Human gastric cancer tissue specimens
Gastric cancer tissues and paired adjacent normal gastric epithelial tissues were obtained from the Third Affiliated Hospital of Soochow University from 2003 to 2015. Tissue specimens were obtained though needle puncture and stored in liquid nitrogen at -80°C. All experiments using the human tissues were conducted under the approval of the Third Affiliated Hospital of Soochow University. All patients gave informed consent.
Cell lines and cell culture
All cells in this study were obtained from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences. HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. Gastric epithelial cell GES-1 and gastric cancer cells AGS, SGC-7901 and HGC-27 were cultured in RPMI 1640 supplemented with 10% fetal bovine serum.
Expression plasmids and siRNA
The full-length human lncRNA GRIK1-AS1 cDNA was cloned into pLVX-IRES-Puro vector (Clontech) to generate GRIK1-AS1 expression plasmids. The siRNA targeting IFIT2 was purchased from GenePharma.
Cell transfection
siRNA duplexes (100nM), miR-375 mimic and corresponding negative control (NC) oligonucleotides were transfected into cells mediated by Lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions.
Lentivirus production and infection
To establish individual stable cells, lentivirus was used. Lentiviral particles in cell culture supernatant were harvested at 72h after lentivirus transfection into HEK293 cells. The AGS and SGC-7901 cells were then infected with lentivirus. Stably transfected cells were selected by puromycin (2mg/mL) for 1 week at 48h after lentiviral infection.
Cell proliferation assays
Cell proliferation assay was performed using CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) kit (Promega) according to the manufacturer’s instructions. Cells (1×104cells/mL) were seeded into a 96-well plate (100 ml/well) and cultured in an incubator with 5% CO2 at 37°C. Each well was added with 10 ml MTS solution and then incubated at 37°C for 2h. The spectrophotometric absorbance at 590 nm was measured for each sample. All the experiments were repeated 3 times in triplicates.
Transwell assay
The cell invasion ability was measured by transwell assay, in which transwell chambers (8-μm pore size; Corning) was applied. Cells (1×105) were seeded into the upper chamber with serum-free RPMI 1640 medium, and RPMI 1640 medium supplemented with 20% fetal bovine serum was added into the lower chamber. After 48h incubation, cells on the top surface of filters in the upper chambers were cleaned up gently with cotton swab. Then, the filters were fixed in 4% PFA for 15 min and stained with 0.1% crystal violet for 10 min. After washed by PBS for 3 times, cells migrated across the filters were imaged and the number of invaded cells was calculated by counting five random views under a microscope. The experiment was performed in triplicates and repeated for 3 times.
Wound healing assay
Wound healing assay was performed to determine the cell migration capability. Cells were seeded in 6-well plates. When the cells were about 90% confluent, the artificial wounds were cut using a 200-ul pipette tube. After 24h, the wound closure was observed and imaged under a microscope. The migration distance was indicated by the fraction of cell coverage across the line. Triplicates were required for each experiment.
RNA isolation and real-time PCR
Total RNA was extracted using TRIzol according to the manufacturer’s instructions. First strand cDNA was synthesized using Superscript II (Invitrogen) and 1μg of total RNA was used in each cDNA synthesis reaction. SYBR green Universal Master Mix reagent (Roche) and primer mixtures were used for the real-time qPCR. GAPDH was used as the internal reference for mRNAs.
miRNAs expression level was measured using the All-in-OneTMmiRNAqRT-PCR Detection Kit (GeneCopoeia)according to the manufacturer’s instructions.U6 small RNA was used as the reference. The primers used for real-time qPCR were as follows: LncRNA GRIK1-AS1, forward: 5’-ATGGAGGATGCAGCAAAAGGG-3’; reverse: 5’-TTCTGTGTCCTGGTTGTTTCTC-3’;IFIT2, forward: 5’-AAGCACCTCAAAGGGCAAAAC-3’; reverse: 5’-TCGGCCCATGTGATAGTAGAC-3’;GAPDH, forward: 5’-CTGGGCTACACTGAGCACC-3’; reverse: 5’-AAGTGGTCGTTGAGGGCAATG-3’;U6, forward: 5’-CTCGCTTCGGCAGCACA-3’; reverse: 5’-AACGCTTCACGAATTTGCGT-3’.
Nuclear mass separation
SurePrepTM Nuclear or Cytoplasmic RNA Purification Kit (Fisher BioReagents) was applied to isolate nuclear and cytoplasmic fractions according to the manufacturer’s instructions. RNA levels of lncRNA GRIK1-AS1, the nuclear control RNU6-1 and the cytoplasmic control GAPDH were analyzed by real-time qPCR.
RNA-Fluorescence in situ hybridization (RNA-FISH)
Cells were fixed in 4% PFA for 15 min and then permeabilized by 0.5% TritonX-100 for 15 min at 4°C.0.5% TritonX-100 was used to permeabilize the cells for 15 min at 4°C. Digoxigenin (DIG) labeled GRIK1-AS1 probe or control probe mix were performed to incubate cells for 4h at 55°C. After 2xsaline-sodium citrate briefly washing 3 times with 5 min every time, signals were detected by Horseradish peroxidase (HRP)-conjugated anti-DIG secondary antibodies (Jackson). DAPI was used to counterstain nuclear. Con-focal laser scanning microscope was used for images.
Western blotting analyses
Total proteins were extracted and separated using SDA-PAGE gels. IFIT2 antibody (Abcam) was used at a concentration of 1:2000. GAPDH antibody (Santa Cruz Biotechnology, 1:2000) was used as a loading control.
Reporter vector construction and luciferase reporter assays
GRIK1-AS1-WT luciferase reporter vector was conducted by cloning GRIK1-AS1 cDNA containing predictive miR-375 binding site into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). GRIK1-AS1-Mut vector was generated by insertion of mutant GRIK1-AS1 containing point mutations in miR-375 binding site. Likewise, wild-type and mutant IFIT2 3’-UTR fragments were cloned into pmirGLO vector to conduct the IFIT2 3’UTR-WT and IFIT2 3’UTR-Mut luciferase reporter vectors. The miR-375 or miR-NC was co-transfected with the reporter vector into HEK293 cells using Lipofectamine 3000 (Invitrogen). The luciferase activity was measured at 48h after transfection using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Triplicates were required for each experiment.
RNA-pull down
3’-end biotinylated miR-375, miR-375-Mut or candidate miRNAs were transfected into cells at a final concentration of 20 nmol/L. After 24h, cells were harvested and incubated in the cell lysate with streptavidin-coated magnetic beads (Ambion, Life Technologies). The biotin-coupled RNA complex was pulled down and analyses of the abundance of GRIK1-AS1 was conducted by real-time qPCR.
Statistical analysis
All experiments were performed using 3 independent repeated experiments with cells. GraphPad Prism 8.0 was applied for statistical analyses. Data in all figures is presented as the mean ± SEM. Statistical significance was determined by Student’s t test, One-way ANOVA and Two-way ANOVA. For all statistical tests, the 0.05 level of confidence (2-sided) was accepted for statistical significance.