HNSCC samples
A total number of 119 tissue samples of patients with primary HNSCC were collected from patients who underwent curative resection (January 2012–September 2015) at the department of oral and maxillofacial surgery, Affiliated Stomatological Hospital of Nanjing Medical University, None of the patients had received adjuvant chemotherapy, radiation or any other treatment before resection, and all patients have detailed demographic, clinical, pathological and follow-up data. 26 normal adjacent oral mucosa samples obtained from donors during non-tumor surgeries were also included. This study was performed in accordance with guidelines outlined in the 1964 declaration of Helsinki and was approved by the Ethic Committee of Nanjing Medical University.
Cell culture
Five HNSCC cell lines including SCC9, SCC25, Cal27, Fadu and a normal human oral keratinocytes (HOK) were obtained from American Type Culture Collection (ATCC, USA). And HN4 and HN6 were generously gifted from Dr. Wantao Chen (Shanghai Jiao Tong University). HNSCC cells were cultured in DMEM/F12 (Invitrogen, USA) with 10 % fetal bovine serum (FBS, Gbico, USA) in a humidified atmosphere with 5% CO2 at 37 °C.
Small interfere RNAs, lentivirus production and cell transfection
The siRNAs against HOXB7 with siNC as a negative control were obtained from GenePharma (Shanghai, China). and sequences of the HOXB7 siRNA including siRNA-1: 5′-GCUAUUGUAAGGUCUUUGUTT, 5′-ACAAAGACCUUACAAUAGCTT; siRNA-2: 5′-CCCUUUGAGCAGAACCUCUTT, 5′-AGAGGUUCUGCUCAAAGGGTT; The final concentration of 100 nM siRNA or siNC pre-coated with Lipofectamine 3000 (Invitrogen, USA) were used for transfection.
Lentiviral vectors encoding the short hairpin RNAs (shRNAs) that target HOXB7 with the sequence of and a scramble shRNA were purchased from GenePharma (Shanghai, China). Transfection processes were conducted according to the instructions provided by the manufacturer. To generate the stable cell line, The transduced cells were then selected in culture medium containing puromycin (5.0 µg/ml).
RNA extraction and quantitative real-time PCR
Total RNA was extracted by trizol reagent according to the manufacturer’s protocol. Two micrograms of RNA was reverse-transcribed into cDNAs and subjected to PCR reactions using the Prime-ScriptTM RT-PCR kit (Takara). Primers used for real-time PCR were as follows: HOXB7, forward: 5′-TTCCCAGAACAAACTTCTTGTGC-3′; reverse: 5′-GCATGTTGAAGGAACTCGGCT-3′. 18sRNA, forward: 5′-ACACGGACAGGATTGACAGA-3′; reverse: 5′-GGACATCTAAGGGCATCACA-3′. All of the determinations were performed in duplicate. The relative expression of HOXB7 mRNA was normalized to the expression level of 18sRNA mRNA using the 2−ΔCt method.
Western blot analysis
Total protein was extracted from tumor cells. Equal amounts of protein were loaded onto a 10 % SDS-PAGE and electrophoresed and transferred onto PVDF membrane for 60–90 min based on the molecular weight of the target protein. After the membranes were blocked with 5 % non-fat milk, they were incubated overnight at 4 °C with the following primary antibodies: HOXB7 (1:1000, H00003217-M03, Abnova), vimentin (1:2000, #5741, Cell signaling, USA), E-cadherin (1:2000, #3195, Cell signaling), N-cadherin (1:1000, #13,116, Cell signaling), CD44 (1:1000, #6024-1-Ig, Proteintech, USA), CD133 (1:1000, #18470-1-AP, proteintech), ALDH1A1(1:1000, #15910-1-AP, proteintech), Bmi1 (1:1000, Bmi1, Cell signaling), SOX2 (1:1000, #4900, Cell signaling) and GAPDH (1:5000, MB001, Bioworld, China) followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive bands on the blots were detected by ECL chemiluminescence kit (Bio-Rad, USA).
CCK‑8 and BrdU assay
Cell Counting Kit-8 (CCK8) assay (Cell Counting Kit-8, Dojindo, Japan) was used to measure cell viability. After transfection, cells were placed in 96-well plates at a density of 2 × 103 cells per well; the absorbance values were detected 0 to 3 days after transfection. 10 µl of CCK-8 solution was added daily to each well flled in the 96-well plates and incubated for another 2 h. Then, a microplate reader (Multiskan MK3, Thermo, USA) was used to measure the absorbance at 450 nm. For BrdU assay, 2 × 105 cells were inoculated into a 6-well culture plate (with a cover slip placed inside) for 24 h, then incubated with 1.0 mg/ml BrdU solution (Applied Biosystems, USA) for 4 h. The culture solution was then discarded, followed by cell fixation in methanol for 10 min and cell staining in diamidine phenyl indoles (DAPI; Thermo Fisher Scientific). BrdU-positive cells were arbitrarily counted in three visual fields through the microscope.
Tumorsphere formation assay
In total, 2 × 104/ml cells were plated onto ultra-low-attachment plates (NUNC, Thermo, USA) and cultured in DMEM/F12 serum-free medium (Invitrogen, USA) supplemented with B27 (BD Bioscience), 10 ng/ml b-fibroblast growth factor (bFGF, BD Bioscience, USA), 20 ng/ml epidermal growth factor insulin (BD Bioscience). The number of spheres was captured and counted under an inverted microscope after 10 days.
Cell apoptosis assessed by flow‑cytometric assay
Cells were harvested and resuspended in 500 µl of binding buffer, and stained with Annexin V-FITC/PI Apoptosis kit (BD Biosciences). Apoptosis percentages were then detected using a FACSC aliber flow cytometer (BD Biosciences) and analyzed by Flowjo V10.1.
In vitro cell invasion and wound healing assay
Cell invasion was determined by a Matrigel transwell invasion assay. Cells (1 × 105/well) were suspended in 200 µl of serum-free DMEM and added to the upper chamber of an insert (8 μm pore size, Millipore, Germany) coated with Matrigel (BD Biosciences, USA). And Then, 600 µl DMEM with 10 % FBS were added to the lower chamber. For wound healing assay, Cells (2 × 105 cells/well.) were seeded onto a 6-well plate overnight. The confluent monolayers were scratched using sterile pipette tips and washed with phosphatebuffered saline (PBS) 3 times to remove detached cells. The wounds were photographed at 0, 6, 12 and 24 h as indicated.
Immunofluorescence assay
Cells were seeded on glass coverslips 18 h prior to the experiment and fixed with 4 % paraformaldehyde and washed thoroughly with PBS. Then permeabilized in 0.1 % Triton X-100 (Sigma-Aldrich). The cells were washed with PBS and blocked with 3 % bovine serum albumin (BSA) for 30 min at 37 °C. Then, incubation with primary antibodies against HOXB7 overnight. Cell were followed by incubation with secondary antibodies for 1 h. DAPI was used to counterstain DNA. Immunofluorescence images for HOXB7 were viewed with Zeiss fluorescence microscope.
Immunohistochemical staining and scoring
Paraffin-embedded tissue samples from HNSCC patients were sliced into 4-µm-thick sections. Tumor tissues from mice were also sectioned at a 4-µm thickness using a thin semiautomatic microtome. All sections were deparaffinized in xylene and rehydrated in a series of graded alcohol dilutions. Antigen retrieval was performed by heating in a microwave oven. Then, the sections were incubated with 3 % H2O2 for 10 min followed by 10 % normal goat serum for 15 min at room temperature to block endogenous peroxidases and non-specific antigens. Histological sections were immunostained overnight at 4 °C using the following primary antibodies: anti-HOXB7 antibody (1:200, #H00003217-M03, Abnova), anti-CD133 antibody, anti-CD44 antibody and anti-Ki67 antibody. Negative controls (only PBS incubation) were included in each staining run. Immunoreactivity in each slide was semi-quantitatively evaluated according to staining intensity and distribution and the immunoreactive score was calculated as intensity score × proportion score. Intensity score was defined as 0, negative; 1, weak; 2, moderate; 3, strong, while the proportion score was evaluated by two independent pathologists via counting positive nucleus with 0, negative; 1, < 10 %; 2, 11–50 %; 3, 51–80 %; 4, > 80 % positive cells. The immunoreactivity of each slide was categorized into three subgroups according to the final score: 0, negative; 1–4, low expression; ≥4, high expression.
HNSCC xenograft animal model
All experiments involving animal subjects were in accordance with the institutional animal welfare guidelines and approved by Institutional Animal Care and Use Committee of Nanjing Medical University. Six-week-old female nude mice were purchased from Model Animal Research Center of Nanjing Medical University and maintained in the specific pathologic-free animal facility. 2 × 106 cells of Fadu in 100 µl PBS then subcutaneously injected into both flanks of each animal (6 animals per experimental group). Sizes of tumors and were measured every 3 days when tumour masses were identified. Tumor volume = [(length) × (width) × (width)]/2. All the mice involved was sacrificed by intraperitoneal injection of a deadly dose of pentobarbital sodium (150 mg/kg) at the 31th day after tumor cell injection. When the vital signs of mice disappeared, the end-point tumor was dissected, weighed and recorded.
Bioinformatics analysis of HOXB7 from public databases data sources
The RNA sequencing and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. The microarray datasets: GSE6631, GSE12452, GSE23036, GSE25099, GSE30784, GSE42743 and GSE9844 were downloaded from the Gene expression Omnibus (GEO) database.
Functional enrichment of HOXB7 co-expression genes
In this study, we used the Pearson correlation coefficient (r) to screen and identify HOXB7-related genes with a P < 0.05 and |r| > 0.3 were identified as HOXB7-related genes. The biological functions of these HOXB7 co-expression genes were comprehensively detected by GO enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. In addition, the protein–protein interactions (PPIs) among all HOXB7 co-expression genes were obtained by Search Tool for the Retrieval of Interacting Genes (STRING; http://string-db.org) and the network was constructed with the Cytoscape 3.7.0 software.
Survival analysis and small molecule targeted drugs screening of HOXB7 in HNSCC
The Connectivity Map (CMap) (http://www.broad.mit.edu/cmap)was used to identify potential small molecule targeted drugs for HOXB7 in HNSCC. Those small molecule drugs with mean connective score < − 0.2 and P < 0.05 were recognized as the possible therapeutic drugs of HOXB7 in HNSCC.
Statistical analysis
Data between two groups were examined using a two-tailed paired Student’s t-test or or ANOVA (Bonferroni post hoc test). The Chi-squared test was applied to assess the correlation between HOXB7 expression and various clinicopathological parameters. Survival data were used to establish Kaplan–Meier curves, and the differences among the groups were analyzed by the log rank test. Univariate and multivariate Cox-regression analysis were employed to determine prognostic factors associated with survival. Two-tailed P values < 0.05 were considered as statistically significance. All statistical analyses were performed by GraphPad Prism 7 or R (4.0.2).