Patient samples. A total of 20 human gastric cancer and corresponding and adjacent normal tissues were obtained from patients admitted to Shaanxi Provincial People’s Hospital (Xi’an, China) between July 2020 and December 2020. All participants provided written informed consent, and the study was approved by the Ethical Committee of Shaanxi Provincial People’s Hospital.
Bioinformatics analysisHuman gastric cancergene expression data were obtained from the Gene Expression Omnibus (GEO) dataset, GSE63288. Data analysis was performed using the DEGseq package of R software(1.12.0)(RStudio, Inc.). Genes withlog2|Fold Change|>1and significance of P<0.05 were considered to be differentially expressed genes.The binding sites between lncRNA TERC and miR-423-5pwere predicted usingstarBase database (http://starbase.sysu.edu.cn).The binding sites betweenmiR-423-5pand SOX12 were predicted using TargetScan 7.1 database (www.targetscan.org).
Cell lines and culture. The human gastric mucosal epithelial cells GES-1 and the human gastric cancer cell lines, NCI-N87,KATO3, Hs-746T, HGC-27 and SNU-1, were obtained from Procell Life Sciecne & Technology Co., Ltd. SNU-1, KATO3 and HGC-27 cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.)supplemented with 10 or 20% FBS(Gibco; Thermo Fisher Scientific, Inc.), respectively, and 1% penicillin-streptomycin solution. . GES-1,NCI-N87 and Hs-746T cell lines were cultured in DMEM medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS(Gibco; Thermo Fisher Scientific, Inc.), respectively, and 1% penicillin-streptomycin solution. Alll cells were maintained at 37˚C in a 5% CO2 humidified incubator.
Cell transfection. A total of 5x105HGC-27 and SNU-1 cells/well were seeded into a 6-well plate overnight at 37˚C. Then, cells were cultured in serum-free RPMI-1640medium for 2 h prior to transfection. Cells were subsequently transiently transfected with small interfering RNA (siRNA/si)-TERC (5’-CCTTCCACCGTTCATTCTA-3’), si-negative control (NC, 5’-UUCUCCGAACGUGUCACGUTT-3’) (both Guangzhou Ribobio Co., Ltd.), miR-423-5p mimic (5’-UGAGGGGCAGAGAGCGAGACUUU-3’)or mimic-NC (5’-UUCUCCGAACGUGUCACGUTT-3’) (both Shanghai GenePharma Co., Ltd.) using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol.
Cell Counting Kit-8 (CCK-8) assay. The proliferative ability of HGC-27 and SNU-1 cells was measured using a CCK-8 assay. Briefly, 5x103HGC-27 and SNU-1 cells/well were seeded into a 96-well plate and incubated overnight. Cells were then transfected for a further 48 h. Following the transfection, 10 μl CCK-8 solution was added to each well and incubated at 37˚C for an additional 4 h. The absorbance of each well was measured at a wavelength of 450 nm using amicroplatereader (Flexstation® 3; Molecular Devices, LLC).The experiments were performed in triplicate.
Cell migration and invasion assays. The migratory and invasive abilities of HGC-27 and SNU-1 cells were determined using Transwell plates (24-well inserts; Corning, Inc.). Briefly, for the migration assay, 5x105HGC-27 and SNU-1 cells/well were seeded into a 6-well plate and incubated overnight. Cells were transfected for 48 h, trypsinized and resuspended in serum-free RPMI-1640 medium at a density of 3x105cells/ml.A volume of 200 µl cell suspension was added intothe upper chambers of the Tranwell plate, while 800 μl growth medium (RPMI-1640 mediumsupplemented with 10 or 20% FBS) was added into the lower chambers. Following incubation for 24 h, the migratory cells were stained with crystal violet and counted using an inverted microscope (ECLIPSE Ts2; Nikon Corporation; magnification, x200). The invasion assay was performed as described, with a minor alteration in that the upper chamber of the Transwell plate was precoated with 100 µl Matrigel (1 mg/ml; Corning, Inc.).
Reverse transcription-quantitative PCR(RT-qPCR). Total RNA was extracted fromgastric cancer tissues, adjacent normal tissues, HGC-27 and SNU-1 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using random primers and Hiscript Reverse Transcriptase(GeneCopoeia Company, USA) for mRNA quantification. For miRNA quantification, the reverse transcription step was performed using an Oligo (dT) 18/miRNA loop and Hiscript Reverse Transcriptase. Primer sequences were listed inTable 1.The PCR reaction conditions were as follows: 10 minute at 95°C, and then 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C.Expression levels were quantified using the 2-ΔΔCq method (26).
Dual luciferase reporter assay. The binding relationships between lncRNA TERC, miR-423-5p and SOX12 were verified using a dual luciferase reporter assay. Briefly, the cDNA fragments of TERC and SOX12 containing the predicted miR-423-5p binding sites were inserted into the pYr-MirTarget luciferase reporter vector (Yingrun Biotechnologies Inc., Chian) to generate pYr-MirTarget-Homo SOX12-wild-type (WT) and pYr-MirTarget-Homo TERC-WT vectors, which are denoted as SOX12-WT and TERC-WT, respectively, henceforth.
A mutant (MUT) site in the miR-423-5p binding site was also designed and cloned into the pYr-MirTarget luciferase reporter vector to generate pYr-MirTarget-Homo SOX12-MUT (SOX12-MUT) and pYr-MirTarget-Homo TERC-MUT (TERC-MUT) vectors.The TERC or SOX12 plasmids (WT or MUT) were co-transfected with the miR-423-5p mimic or mimic-NC into 293T cells.Following 48 h of transfection,aDual Luciferase Reporter Gene assay kit(Beyotime Institute of Biotechnology) was used to determine the relative luciferase activity.
RNA immunoprecipitation (RIP) assay. An RNA-Binding Protein Immunoprecipitation kit (MilliporeSigma) was used, according to the manufacturer’s protocol, to determine the relationship between lncRNA TERC and miR-423-5p. Anti-argonaute 2 (AGO2)(SAB4200085, MilliporeSigma) and control IgG (R9255, MilliporeSigma) antibodies were used to performed the RIP assay, and the expression levels of lncRNA TERC and miR-423-5p were subsequently evaluated using RT-qPCR.
Western blotting. Relative protein expression levels were examined using western blotting as previously described[18]. Briefly, total protein was extracted from tissue samples and HGC-27 and SNU-1 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Proteins were separated via SDS-PAGE and then transferred to nitrocellulose membranes (MilliporeSigma). After blocking with 5% non‐fat milk for 2 hat room temperature,the membranes were incubated with the following primary antibodies at 4˚C overnight: Anti-N‐cadherin (cat. no.13116,1:1000 dilution), anti-E‐cadherin(cat. no.3195,1:1000 dilution), anti-MMP9 (cat. no.13667,1:1000dilution), anti-proliferating cell nuclear antigen (PCNA) (cat. no.13110,1:1000dilution), and anti-β-actin (cat. no.4970,1:1000 dilution) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-SOX12 (cat. no.13116,1:1000dilution)was purchased fromProteintech (Chicago, USA).Following the primary antibody incubation, the membranes were incubated withthe appropriate secondary antibodies(Cell Signaling Technology, Inc., cat. no.7074,1:1000dilution) for 2 hat room temperature. Protein bands were visualized using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.).
Statistical analysis. All data are presented as the mean ± SD. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, Inc.). Statistical differences between groups were determined using a Student’s t-test or one-way ANOVA followed by a Dunnett’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.