Patients and samples
A total of 36 paired GC and adjacent non-cancerous tissues (> 5 cm from cancer tissue) used in the study were obtained from the Fourth Affiliated Hospital of China Medical University. All samples were confirmed by histopathological examination and were stored at -80 °C immediately after surgical resection. The study was authorized by the Research Ethics Committee of the Fourth Affiliated Hospital of China Medical University and informed consent was obtained from all patients.
Cell Culture
Human GC cell lines ((BGC-823, AGS, MGC-803, and SGC-7901) and normal gastric epithelial cell line (GES-1) were purchased from the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI 1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were incubated in humidified air at 37 °C with 5% CO2.
Rna Isolation And Quantitative Real‑time Polymerase Chain Reaction (qrt‑pcr)
Total RNA samples were extracted from tissues and cell lines using RNAios Plus kits (TaKaRa Bio Technology, Dalian, China). RNA reverse transcription and quantitative PCR were performed with the ABI 7500 system (Applied Biosystems) using lnRcute lncRNA First-Strand cDNA Synthesis kit and SYBR Green qPCR kit (TIANGEN, Beijing, China). The primers used for qRT-PCR were listed in Table 1. Relative gene expression was determined using the 2−ΔΔCt method.
Cell Transfection
Small interfering RNAs (siRNAs) designed and synthesized for decreasing gene expression by RiboBio were siRNA-LINC01235 #1, #2, #3, and negative control siRNA-NC. The siRNA sequences are shown in Table 1. The siRNAs were transfected into GC cell lines (SGC-7901 and MGC-803) with riboFECT™ CP transfection kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. Gene silencing effect was detected by qRT‑PCR.
Transwell Migration And Invasion Assay
The numbers of SGC-7901 and MGC-803 cells were counted after transfection for 24 h. Cell invasion and migration assays were carried out in 24-well plates with or without pre-coated Matrigel Basement Membrane Matrix (BD Biosciences). A total of 2.0 × 104 cells with 200 mL of FBS-free RPMI 1640 medium were plated into the upper chambers of the Transwell plates (8.0-µm pore size, Corning, New York, NY, USA). Lower chambers were filled with 500 mL of medium with 10% FBS. After a 24-h incubation, cells adhered to the lower surface of the upper chambers were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Solarbio) for 30 min. Then, cell numbers per field of view were counted and photographed under a microscope.
Western Blot Analysis
The samples were lysed with RIPA buffer (Solarbio). Equivalent amounts of total protein were separated on 8% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk for 2 h by gentle shaking. The primary antibodies were then incubated at 4 °C overnight according to the following dilution ratio: E-cadherin (1:400; Wanleibio), N-cadherin (1:500), vimentin (1:500), Slug (1:1000), and β-actin (1:1000). The membranes were washed the following day and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:5000; Wanleibio). Finally, the signals were analyzed using enhanced chemiluminescence (ECL) substrate (Wanleibio) on a chemiluminescence detection system (Tanon, Shanghai, China).
Data Source And Bioinformatics Analysis
The RNA-Seq data and clinical information for GC were obtained from the UCSC Xena platform 14, which stores various datasets from TCGA. The lncRNA expression profiles were selected and used for screening differentially expressed lncRNAs with a limma package 15 in R software. Relative LINC01235 expression values in GC and normal tissues were also collected from three microarray datasets (GSE65801, GSE51575, and GSE63089) and the GEPIA platform 16. LINC01235 coding potential was calculated and predicted using CPC 2.0 project 17. Gene set enrichment analysis (GSEA) was carried out to explore potential molecular functions that can be influenced by the LINC01235 expression level 18. The hallmark gene sets and KEGG gene sets in the Molecular Signatures Database (MSigDB) were selected as the background during GSEA 19.
Statistical analysis
The SPSS software V19.0 and GraphPad Prism 7.0 were used for statistical analysis. LINC01235 expression levels between groups were analyzed by paired or unpaired test. Overall survival analysis was performed using the Kaplan-Meier method. The univariate and multivariate Cox regression analyses were used to test the effects of variables on survival. Pearson correlation analysis was utilized for calculating the expression correlation between genes. P-values < 0.05 were considered statistically significant.