Cell culture
HIOEC cells were purchased from BeNa Culture Collection (BNCC, Beijing, China). CAL-27, TCA-8113, SCC-4, SCC-9 and SCC-15 cells were all obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HIOEC cells were cultured in Dulbecco's modified Eagle's medium: nutrient Mixture F-12 (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Other cells were cultured in DMEM medium containing 10% FBS, 100U/mL penicillin and 100U/mL streptomycin. All the cell lines were incubated in a fully humidified atmosphere with 5% carbon dioxide at 37˚C. Change the medium regularly and observe the growth of cells on time. Cell passage are carried out when the cell confluence reach to 90%. Logarithmic phase cells were taken for follow-up experiment.
Cell transfection
Short-hairpin RNA (shRNA) against HOXA-AS2 (shRNA HOXA-AS2-1, shRNA HOXA-AS2-2) and corresponding empty vector (shRNA NC) were purchased from GenePharma (Shanghai, China). Liposome 3000 transfection reagent (Thermo Fisher Scientific) were used to transfect into TCA-8113 cells following the product instruction. After 48 hours, qRT-PCR was used to detect.
qRT-PCR analysis
Total RNA was extracted by Trizol. The optical density (OD) at 260 nm and 280 nm was measured by ultraviolet spectrophotometer respectively. Meanwhile, the concentration and purity of RNA were calculated. According to the All-in-One™ qRT-PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) instruction, RNA was reverse transcribed into cDNA and examined by the qRT-PCR. GAPDH was taken as the internal reference, the relative expression of target gene was calculated by 2−ΔΔCt method.
CCK-8 assay
Cells in the logarithmic growth period were seeded into 96-well plate at a density of 2 × 103/well for routine culture. After 24 h, 48 h, 72 h and 96 h, 10 µL CCK-8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) was added to each well and incubated continued for 2 h. The absorbance of each well was measured by a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm wavelength.
EdU staining assay
Cells concentration were adjusted to 1 × 105 cells/ml, seeded into 96-well plates and cultured continued overnight at 37˚C in 5% carbon dioxide. According to the EdU immunofluorescence staining kit (Ribobio) instructions, the concentration of EdU solution was adjusted to 10 µM and 100 µL diluted solution was added to each well. Culture medium was discarded after incubation for 4 hours and cleaned with PBS twice. Cells were fixed with paraformaldehyde and stained with Apollo. After staining was completed, observe and take pictures under a fluorescence microscope (Olympus BX 60 fluorescence microscope, Japan).
Tunel staining assay
The apoptotic cells were stained according to the operation of the TUNEL staining kit (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, Jiangsu, China) and were observed by phase-contrast microscopy. Apoptotic nuclei were stained brownish yellow.
Flow cytometry analysis
Cell-cycle distribution was detected by flow cytometry (FACScan, Becton 4 Dickinson). Apoptosis detection refers to the instruction manual of the Annexin V-FITC Apoptosis Detection Kit (Sangon, China). Treated cells were diluted into 1 × 106 cells/ml suspension and resuspended in 500 µl buffer solution. 5 µl PI and 5 µl Annexin V-FITC were added to incubate 30 min in the dark at room temperature. Analysis of apoptotic cells was performed by flow cytometry.
Cell migration and invasion assay
The ability of cell migration was analyzed by a wound scratch test. According to the experimental requirements, cells were seeded into 6-well plates, after the cells grew to complete fusion, the layer of cells was scratched to form wounds using a sterile 20 µl pipette tip and cultured for 48 hours. The image of the scratched area was captured under the phase contrast microscope and the cell healing area was analyzed by ImageJ software (NIH, USA). Transwell invasion assay was used to evaluate cell invasion. 8 µm transwell chamber (Corning, cat:354578) was applied to this experiment. Apply 5 µl Matrige (Becton Dickinson, Franklin Lakes, NJ, USA) to the chamber and adjust the cell concentration, then continuing to culture normally for 4 hours, the excess cells in the inner side of the chamber were washed with PBS twice, fixed with polyformaldehyde. Finally, the images were observed by the inverted microscope (Leica).
RNA immunoprecipitation (RIP) assay
RNA immunoprecipitation assay was conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (EMD Millipore) following the manufacturer’s protocols. RIP was treated with anti-EZH2 antibody (Abcam, Cambridge, MA, USA) and IgG was used as control. Coprecipitated RNAs were detected by qRT-PCR.
Western blot analysis
Total protein was extracted with RIPA lysis and protein concentrations were determined with a Pierce® BCA Protein Assay Kit (Thermo). Proteins on the SDS-PAGE gel were transferred to the PVDF membrane (Millipore, USA) and 5% skimmed milk powder was sealed for 1 h at 37℃. These membranes were treated with primary antibodies against CDK2 (1:1000; cat. no. #2546; Cell Signaling Technology Inc., USA), cyclinE1 (1:1000; cat. no. #20808; Cell Signaling Technology Inc., USA), P27 (1:1000; cat. no. #3686; Cell Signaling Technology Inc., USA), MMP-2 (1:1000; cat. no. #40994; Cell Signaling Technology Inc., USA), MMP-9 (1:1000; cat. no. #13667; Cell Signaling Technology Inc., USA), Bcl-2 (1:1000; cat. no. #3498; Cell Signaling Technology Inc., USA), Bax (1:1000; cat. no. #5023; Cell Signaling Technology Inc., USA), cleaved caspase-3 (1:1000; cat. no. #9661; Cell Signaling Technology Inc., USA), P21 (1:1000; cat. no. #2947; Cell Signaling Technology Inc., USA) and GAPDH (1:1000; cat. no. #5174; Cell Signaling Technology Inc., USA) at 4 ℃ overnight, respectively. Blots were then washed 3 × 10 min with TBST and incubated with secondary antibodies conjugated to horseradish peroxidase (1:500; sc2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. The immunoreactive bands were detected using the ECL kit (GE Healthcare). GAPDH was taken as an internal reference.
Statistical analysis
All data analyses were performed using GraphPad Prism6.0 software (GraphPad Software Inc., San Diego, CA, USA). The data were expressed as mean ± standard deviation (SD) and the differences between samples were analyzed by Student's t test or one-way ANOVA. P༜0.05 indicated that the difference was statistically significant. All experiments were carried out for three times or more, independently.