (Corning, USA), while the bottom chambers were supplied with complete medium with 20% FBS as a chemoattractant. After incubating for 24 h at 37 °C, non-migrated cells on the top surface were gently eliminated with a cotton swab. Migrated cells on the bottom surface underwent fixing with 4% methanol, staining with 0.1% crystal violet (Solarbio, Beijing, China), and counting via an inverted microscope. For the invasion assay, the top chambers were pre-coated with Matrigel (Corning, USA), and all subsequent steps were identical to the migration assay. Trials were conducted at least three times independently.

Cytoskeletal/nuclear isolation

GC cell lysis was conducted on ice via a nuclear-cytoplasmic separation buffer (Beyotime, China) for 10 min, and then spinning was conducted at 800 × g for 10 min to acquire cytoplasmic (supernatant) and nuclear (pellet) fractions. Total RNA isolation from each fraction was conducted via the RNA Isolation and Extraction Kit (Fastagen Bio, China) and analyzed by RT-qPCR to estimate the subcellular localization of Linc-ROR. GAPDH and U6 served as internal controls for the cytoplasmic and nuclear fractions, respectively. Experiments were conducted in triplicate.

Dual luciferase assay

Plasmids with wild-type (WT) or mutated (MUT) Linc-ROR and CARMIL1 3′UTR sequences with predicted miR-145-5p binding sites were constructed by GeneCopoeia (MA, USA). For the dual-luciferase reporter assays, CS-MiT0036-MT06-01 was used as the WT Linc-ROR reporter. To validate the specific interaction between Linc-ROR and miR-145-5p, two single-site mutant reporters (Linc-ROR MUT1 and Linc-ROR MUT2) were generated, each harboring distinct mutations within one predicted miR-145-5p binding site. In addition, a double-mutant reporter (Linc-ROR MUT), in which both miR-145-5p binding sites were simultaneously mutated, was created. Specifically, GC cells were plated in plates with six wells and transfected at ~ 75% confluence with WT or MUT reporter plasmids together with miR-145-5p mimics or NC via Lipofectamine™ 3000 (Invitrogen, USA). Following 48 h, luciferase activities (Firefly and Renilla) were assessed via a dual-luciferase assay kit (Vazyme, Nanjing, China), and the Firefly/Renilla ratio was assessed for normalization. Trials were conducted at least three times independently.

Autophagy flux detection

GC cells were transduced with a tandem mRFP-GFP-LC3B lentiviral construct and plated on glass coverslips in plates with 24 wells. Following the suggested treatments, autophagic flux was assessed by confocal microscopy (Leica, Germany). For each condition, puncta were quantified and averaged from a minimum of five randomly selected fields per experiment using ImageJ (NIH, USA), with analyses performed from three independent biological replicates. Autophagosomes were defined as yellow puncta (mRFP⁺/GFP⁺), while autolysosomes were determined as red-only puncta (mRFP⁺/GFP⁻). Autophagic flux was considered enhanced when red-only puncta were elevated, or the ratio of yellow-to-red puncta was reduced, and impaired when red-only puncta were reduced, or the ratio of yellow-to-red puncta was elevated.

Transmission electron microscope scanning

To evaluate autophagosome and autolysosome dynamics, GC cells stably overexpressing Linc-ROR or transduced with an empty vector were cultured in 75 cm2 flasks until 75% confluence. Then, cells were exposed to 5 μM Everolimus (MCE, USA) for 24 h. Cells were collected, pelleted, and fixed in 0.5 mL electron microscopy-grade fixative (Servicebio, Wuhan, China) at room temperature for 30 min in the dark. Fixed samples were stored at 4 °C until further processing for TEM imaging, which was performed following standard protocols. For quantitative analysis, five randomly selected fields per condition were assessed in each experiment. The autophagosomes were quantified on a per-cell basis and averaged for each field. Analyses were performed from three independent biological replicates.

Xenograft mice experiment

Male BALB/c nude mice (four weeks, 18–20 g) were brought from Beijing Vital River Laboratory Animal Technology Co., Ltd. and kept in specific pathogen-free (SPF) environments. The sample size was determined using variability and effect‐size estimates derived from pilot xenograft experiments (Zhang and Hartmann 2023). Depending on the 3R principles of the institutional animal ethics committee, five animals per group were deemed sufficient to achieve adequate statistical power at α = 0.05. To establish subcutaneous xenografts, a suspension of AGS cells (1 × 10⁷) stably overexpressing Linc-ROR or vector control in 150 μL of PBS was introduced with a subcutaneous injection into the flanks of the mice. Tumor growth was observed every three days, and when tumors reached approximately 2 × 2 mm in size, mice were randomized to receive Everolimus (2.5 mg/kg/day) or vehicle control (PBS, 100 μL/day). Mice were sacrificed upon reaching the largest tumor diameter of 15 mm. Tumors were excised, weighed, and their volumes measured as: volume = (length × width2)/2. The harvested tumor tissues were subsequently processed for immunohistochemical (IHC) analysis.

Bioinformatics analysis

Gene expression profiles of GC, accompanied by matching clinical data, were acquired from The Cancer Genome Atlas (TCGA, TCGA-STAD; https://portal.gdc.cancer.gov) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo), including datasets GSE106817, GSE59856, GSE26253, and GSE34942. Raw count data from TCGA and GEO were preprocessed and normalized using standard pipelines in R (version 4.2.0). Differentially expressed lncRNAs between GC and healthy tissues were determined via DESeq2 with thresholds of |log₂ fold change (FC)|≥ 1 and P.adj < 0.05 (Benjamini–Hochberg correction for various testing). Spearman’s rank correlation coefficient was measured to estimate associations between lncRNAs, mRNAs, and miRNAs. Kaplan–Meier survival analyses were conducted to estimate the predictive significance of selected genes, and survival curves were visualized via the R survival and survminer and UALCAN (https://ualcan.path.uab.edu/index.html). P < 0.05 was deemed significant.

Statistical methodology

Continuous variables were reported as mean ± standard deviation (SD) from at least three independent trials. Two-group comparisons were conducted via Student’s t-test or the Mann–Whitney U test. Categorical variables were shown as counts or proportions, and comparisons were conducted via the Chi-square or Fisher’s exact tests. GraphPad Prism 9.0 was utilized for statistical analyses. The diagnostic performance of Linc-ROR was estimated by the measurement of the area under the receiver operating characteristic (ROC) curve (AUC) via the “pROC”. P < 0.05 was deemed significant.