Cell lines
The in vitro experiments were carried out in three representative NPC cell lines expressing low levels of Snail (S26, 6-10B, HK1) and two representative cell lines expressing high levels of Snail (S18, 5-8F) [2,3,4]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco). All cell lines were authenticated using short-tandem repeat profiling within 6 months prior to the experiments. All five cell lines were obtained from Sun Yat-sen University Cancer Center (SYSUCC).
Plasmids
The full-length cDNAs of human TEL2 and Snail were cloned into pBABE-puro vector (Cell Biolabs, INC). An HA tag was inserted to the N-terminus of TEL2. The fusion protein Snail-T2A-TEL2, in which Snail and TEL2 were linked by a T2A linker, was also expressed using the pBABE-puro vector. Mutations were introduced using the Quick-Change Site-Directed Mutagenesis Kit (Stratagene), and verified with DNA sequencing.
Antibodies
Antibody against human TEL2 (Dilution, 1:1000) was obtained from Sigma (HPA029033). Antibodies against human Snail were obtained from R&D (AF3639) for chromatin immunoprecipitation (ChIP) assays and from Cell Signaling Technology (#3895) for Western blotting. Anti-E-cadherin antibody was obtained from BD Company. Antibodies against HA and Tubulin were obtained from Cell Signaling Technology. Antibody against human SERPINE1 was obtained from Santa Cruz (sc-5297).
RNA interference
Cell lines stably expressing short hairpin RNA (shRNA) targeting Snail transcripts or negative control scrambled shRNA were established using kits from Sigma. The sequences of the 2 human Snail shRNAs are: 5′-ATGCTCATCTGGGACTCTGTC-3′ and 5′-TGCTCCACAAGCACCAAGAGT-3′. Sequence of the short interfering RNA (siRNA) targeting human TEL2 is: 5′-GCCAGATGTGAAGCTCAAATTA-3′.
RNA extraction and qRT-PCR
These procedures were performed as described previously [3, 17, 18]. Briefly, total RNA was isolated using Trizol reagent (Invitrogen). First-strand cDNA was synthesized using the Revert Aid™ First Strand cDNA Synthesis Kit (MBI Fermentas). The primers were used to amplify target sequences (Additional file 1: Table S1).
Migration and invasion assays
For transwell migration assays, 1.5 × 104 cells (S18, 5-8F) or 3.5 × 104 cells (S26, 6-10B) in 200 μl of serum-free DMEM were added to cell culture inserts with an 8-μm microporous filter without extracellular matrix coating (Becton–Dickinson Labware). DMEM medium containing 10% FBS was added to the bottom chamber. After 24-h incubation, the cells in the lower surface of the filter were fixed, stained, and examined using a microscope. The numbers of migrated cells from triplicate filters were counted in three random optical fields (×100 magnification) and averaged.
Transwell invasion assays were carried out in the same way, except that the chamber inserts were precoated with Matrigel (Becton–Dickinson Labware).
Chromatin immunoprecipitation (ChIP) assay
The assay was conducted using a commercial ChIP kit from Active &Motif (cat#: 53040). Briefly, cells were seeded onto 15-cm plates and allowed to grow to 70%–80% confluence. Cells were fixed and collected, and the nuclear pellet was resuspended in ChIP Buffer, subjected to sonication and incubated overnight with 5-μg antibody, followed by incubation with protein G agarose beads for 3 h at 4 °C. DNA–protein complexes were eluted and de-cross-linked through a series of washes. Purified DNA was resuspended in TE buffer and analyzed with PCR using the following primers: E-cadherin-ChIP-F, 5′-ACTCCAGGCTAGAGGGTCACC-3′; E-cadherin-ChIP-R, 5′-CCGCAAGCTCACAGGTGCTTTGCAGTTCC-3′; TEL2-ChIP-E1-F, 5′-TGAATGTGCATTAGTTTATCAAGCC-3′; TEL2-ChIP-E1-R, 5′-CAATCTGCCTACCAGAAATTTATTC-3′; TEL2-ChIP-E2-F, 5′-CACAGTCACGGCTCACTGCAG-3′; TEL2-ChIP-E2-R, 5′-GAGTTGGACACCAGTCTGAACAAC-3′; TEL2-ChIP-E3-F, 5′-GGAGCGCTCAAGACAGAAAGC-3′; TEL2-ChIP-E3-R, 5′-AAAATAGGTTTGGAAATCTAGGTGG-3′; TEL2-ChIP-E4-F, 5′-AGGCAGTAGAGTGGTTAACACAAAC-3′; TEL2-ChIP-E4-R, 5′-TTTATGGAGTTCTCTGTGGATCATG-3′; GAPDH-ChIP-F, 5′-TTCTTGCCTTGCTCTTGCTACTC-3′; and GAPDH-ChIP-R, 5′-AGCCTGCCTGGTGATAATCTTTG-3′.
Luciferase assay
The assay was carried out as described previously [2, 4]. Briefly, cells were plated in 12-well plates at a density of 2 × 105 per well, and transfected with 0.8-μg promoter-luciferase plasmid. To normalize transfection efficiency, cells were co-transfected with 8-ng pRL-CMV encoding Renilla luciferase. After transfection for 48 h, luciferase activity was measured using the Dual-Luciferase Assay kit (Promega). Three independent experiments were performed, and means and standard deviations are presented.
Animal experiments
Experiments involving animal subjects and all protocols for animal studies were approved by the Research Animal Resource Center of Sun Yat-sen University, in full compliance with the guidelines of the Institutional Animal Care and Use Committee at Sun Yat-sen University Cancer Center. Male athymic mice aged 5–6 weeks were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China). Human NPC cells were resuspended in 100-μl phosphate-buffered saline (PBS; Biological Industries) and injected into the lateral tail vein of mice (3 × 106 cells/animal). At 6 weeks after injection, mice were euthanized. Metastatic nodules were counted with the naked eyes.
Clinical samples
Experiments involving human tissue samples were approved by the Institutional Review Board of Sun Yat-sen University Cancer Center (YB2015-010). Written informed consent was obtained from all subjects prior to sample collection. Tissue blocks prepared from NPC tissues (10 cases) and lymph node metastases (4 cases) were stored for qRT-PCR.
Statistical analysis
Differences between two groups were assessed using Student’s t-test. Differences among three or more groups were assessed using parametric ANOVA and the least significant difference (LSD) test. Statistical significance was set at P < 0.05 (2-sided).