Cell culture and clinical specimens
Human NPC cell lines (CNE-1, CNE-2, SUNE-1, HNE-1, HONE-1, 5-8 F, 6-10B) were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The human immortalized nasopharyngeal epithelial NP69 cell line was grown in keratinocyte serum–free medium (Invitrogen, Carlsbad, CA, USA) supplemented with bovine pituitary extract (BD Biosciences, San Jose, CA, USA). We maintained 293FT cells in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10 % FBS. In addition, 22 freshly frozen NPC biopsy samples and 12 normal nasopharyngeal epithelium samples were collected from Sun Yat-sen University Cancer Center. This study was approved by the Institutional Ethical Review Boards of our center, and written informed consent was obtained from each patient.
RNA isolation and reverse transcription–PCR (RT-PCR)
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and quantified at 260 nm by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA (2 μg) was reverse-transcribed to complementary DNA (cDNA) using an RT kit (Promega, Madison, WI, USA). Quantitative PCR was performed in triplicate using Platinum SYBR Green qPCR Super Mix-UDG reagents (Invitrogen, Carlsbad, CA, USA) on a CFX96 Touch™ sequence detection system (Bio-Rad, Hercules, CA, USA). The following primer sequences were used for amplification: YPEL3 forward, 5′-CCACGACGACCTCATCTC-3′; reverse, 5′-CATATTTCCAGCCCAAAGT-3′; E-cadherin forward, 5′-GAAGAGGACCAGG ACTTTGAC-3′; reverse, 5′- GTAGTCATAGTCCTGGTCTTTGTC-3′; Vimentin forward, 5′- TCAGACAGGATGTTGACAATGC-3′; reverse, 5′- TCATATTGCTGACGTACGTCAC-3′. GAPDH was used as the endogenous control, and the comparative threshold cycle (2-ΔΔCT) equation was used to calculate the relative expression levels.
Cultured cells were washed twice with ice-cold phosphate-buffered saline (PBS), solubilized in a lysis buffer containing 1 mmol/L protease inhibitor cocktail (FDbio Science, Hangzhou, China) on ice, and quantified using the bicinchoninic acid method. Cell lysate protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % skim milk in Tris-buffered saline–Tween (TBST) buffer (10 mmol/L Tris–HCl [pH 7.4], 150 mmol/L NaCl, 0.1 % Tween 20) for 2 h. Protein expression was detected following overnight incubation at 4 °C using primary antibodies against HA (1:2000, Sigma-Aldrich, USA); YPEL3 (1:100, Abcam, Cambridge, MA, USA), β-catenin (1:500, Proteintech, Wuhan, China), c-MYC (1:2000, Proteintech, Wuhan, China), cyclin D1 (1:500, Proteintech, Wuhan, China), α-catenin (1:500, BD Biosciences, San Jose, CA, USA), E-cadherin (1:500, BD Biosciences, San Jose, CA, USA), vimentin (1:500, BD Biosciences, San Jose, CA, USA), GSK3β(1:1000, Proteintech, Wuhan, China), TBP (1:800, Proteintech, Wuhan, China), and GAPDH (1:500, Proteintech, Wuhan, China). Thereafter, the membranes were washed and incubated for 1 h at room temperature with the appropriate horseradish peroxidase–conjugated secondary antibody. After the membranes were washed with TBST buffer three times, the proteins were visualized with an enhanced chemiluminescence reagent (Beyotime, Shanghai, China). The bands were analyzed using Image J software.
Stable cell line establishment and YPEL3 small interfering RNAs (siRNAs)
The pSin-EF2-puro-YPEL3-HA or pSin-EF2-puro-vector plasmids were obtained from Land. Hua Gene Biosciences (Guangzhou, China). All plasmids were verified by DNA sequencing before use; the pSin-EF2-puro-vector plasmid was used as the control. Stably transfected cells were selected using puromycin and were confirmed using quantitative RT-PCR. SiRNA#1 targeting YPEL3-Homo-974 (siYPEL3), which was obtained from GenePharma Co., Ltd (Shanghai, China), was a pool of siRNAs for the YPEL3 gene (sense strand: 5′-GCCACCUCUUCAACUCAGTT-3′; antisense strand: 5′-CUGAGUUGAAGAG GUAGGCTT-3′); siRNA#2 targeted YPEL3-Homo-838 cDNA (sense strand: 5′-GCGGAU UUCAAAGCCCAAGTT-3′; antisense strand: 5′-CUUGGGUUUGAAUCCGCTT-3′).
Wound healing assay
CNE-2 and SUNE-1 cells were seeded onto a 6-well culture plate and cultured to a subconfluent state in complete medium. After 24-h starvation in serum-free medium, cell monolayers were linearly scraped with a P-200 pipette tip. Cells that had detached from the bottom of the wells were gently aspirated and incubated in serum-free medium for 24 h. The width of the scratch was monitored under a microscope and quantified in terms of the difference between the original width of the wound and the width after cell migration.
Transwell migration and invasion assays
Transwell migration and invasion assays were carried out using Transwell chambers (Corning, Tewksbury, MA, USA) with 8-μm pore polyethylene membranes. For the migration assay, cells were placed in the upper chamber of each insert that had not been coated with Matrigel (BD Biosciences, San Jose, CA, USA). For the invasion assay, cells were placed in the upper chamber of inserts that had been pre-coated with Matrigel. Plasmid- or siRNA-transfected CNE-2 and SUNE-1 cells (5 × 104 for the migration assay;1 × 105 for the invasion assay) were added to the upper chamber in serum-free medium, and the lower chamber contained culture medium with 20 % FBS to act as a chemoattractant. The cells were incubated for 12 h or 24 h at 37 °C in 5 % CO2, and then they were fixed and stained. Cells on the undersides of the filters were observed and counted under × 200 magnification.
CNE-2 and SUNE-1 cells were cultured on coverslips in 6-well plates, washed with PBS, fixed with 4 % paraformaldehyde for 30 min, and permeabilized with 0.5 % Triton X-100 for 10 min. After blocking with 1 % bovine serum albumin for 1 h, the cells were immunostained with antibody against E-cadherin or vimentin at a 1:500 dilution. The cells were washed with PBS and incubated with Alexa Fluor–conjugated goat anti-mouse secondary antibody (1:200, A11011; Invitrogen). After washing with PBS, the nuclei were stained with DAPI (Invitrogen,) for 15 min, and fluorescence images were obtained using a confocal scanning microscope (OLYMPUS FV1000; Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus 6.0 software.
Female, pathogen-free, athymic nude mice were purchased from Charles River Laboratories (Beijing, China) (n = 9mice per group). SUNE-1 cells (1 × 106) stably overexpressing vector or YPEL3 that had been resuspended in 200 μL serum-free medium were injected intravenously into the tail veins of the mice. After 8 weeks, the mice were sacrificed and the lung tissues were fixed for calculating the numbers of macrometastatic nodes formed on the surface of lungs. Then, the tissues were paraffin-embedded, serial 5-μm tissue sections were cut, and one of every ten sections was stained with hematoxylin–eosin (HE) for examination of micrometastatic nodes formed in the lungs as previously described [18, 19]. All animal research was conducted in accordance with the detailed rules approved by the Animal Care and Use Ethnic Committee of Sun Yat-sen University Cancer Center and all efforts were made to minimize animal suffering.
All statistical analyses were carried out with SPSS software (standard version 17.0, Chicago, IL, USA). All experiments were performed in three independent experiments and all data are presented as the mean ± SD. Significant differences between two groups were analyzed using two-tailed unpaired Student’s t-test and a P-value <0.05 was considered statistically significant.