Patients and Tissue Samples
Tumor microarrays were assessed in this study. A total of 233 patient samples were collected from the Stomatological Hospital of Jiangsu Province (In 2014-2019), including 206 primary OSCC samples and 27 normal oral mucosae. Clinical and pathological data were listed in Table 1. Patient clinical information included age, gender, location, tumor size, histological grade, metastatic lymph node, clinical stage (defined by the American Joint Committee on Cancer 7th edition), and postoperative survival rate (as of April 2021). Besides, we collected 8 samples of tumor tissues and adjacent normal tissues from patients diagnosed with OSCC from the Stomatological Hospital of Jiangsu Province (In 2020). Moreover, all samples were stored in -80℃ liquid nitrogen for subsequent experiments. Corresponding clinicopathological data are shown in Table S1.
This study was approved by the Ethics Committee of Nanjing Medical University. Informed consent was obtained from all patients.
Tissue microarrays were stained with primary antibodies against eIF6 (diluted 1:200, Abcam) overnight following secondary antibody incubation for 30 minutes. All of the sections were counterstained using haematoxylin, dehydrated, cleared and mounted before examination using a microscope (DM4000B, Leica, Germany). eIF6 immunoreactivity in microarray samples was calculated according to staining concentration and proportion semi-quantitatively. The score for the scale of positive cells was demonstrated as follows: 0, negative, 1, < 20%, 2, 20- 50%, 3, 51-75%, and 4, > 75% positive cells. For staining strength, grading system was classified as below: 0, no staining, 1, light yellow, 2, brownish yellow, 3, dark brownish yellow. The result was calculated by multiplying the two scores as mentioned above. Scores for > 4 points were regarded as positive.
Cell culture and inhibitor
Human OSCC cell lines HN4 and HN6 were obtained from the Shanghai Ninth People's Hospital (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (FBS, HyClone, USA) in a humid environment at 37°C with 5% CO₂. LY294002 was purchased from Selleck (Selleck Chem, Houston, USA) and was dissolved in Dimethyl Sulphoxide (DMSO). DMSO was used for control.
eIF6, negative control (NC), sh-NC and sh-eIF6 (sh-eIF6-1, sh-eIF6-2) were all purchased from GemmaPharma (Suzhou,China). According to the manufacturer's protocol, plasmids were transfected into HN4 and HN6 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). The transfected cells were cultured in a complete medium for at least 48 hours before performing the next experiment.
Total protein was lysed with lysis buffer (Beyotime, China) containing phosphatase inhibitor and protease inhibitor cocktails. Utilizing Coomassie Brilliant Blue as the standard, protein lysate was quantified with the bovine serum albumin (BSA). The total proteins were loaded into SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) with 5% BSA at room temperature for 2h. Next, the membrane was incubated with primary antibodies (diluted 1:1000) specific for eIF6 (Abcam), E-cadherin, Vimentin, N-cadherin, AKT, p-AKT, and PI3K (CST), EGFR, p-EGFR, and β-actin (Bioworld, China), ZEB1 and ZEB2 (Proteintech, USA) and incubated overnight at 4℃. Samples were then washed three times with PBST for 10 minutes and incubated with anti-goat IgG HRP-conjugated secondary antibodies (Zhongshan Goldenbridge Bio, China) for 1 hour at room temperature. Finally, the immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized with the ImageQuantLAS4000 mini imaging system (General Electric). ImageJ software was used for gray value analysis and β-actin was used as an internal control. Each experiment was independently repeated three times and quantitatively analyzed.
RNA extraction and quantitative reverse transcription PCR (qRT-PCR)
According to the reagent instructions, total RNA was obtained using TRIzol reagent (Invitrogen) and then reverted to cDNA using 5 × PrimeScript RT Master Mix (TaKaRa) after 15 minutes at 37℃ and 5 seconds at 85℃. Quantitative Real-Time PCR in a 7900HT Real-Time PCR System (Applied Biosystems). The RNA levels of eIF6 and GAPDH were determined with the following primers: eIF6: F: 5′- CCGACCAGGTGCTAGTAGGAA-3′, R: 5′- CAGAAGGCACACCAGTCATTC-3', AKT: F: 5′- AGCGACGTGGCTATTGTGAAG-3′, R: 5′- GCCATCATTCTTGAGGAGGAAGT-3′, EGFR:F: 5′- AAAGTTAAAATTCCCGTCATCAG-3′, R: 5′-TCACGTA GGCTTCATCGAGATTTC-3′ , PIK3CA:F: 5′- CCACGACCATCATCAGGTGAA-3′, R: 5′- CCTCACGGAGGCATTCTAAAGT-3′, PIK3CB: F: 5′- TATTTGGACTTTGCGACAAGACT-3′, R: 5′- TCGAACGTACTGGTCTGGATAG-3′, GAPDH: F: 5′- GAAGGTGAAGGTCGGAGT C-3′, R: 5′- GAGATGGTGATGGGATTTC −3'. The result was quantified by the delta-delta Ct method to quantify the relative gene expression. The average expression of each gene was normalized to the geometric mean of GAPDH.
Cell viability and colony formation assay
For cell viability experiment, cells were seeded in a 96-well plate at a density of 2000 cells per well and cultured at 37℃ for 0-7 days. After each time point, 10% CCK-8 reaction solution (DOJINDO, Japan) was added to each well and incubated for another 2 h at 37°C medium to culture the cells to be measured for 2 hours. The absorbance was quantified on a spectrophotometer microplate reader (Multiskan MK3, Thermo) with 450 nm wavelength. Eight experiments were independently conducted every day.
For the colony formation experiment, cells were cultured in 60-mm dish (Corning) with 2000 cells per dish for 14 days. Cells were then fixed with 4% paraformaldehyde (PFA), stained with crystal violet, and analyzed by microscopy.
Cell invasion ability was analyzed through Transwell filters (8 mm pore size, Millipore) coated with 50 mL Matrigel Basement Membrane Matrix (BD Biosciences). The cells (40000 cells) were seeded in the upper chamber containing 200μL of serum-free medium, while 800μL of 10% serum medium was placed in the lower chamber. After indicated time point, cells in the upper chamber were fixed with 4% PFA for 30 minutes, stained with crystal violet, and analyzed under the microscope (ZEISS, Germany).
Wound healing assays
The cell migration was performed using a wound-healing assay. Briefly, cells were seeded in a 6-well plate at 2000 cells per well for 24 hours. A line was then drawn using a marker on the bottom of the dish, after which a sterile 10µL pipet tip was used to scratch three separate wounds through the cells, moving perpendicular to the line. The cells were gently rinsed twice with PBS to remove floating cells and incubated in cultured in a serum-free medium. Images of the scratches were taken by using an optical microscope (Leica, Germany) at ×10 magnification at indicated time of incubation, and the healing area was analyzed with ImageJ software (Wayne Rasband National Institutes of Health, USA).
HN6 transfected cells were cultured on the sterile glass-coverslips in 24-well plates for 12 hours. Cells were then fixed with 4% PFA and permeabilized with 1% Triton and blocked with goat serum for 30 minutes. In the shaded condition, the cells were incubated with the eIF6 (diluted 1:100, Abcam) or AKT (diluted 1:100, CST) or p-AKT (diluted 1:200, CST) antibodies at 4℃ overnight and then stained with goat anti-rabbit IgG antibody Cy3 (Proteintech, China) for 1 hour at 37℃. After staining with DAPI (Sigma, St Louis, MO), cells were analyzed using fluorescence microscopy (ZEISS, Germany).
Subcutaneous tumor model
Twenty male BALB/c athymic nude mice (five-week-old) were purchased from the Animal Core Facility of Nanjing Medical University (Nanjing, China). All the animals were housed in an environment with a temperature of 22 ± 1 ºC, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 hr. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Nanjing Medical institutional animal care and conducted according to the AAALAC and the IACUC guidelines.
Animals were randomly divided into 4 groups (5 mice per group): NC, eIF6, sh-NC, and sh-eIF6. Stably transfected HN6 cells resuspended in 50% matrigel were subcutaneously injected into the nude mice flank (1 × 107 cells/100 μL). The xenograft tumor size was checked every three days and measured with a vernier caliper. The formula that was used to measure the tumor volume was: [volume = (length × width²)/2]. Twenty days after the injection, the nude mice were executed, and the tumor tissues were dissected out, imaged, and weighed up.
Statistical analysis is processed using graphing software GraphPad Prism version 8.0.1 (Graph Pad Software Inc., La Jolla, CA, USA). All experiments are repeated at least 3 times. The data index of each experiment represents the mean ± SD from 3 independent experiments. Use the Student's t-test to calculate the statistical significance of experimental data. All P values represent statistically significant two-sided tests (*P < 0.05, **P < 0.01, ***P < 0.001).