Tissue Samples
32 pairs of human primary NPC tissues and their matched normal adjacent tissues were obtained from January 2017 to July 2018 in The Second Hospital of Anhui Medical University. None of them had received any preoperative radiotherapy, chemotherapy or other medical intervention. Written informed consents from all patients were obtained from the patients prior to experiments, and this study was approved by the Ethics Committee of The Second Hospital of Anhui Medical University.
Cell Culture and Transfection
The C666-1 and CNE2 cell lines were provided by the Shanghai cell bank of the Chinese Academy of Sciences (Shanghai, China). DDP-resistant NPC cell strains (C666-1/DDP and CNE2/DDP) were established from the parental cell lines C666-1 and CNE2 by using an intermittent stepwise selection protocol over 6 months, and finally exposed to 1.2 mg/mL DDP. Cells were seeded into 1640 culture medium containing 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin, and 100 IU/ml streptomycin in 5% CO2 incubator at 37 °C and 95% humidity. The siRNAs against HOTAIR and SOX4, miR‑106a-5p mimic, miR‑106a-5p inhibitor and their respective controls were synthesized by Gene Pharma (Shanghai, China). Full-length cDNA of HOTAIR was inserted into the pcDNA3.1 empty vector. The cells were transfected using Lipofectamine® 3000 with HOTAIR siRNA, SOX4 siRNA, miR‑106a-5p mimic, miR‑106a-5p inhibitor, pcDNA3.1-HOTAIR or their respective controls.
Apoptosis assay
Apoptosis analysis was performed by using flow cytometer. Cells were collected and washed with PBS. Then they were resuspended, and 5 μL Annexin V-FITC and 5 μL PI were sequentially added. Cell apoptosis was detected by flow cytometry.
Cell Proliferation Assay
The cell concentration was adjusted to 3×104 / mL, and 100 μl per well was seeded in a 96-well culture plate. Each group was provided with 5 duplicate wells, which were cultured at 37°C for 48 hours. After 48 h of transfection, 96-well plates were removed, 10μl of CCK-8 solution was added to each well, and incubation was continued for 1 h. At 450 nm, the absorbance (A value) of each well was measured on a microplate reader, and the results were recorded. The experimental results were calculated as follows: cell viability (%) = 100% × (A value of each experimental group / A value of the cell control group).
Quantitative polymerase chain reaction (qPCR)
Total RNA extraction was conducted using Trizol Reagent (Shanghai Pufei Biotech Co., Ltd., Shanghai, China) to detect the relative expression of genes. The prepared cDNA was amplified using SYBR Green Master Mixture (Takara, Otsu, Japan), of which the results were calculated by LightCycler® 480 real-time PCR system (Roche, Indianapolis, Ind). The thermocycling conditions were applied as follows: DNA regeneration at 95°C for 5 min, 40 cycles at 95°C for 30 sec, followed by primer annealing at 60°C for 30 sec and primer extension at 72°C for 5 min.
Transwell Assay
Before the experiment, the cells were starved for 24 h and then collected. 100 μl of cell (6×105/ml) suspension was placed in the upper chamber of Transwell chamber. After incubated for 48 h, the cells adhering to the lower surface were fixed in methanol for 15 min and stained using 0.1% crystal violet for 30 min. The cells were counted under a light microscope.
Luciferase assay
The wild-type HOTAIR-3’-UTR (WT-HOTAIR-3’-UTR) or SOX4-WT (WT-SOX4-3’-UTR) and mutant HOTAIR-3’-UTR (MUT-HOTAIR-3’-UTR) or SOX4-MUT (MUT-SOX4-3’-UTR) containing the miR-106a-5p binding sites were cloned into the firefly luciferase-expressing psicheck2 vector (Promega, WI, USA) for luciferase reporter experiments and then co-transfected into cells. Luciferase assay was performed the firefly luciferase 48h post-transfection.
Western blotting
After washed with PBS, NPC tissues and cells were dissolved in commercial RIPA buffer. We got the supernatant after centrifugation at 12,000 rpm for 20 minutes. And then about equal amount of protein was loaded with 10% SDS-PAGE and transferred onto the PVDF membranes. The PVDF membranes were blocked and they were incubated overnight with primary antibodies at 4°C and then incubated for 2h at room temperature with a second anti-body temperature bound by horseradish peroxidase. Finally, the PierceTM ECL Western Blotting Substrate was added to completely cover the membrane and the collected images were analyzed by Image J.
Statistical Analysis
Values are expressed as mean±standard deviation. SPSS 20.0 was applied to analyze statistical data. Student t-test was used to indicate the difference between two groups while one-way ANOVA was performed for the difference between three or multiple groups. Each assay was repeated at least three times and P<0.05 was accepted as statistically significant.