GEPIA database analyses
The GEPIA online database includes RNA-seq data on 9736 tumor and 8587 control tissue samples derived from the TCGA and GTEx projects, providing tools to enable gene expression profiling as a function of disease type or stage and to conduct corresponding analyses[45]. Herein, the GEPIA database was utilized to assess LINC00467 expression profiles in 33 cancers including GC, with p < 0.05 as the significance threshold.
Cell culture and transfection
The AGS, HGC27, and MKN45 GC cell lines and the control GES-1 human cell line were from bnbio (China), and were grown in DMEM (Gibco) containing 10% FBS (Invitrogen) at 37°C in a humidified 5% CO2 incubator. Lipofectamine 3000 (Invitrogen) 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, miR-NC inhibitor) from GenePharma (Shanghai, China).
Quantitative Real-Time PCR (qPCR).
The methods used for qPCR have been previously described [46]. An RNAiso kit (TaKaRa) was used to extract total RNA based on the provided directions, after which cDNA was prepared from 1 µg of RNA with a TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) for miRNA quantification assays, after which a TaqMan miRNA kit (Applied Biosystems) was used for qPCR analyses with U6 as a normalization control. For mRNA expression analyses, 2 µg of total RNA was reverse-transcribed using a PrimeScript first-strand cDNA synthesis kit (Applied Biosystems ), after which SYBR® Premix Ex Taq™ II (Applied Biosystems) was used to conduct qPCR, based on the provided directions. The primers used are shown in Supplementary Table 1. All analyses were conducted with LightCycler 480 (Roche). GAPDH was used to normalize mRNA expression, with the 2−ΔΔCt method used to calculating relative gene expression.
RTCA proliferation assay
An xCELLigence system E-Plate Real-Time Cell Analyzer (RTCA) was used to monitor proliferation (Roche Diagnostics GmbH, Mannheim, Germany)[47] by assessing impedance values in each well and using these results to calculate cell index (CI) values. Briefly, cells were plated at 1.0 × 104 cells/well and grown for 24 h, after which they were treated and the CI was monitored for 15 h. CI values were used for normalization of the proliferation data.
Clonogenic assay
GC cells were plated in 6-well plates (4000 cells/well) and were treated for 24 h in appropriate assays. The media were then replaced with fresh media, after which cells were cultured for 10 days. Colonies were then fixed for 5 min with 70% ethanol and stained for 10 min with 0.05% crystal violet, after which colonies were counted and the diameters of the colonies were determined.
EdU incorporation assay
An EdU kit (BeyoClick™ EdU Cell Proliferation Kit) was used to assess GC cell proliferative activity. Briefly, cells were added to 24-well plates (2 x 104/well) and were treated as appropriate for 72 h, after which EdU was added for 3 h. Cells were then fixed for 15 min with 4% paraformaldehyde, 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.
Wound healing assay
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. At 24 and 48 h post-wounding, the monolayer was imaged with a Nikon microscope.
Transwell assays
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 with or without Matrigel coating. The plates were then incubated for 24 h 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 crystal violet and imaged under microscopy.
Luciferase reporter assay
GC cells were added to 24-well plates overnight, and were then transfected with luciferase reporter vectors containing WT or mutated (Mut) versions of the DPYSL3 3-UTR or the LINC00467 binding region (GeneChem, China) along with pre-miR-141-3p or a corresponding control construct control plasmids. At 48 h post-transfection, a dual-luciferase kit (Promega) was used to assess luciferase activity based on the provided instructions.
Patient samples
Samples of GC patient tumor and para-cancerous tissue samples were collected from 60 patients treated in the Department of Pathology between 2013 and 2015. Both primary tumors and metastatic lymph nodes were used to prepare tissue microarrays from formalin-fixed paraffin-embedded (FFPE) tissues. Patient follow-up data were obtained from the hospital and through direct follow-up.
Fluorescence in situ hybridization (FISH)
GC cells were first treated under non-denaturing conditions and were then hybridized with LINC00467 probes (GenePhama, China). DAPI (1:1000) was then used to stain nuclei for 5 min, after which a confocal microscope (SPE, Leica) was used to image the cells. ImageJ was used to analyze the images to assess the subcellular localization of LINC00467.
Western blotting
Protein samples were isolated 48 h after transfection, and equal amounts of protein (30 µg/sample) were separated on SDS-PAGE and transferred to PVDF membranes (Millipore). Blots were blocked with 5% non-fat milk for 1 h, after which they were stained with anti-DPYSL3 (Abcam, Cambridge, UK), and anti-GAPDH (CST), followed by a 1-h incubation with HRP-linked goat anti-mouse IgG (1:2000, CST). An enhanced chemiluminescence kit (Amersham Pharmacia Biotech) was used to detect protein bands, with Image Lab 2.0 (Bio-Rad) used for data analysis. GAPDH served as a loading control.
Biotinylated miRNA pulldown assay
Cells were transfected with biotinylated miR-141-3p mimics or miR-141-3p mutants using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). 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) at 4°C for approximately 3.5 hours. Subsequently, the bound RNA was eluted using wash buffer, and TRIzol (Beyotime Biotechnology, Shanghai, China) was used to purify the RNA for subsequent analysis. The biotinylated miR-141-3p mimics and mutants were synthesized by RiboBio (Guangzhou, China).
RNA immunoprecipitation (RIP) assay
Cells were transfected with miR-141-3p mimics and the miR-141-3p mutant using Lipofectamine 3000 (Invitrogen). After 48 hours, AGO2 immunoprecipitation was performed using an RNA immunoprecipitation kit (GeneSeed, Guangzhou, China) according to the kit’s instructions. Antibodies against IgG (ab172730) and anti-AGO2 (ab32381) were obtained from Abcam). 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 qRT-PCR for further analysis.
Glucose uptake, lactate production, and ATP production assays
GC cells were seeded in 96-well plates and analyzed using a colorimetric glucose uptake assay kit (AAT Bioquest, Sunnyvale, CA, USA) and a colorimetric L-lactate assay kit (AAT Bioquest) following the manufacturer’s instructions. For the ATP assay, 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 (Beyotime Biotechnology, Shanghai, China) according to the recommended protocol. The content was normalized to the cell number. Each experiment was performed in quintuplicate.
Assays of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)
The ECAR and cellular OCR were assessed using the Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s instructions. ECAR and OCR were determined using Seahorse XFe Glycolysis Stress Test Kit (103020–100; Agilent) and Seahorse XF Cell Mito Stress Test (103015–100; Agilent) Kit, respectively. Briefly, 1 × 104 cells per well were seeded into a Seahorse XFe 96 cell culture microplate. 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 FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone), and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rote/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-deoxy-glucose (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, resulting 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, one can determine the extent of the glycolytic reserve.
The initial introduction of oligomycin (2 µM final concentration per well), an ATP synthase inhibitor, leads to a reduction in oxygen consumption rate (OCR) associated with mitochondrial respiration and ATP production. Subsequently, the administration of FCCP (1 µM final concentration per well), an uncoupling agent, causes the collapse of the proton gradient and mitochondrial membrane potential. This disruption allows for unimpeded electron flow through the electron transport chain (ETC), maximizing oxygen utilization by complex IV and yielding peak respiration. The disparity between the basal respiration and the maximal respiration levels determines the spare respiratory capacity, which represents the respiratory capability available to meet increased energy demands. In the last injection stage, a combination of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor (0.5 µM final concentration per well), is introduced. This combination completely halts mitochondrial respiration, facilitating the calculation of non-mitochondrial respiration attributed to processes occurring outside the mitochondria. Data were analyzed by Seahorse XFe 96 Wave software. ECAR in mpH/minute and OCR is indicated in pmols/minute.
Xenograft model studies
HGC27 cells that had been transduced with sh-LINC00467 or sh-NC were subcutaneously implanted into the right flanks of BALB/c nude mice (4 weeks old, Viltalriver Inc., Beijing, China), with each mouse receiving 5 × 106 cells in 150 µL of FBS-free culture medium. Mice were housed in a climate-controlled facility (12 h light/dark cycle) with free food and water access, and tumors were measured twice per week with tumor volume being defined as tumor volume = (width × width × length)/2. Six mice were included per experimental group. On day 22 post-implantation, 1% pentobarbital sodium (50 mg/kg, Sigma) was used to euthanize mice, after which tumors were collected and weighed. All studies were approved based upon the protocols included in the blinded for peer review guide for the Care and Use of Animals.
Statistical analysis
SPSS 19.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) were used for all statistical analyses. Correlations between patient clinicopathological findings and LINC00467 expression were assessed via χ2 tests, while other differences between groups were compared using Student’s t-tests and ANOVAs. Spearman correlation analyses were used to evaluate the association between LINC00467 and miR-141-3p or DPYSL3 in tumors, with p < 0.05 as the significance threshold.