Patients and follow-up
A total of 41 HNSCC patients underwent surgical resection at the Department of Craniofacial Surgery, GuanghuaSchool of Stomatology, Sun Yat-sen University (Guangzhou,China) from January 2017 to December 2017 were enrolled in the study. Before the surgery, no one had received radiotherapy or chemotherapy. Primary HNSCC tissues were obtained rom the representative area of each case, verified by pathological examination. The paraffin-embedded HNSCC and matched peritumor tissues were collected for immunohistochemistry. Informed consent from patients were obtained and their medical records were reviewed for clinical information. Ethical approval of the study was obtained from the Ethics Committee of Guanghua School of Somatology, Sun Yat-sen University.
GEPIA (Gene Expression Profiling Interactive Analysis) dataset
GEPIA (http://gepia.cancer-pku.cn/) is an interactive web used for analyzing RNA sequencing expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression project (GTEx), with a standard processing pipeline. The website provides customizable functions like tumor/normal tissue differential expression analysis and patient survival analysis. The raw data of unpaired normal and tumor tissues were compared and filtered based on the cutoffs |log2FC| >1 and P < 0.01. The log2(TPM + 1) transformed expression data were shown in plots or used for one-way ANOVA analysis. While analyzing the overall survival (OS) and disease-free survival (RFS) of patients with HNSCC, patient samples were divided into two groups by median expression (high/low expression) and then shown in a Kaplan-Meier survival plot, with the hazard ratio (HR) with 95% confidence intervals (CI) and logrank p value.
Tissue sections from paraffin-embedded HNSCC patient tissue or SAS xenograft model were dewaxed in xylene and rehydrated in a graded alcohol series. Next the sections were soaked with 0.1% Trion X-100, incubated in 3% H2O2 in order to eliminate endogenous peroxidase activity and then heated in sodium citrate buffer (pH 6.0) for antigen retrieval. After blocking in 10% goat serum for 1h at room temperature, the sections were incubated with CCT2 primary antibody overnight at 4°C. Washed with PBST, the sections were incubated with secondary antibody for 1h at room temperature and visualized with DAB solution and counterstained with hematoxylin. The expression of CCT2 was scored by two pathologists respectively. The ratio of positively stained cells<25 was negative, 25%-50% was +,50%-75% was ++ and >75% was +++.
Cell culture and transfection
The human HNSCC cell lines CAL-27 were purchased from ATCC(Rockville, MD, USA), and the cell lines HSC-3 and HSC-6 were provided by J. Silvio Gutkind (NIH, Bethesda, MD, USA). Cell lines SAS and NOK was purchased from Fuheng Biology (Shanghai,China). Cells were mycoplasma-free and were cultured under the recommended conditions.
The vectors of pLKO.1-TRC-puromycin-shRNA-CCT2 and pLKO.1-TRC-puromycin were constructed and transfected into SAS and HSC-3 cells, according to the manufacturer’s instruction. The shRNA1 sequence is TTATCGAGGAAGTCATGATTG. The shRNA2 sequence is GCCTCTCTTATGGTAACCAAT. The transfected cells were screened with puromycin at a concentration of 3μg/ml for SAS and HSC-3 cell lines. The transfection efficiency was verified by qRT-PCR and western blotting.
Real-time reverse transcription PCR(qRT-PCR) and western blot analysis
For total protein extraction, cells lysates were collected using RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors. 20μg of total protein were loaded into 10% SDS-PAGE gel for separation and transferred to a PVDF membrane (Millipore, MA, USA). The PVDF membranes were blocked with 5% milk for 1h and then incubated with primary antibodies at 4 ˚C overnight. Next the membranes were incubated with the secondary antibody for 1h. The bands was visualized using the enhanced chemiluminescence (ECL) detection system (Millipore, MA, USA). All the primary antibodies are listed in Supplementary Table S1.
Total RNA was extracted from cell lines with TRIzol (Invitrogen, Carlsbad,
CA, USA) and reverse-transcribed to cDNA with HifairTM III 1st Strand cDNA Synthesis SuperMix for qPCR(Yeasen, SH, China). qRT-PCR was performed using SYBR Green Master Mix Kit (Yeasen) on a Light Cycler 480 system (Roche).The qRT-PCR primers are shown in Supplementary Table S2. The results were normalized to those for GAPDH.
CCK-8, EdU and colony formation assays
shCCT2 cells and control cells were inoculated into 96-well plates(3000 cells/well). 10μl of CCK-8 solution (Dojindo, Kumamoto, Japan) was added itno the sextuplicate wells At each time point(24, 48, 72 and 96 h). After incubating for 1h, the absorbance was detected at 450nm.
The EdU incorporation assay is designed to quantitate cell proliferation based on the measurement of EdU incorporation during DNA synthesis in proliferating cells. The developed color correlates with the number of proliferating cells in the respective microcultures. The assay was performed with the Cell Proliferation EdU Image Kit (Green Fluorescence) (Abbkine, CA, USA) according to the manufacturer’s instruction.
The colony formation assay was used to test the cell proliferation capacity as well. Cells were seeded in 6-well plates(1000cells/well) with culture medium refreshed twice a week. After 10-14 days, the cells was fixed with 4% formaldehyde and then stained with crystal violet. Colonies containing >10 cells was counted manually.
Transwell and wound healing
Transwell assay was performed to detect cell migration and invasion. In brief, SAS and HSC-3 cells (1x105) were suspended in 200μl serum-free medium and added into the upper compartments of 24-well Transwell chambers (8 μm pore size; Corning, NY, USA) after transfection. 600μl of complete medium containing 10% FBS was filled into the lower compartment. After co-culturing for 24-48h, the cells remained in the upper compartments were removed with cotton buds and the cells translocated to the lower compartments were fixed in 4% paraformaldehyde and stained 0.1% crystal violet. Under an optical microscope (Carl Zeiss), photographs of randomly selected five visual fields of each well were captured for analysis. For Boyden assays, the procedure was similar except the Transwell membranes were pre-coated with 24 µg/ml Matrigel (R&D Systems,Minneapolis, MN, USA).
As to the wound healing assay, HNSCC cells were seeded in 6-well plates, cultured overnight and then scratched by a sterile plastic tip with 95% confluence rate. After washing with PBS for three times, the cells were cultured with serum-free medium for 24-48 h. Under an optical microscope (Carl Zeiss), photographs of randomly selected fields across three replicate wells were captured for analysis.
Flow cytometric analysis
Flow cytometric analysis was used to detect cell cycle and apoptosis. Cells were stained with Cell Cycle Staining Kit (Lianke, HZ, China) according to the manufacturer’s instructions. Stained cells were detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FlowJo software version 10.
Total RNA of transfected SAS cells was purified was described above. mRNA was enriched with oligo (dT)-attached magnetic beads and fragmented into pieces. First-strand cDNA was developed using random hexamer-primed reverse transcription. Then second-strand cDNA was synthesized and sequenced on an MGI 2000 platform (BGI, Shenzhen,China). The clean reads were then mapped to the human reference genome (mm10) with STAR v1.5.1. Gene expression level was quantified with HTSeq-count v0.11.3. DEGs were analyzed with the default settings of the DEseq2 v1.28.1 algorithm and the significant ones were set a false discovery rate of <0.05 and a fold change of >2. The following processing and visualization of the data were performed by Dr.Tom platform(BGI, Shenzhen,China). The RNA-seq data are deposited in GEO database (Access No. GSE195850)
In vivo tumorigenesis
Xenograft experiments in nude mice were approved by the Ethics Committee of the Hospital of Stomatology, Sun Yat-sen University. The animal experiments were complied with the stated guidelines of 3Rs (replacement, reduction, and refinement). Male mice aged from 4-6 weeks of BALB/c nude mice were randomly divided into two subgroups (n=7 for each group): control group (Vector) and treatment group (shRNA). 5ⅹ106 cells (per mouse) suspended in 100μl PBS were injected to subcutaneous space of left waist of nude mice. Tumor growth was monitored every three days. Animals were sacrificed after 21 days. Tumor tissues were fixed in formalin and embedded in paraffin for immunohistochemistry. The tumor volume was measured following the formula :V= π/6× (larger diameter) × (smaller diameter) 2.
All experiments were performed in triplicate. Data are presented as the mean ±SD. Student’s t-test was used for comparison between two groups. One-way ANOVA (no matching) was used for comparison between multiple groups. Categorical data were analyzed with the chi-square test or Fisher’s exact test. The data was analyzed using SPSS 23.0 software (IBM,Armonk, NY, USA) and performed by GraphPad Prism 5.0 (GraphPad software, San Diego, CA, USA). A two-tailed P value <0.05 was considered statistically significant.