The RNA-Seq and clinical data of stomach cancer patients from UCSC Xena datasets 11, RNA-Seq data of multiple human normal tissues from The Genotype-Tissue Expression (GTEx) project 12, and protein staining data from The Human Protein Atlas 13 were downloaded and used for further analyses.
Patients and tissues
Thirty GC tissue specimens were obtained from patients who underwent stomach surgery at the Fourth Affiliated Hospital of China Medical University (Shenyang, China). All specimens were confirmed by histopathological examination and were stored at -80°C immediately after surgical resection. All patients provided written informed consent and the study protocol was approved by the Research Ethics Committee of the Fourth Affiliated Hospital of China Medical University.
Cell Culture and quantitative real-time RT-PCR
Detailed protocols were performed as described in our previous study 10. Specific primers for qRT-PCR are shown in Supplementary Table 1.
The specific siRNA against LINC02532 (Si-LINC02532 #1 and #2), FOXF2 (Si-FOXF2 #1 and #2), SOX7 (Si-SOX7), and the corresponding negative control (Si-NC), together with the pcDNA3.1 vector targeting LINC02532, FOXF2, SOX7, and the corresponding empty vector, were constructed by RiboBio (Guangzhou, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was utilized to transfect these vectors into SGC-7901, MGC-803 and HEK293T cells. The primer sequences are shown in Supplementary Table 1.
RNA-Fluorescence in situ hybridization (FISH) and protein coding potential
The subcellular location of LINC02532 was first predicted by the bioinformatics tools, lncLocator 14 and iLoc-LncRNA15. The FISH assay was carried out to confirm the subcellular location of LINC02532 using the FISH Kit (GenePharma, Shanghai, China) with Cy3-labeled LINC02532 probes according to the manufacturer’s protocol (Supplementary Table 1). The protein coding potential of LINC02532 was predicted by LNCipedia version 5.2 16, which integrates different algorithms from PRoteomics IDEntifications (PRIDE) 17, translation initiation sites18, PhyloCSF19, Coding Potential Assessing Tool (CPAT) 20 and small ORFs21.
Cell migration and invasion assays
The Transwell assays were performed as described in our previous study 10.
Western blot analysis
Samples were lysed using the RIPA buffer (Solarbio, Beijing, China). 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. Then, the primary antibodies were incubated at 4°C overnight according to the following dilution ratios: E-cadherin (1:200; Abcam, Cambridge, MA, USA), N-cadherin (1:2000; Abcam), Vimentin (1:1000; Abcam), Slug (1:2000; Abcam), SOX7 (1:1000; Abcam), and β-actin (1:2500; Abcam). The membranes were washed the following day and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:5000; Wanleibio). Finally, enhanced chemiluminescence (ECL) substrate was used to analyse the signals with a chemiluminescence detection system (Tanon, Shanghai, China).
Prediction of target binding sites and dual-luciferase reporter assay
The sequence from -2000 to +100 bp relative to the transcription start site (TSS) of LINC02532 was regarded as the promoter region. The transcriptional binding sites of FOXF2 on the gene promoter region of LINC02532 were predicted by the JASPAR CORE database 22. The RNA-RNA interaction binding bites between LINC02532 and the 3’UTR region of SOX7 mRNA were obtained using the IntaRNA program 23.
Further verification of target binding sites was conducted using the dual-luciferase reporter assay. For the LINC02532-SOX7 3’UTR luciferase reporter assay, sequences containing the predicted RNA-RNA interaction binding bites (pGL3-SOX7 3’UTR-WT/Mut) were inserted into the pGL3-basic firefly luciferase reporter (Promega, Madison, WI, USA), then co-transfected with pcDNA3.1-LINC02532 or empty vector into HEK293T and MGC-803 cells. For the LINC02532 promoter luciferase reporter assay, sequences containing the predicted FOXF2 binding sites (pGL3-LINC02532-WT/Mut) were also inserted into the pGL3-basic firefly luciferase reporter, then co-transfected with pcDNA3.1-FOXF2 or empty vector into HEK293T and MGC-803 cells. After 24 h of transfection, relative luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) and normalized to the control Renilla.
mRNA stability assay
SGC-7901 and MGC-803 cells (1 × 106) transfected with Si-LINC02532 #1, #2 and Si-NC were seeded into six-well plates. Then, 5 μg/ml of Actinomycin-D (ActD, MedChem Express) was added to the medium to block the de novo RNA synthesis. After treatment for 3, 6, and 9 h, cells were harvested and total RNA was isolated for measuring SOX7 mRNA levels by qRT-PCR.
Subcutaneous xenograft models
In vivo assays were conducted using BALB/C nude mice (female, 4 weeks old). A total of 1 × 106 MGC-803 cells transfected with LINC02532 overexpression or negative control were injected into the dorsal right flank, respectively (n = 4 per group). Tumour diameter and volume were measured and calculated every 3 days for 3 weeks. All experiments were approved by the Research Ethics Committee of the Fourth Affiliated Hospital of China Medical University and carried out based on the guidelines of the Institutional Animal Care and Use Committee.
Lung metastasis assay
For the lung metastasis model, the corresponding GC cells were injected into the tail veins of nude mice. Then, a total of 10 nude mice were randomly separated into two groups (5 mice in each group). Six weeks after the injection, the nude mice were euthanatized. Next, all the lung tissues were dissociated and metastatic nodules were counted.
Gene set enrichment analysis (GSEA)
GC patients from The Cancer Genome Atlas (TCGA) were divided into high or low expression groups according to the median value of LINC02532 expression. GSEA was performed on the RNA-Seq data to explore differences in biological pathways between high and low LINC02532 expression groups.
SPSS version 20.0 (SPSS, Inc., Chicago, IL, USA) and Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) statistical software were used for statistical analysis. The difference between two groups was compared with two-tailed Student’s t-test, and data were presented as the mean ± standard deviation. A chi-squared test was used to compare the clinicopathologic characteristics of GC patients and LINC02532 expression level. Pearson correlation analysis was used to assess gene expression relationships. The optimal cut-off value for dividing patients into high or low LINC02532 expression groups was calculated using X-tile software 24. The univariate and multivariate Cox regression analysis were carried out to assess the prognostic value of LINC02532 expression. A nomogram for the 1-, 2-, and 3-year OS prediction was constructed using LINC02532 expression and clinical factors (p < 0.2 was used to select variables to construct the model). Kaplan–Meier survival curve and log-rank tests were performed to evaluate the overall survival in relation to LINC02532 expression. P < 0.05 indicated a significant difference.