We recruited melanoma patients who underwent surgical removal of melanoma at Affiliated Hospital of Qingdao University. Melanoma tissues and the matched tumor-adjacent normal tissues (n = 60) were obtained from the surgery and stored further use. These patients with melanoma did not receive any treatment before surgery. All melanoma patients were informed and gave written the consent. All protocols were performed according to the Declaration of Helsinki and authorized by the Ethics Committee of Affiliated Hospital of Qingdao University.
Normal human dermal fibroblast cells (NHDF, ATCC, Manassas, VA, USA) and melanoma cell lines (A2058, A375, WM115, MV3, ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Waltham, MA, USA) at a constant temperature (37˚C) with 5% CO2. The medium was contained 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Solarbio, Beijing, China).
The vector pcDNA3.1 containing HOXC10 (pcDNA3.1-HOXC10) or Slug (pcDNA3.1-Slug) were constructed for HOXC10 or Slug overexpression (GenePharma, Shanghai, China). The vector pcDNA3.1-NC was served as negative control (NC). For HOXC10 knockdown, HOXC10 (sh-HOXC10) targeted short hairpin RNA (shRNA) and the scramble shRNA (shNC) were synthesized by GenePharma. Cells were transfected with the vectors or oligonucleotides using Lip2000 (Invitrogen, Carlsbad, CA, USA).
Total RNA was extracted applying Trizol reagent (Invitrogen). 1.0% agarose gel electrophoresis was used to examine RNA integrity. The relative expression of genes in cells and tissues was examined through quantitative real-time PCR (qRT-PCR) using TransScript® II Two-Step RT-PCR SuperMix (Transgen, Beijing, China) on a Real-Time PCR Instrument. The 2-ΔΔCt method was used to analyze the data. Primer sequences (5’-3’) were as follows:
Protein extraction and the detection of protein concentration were carried out using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) and BCA Protein Assay Kit (Sangon Biotech). Proteins (25 μg) were used for 10% SDS-PAGE gel electrophoresis, and then blotted onto the nitrocellulose membranes (Whatman, Maidstone, Kent, UK). The membranes were stained with the primary antibodies, HOXC10 (1:1000, #ab153904), Bax (1:2000, #ab182733), BCL2 (1:1000, #ab32124), Cleaved Caspase-3 (1:500, #ab2302), E-Cadherin (1:1000, #ab133597), N-Cadherin (1:5000, #ab76011), snail (1:1000, #ab216347), Slug (1:1000, #ab183760), YAP1 (1:5000, ab52771) and TAZ (1:1000, #ab224239) at 4°C for 12 h. Subsequently, the membranes were stained with HRP-IgG (1:2000, #ab6721). β-actin antibody (1:1000, # ab5694) was used as an reference protein. The data were analyzed by Image J software. These antibodes all obtained from Abcam (Cambridge, MA, USA).
Immunohistochemical (IHC) staining
Paraffin tissue sections were prepared through fixation and embedding. After deparaffin and rehydration, the sections were incubated with Target Retrieval Solution (Dako, CA, USA). The sections were stained with the primary antibodies, HOXC10 (1:1000, #ab153904, Abcam), anti-Slug (1:100, #ab85936, Abcam), anti-YAP1 (1:50, #ab52771, Abcam) and anti-TAZ(1:500, #ab224239, Abcam), and then labeled with HRP-IgG (1:2000, #ab6721, Abcam). Finally, the sections were stained successively with DAB and hematoxylin. The sections were observed under a microscope (Olympus, Tokyo, Japan).
Cell viability was examined using Cell Counting Kit-8 (Beyotime Biotechnology, Shanghai, China). Cells (2000 cells/100 μL) were seeded into 96-well plate, and incubated with 10 μL CCK-8 reagent at 37°C for 72 h. The absorbance value of each well was detected at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
Cell proliferation was examined using Yefluor 488 EdU Imaging Kit (Yeasen, Shanghai, China). Cells were first incubated with EdU at 37°C for 4 h. After fixation and permeabilization, the cells were reacted with 1 mL Click-iT reaction cocktail in darkness for 30 min. For nuclear staining, cells were incubated with DAPI. Finally, the fluorescence was observed under a fluorescence microscope (Nikon, Tokyo, Japan).
Cell clone formation assay
Cells at logarithmic phase were treated with 0.25% trypsin/0.02% EDTA to obtain the single-cell suspension. The cells (1000/well) were seeded into a six-well plate, and cultured at 37°C. Until the cell clones could be observed with the naked eye, the cells were stopped. After fixation, the cells were stained with Giemsa solution (Beyotime Biotechnology) for 30 min. Finally, the number of cell clones was counted.
Cell apoptosis was examined using Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology). After washing with PBS, cells were mixed with Annexin V-FITC binding buffer, and then stained with Annexin V-FITC and PI at darkness for 20 min. Finally, cell apoptosis was examined on a FACSCalibur (BD Biosciences, San Jose, CA, USA) in an hour.
Migration and invasion
Cell migration and invasion were estimated using a 24-well Transwell insert system (Corning, NY, USA). For migration assay, cells were cultured in the DMEM-contained upper chamber. For invasion assay, cells were cultured in the upper chamber containing DMEM and coating with Matrigel. The bottom chamber was supplemented with DMEM and 10% FBS. After 24 h of culture, the migrating or invading cells on the bottom surface of the chamber were collected, and then were fixed with paraformaldehyde and dyed with 0.1% crystal violet. Images were photographed from each membrane under Olympus microscope from five randomly selected fields.
The relationship between HOXC10 and Slug in the HOXC10-overexpressed A375 and A2058 cells was determined by Co-Immunoprecipitation (Co-IP) assay. The washed cells were lysed with RIPA Lysis Buffer (Beyotime, Shanghai, China) at 4°C for 30 min. Subsequently, the cell lysates were incubated with the primary antibodies, HOXC10 (1:100, #A303-177A, Thermo Fisher Scientific) or Slug (1:50, #9585, Cell Signaling Technology, Danvers, MA, USA), and then incubated with the Protein A/G Plus-Agarose (Thermo Fisher Scientific). Immunoprecipitates and a portion of the cell lysates (Input group) were analyzed by WB assay using the primary antibodies, HOXC10 and Slug.
Luciferase reporter assay
The interaction between HOXC10 and Slug in A375 and A2058 cells was examined by luciferase reporter assay. The vector pGL3-basic containing slug promoter (pGL3-basic-slug promoter) was transfected into cells using Lip2000 Reagent. Cells were co-transfected with pGL3-basic-slug promoter and different concentration of pcDNA3.1-HOXC10 (0, 50, 100, 200 ng). The activities of luciferase were measured using Dual luciferase assay kit (Promega, Madison, USA) on luciferase assay system (Ambion, Austin, TX, USA).
Xenograft mouse model
Female BALB/c nude mice (SLAC, Shanghai, China) (weighting 18 ± 2 g, aged 4 weeks) were housed at 18‑25˚C. All the animal protocols were carried out according to the Guide for the Care and Use of Laboratory Animals, and authorized by the Ethics Committee of Affiliated Hospital of Qingdao University. For tumor xenograft mouse model, HOXC10-silenced A375 cells (200 µL, 2.0 × 106/mL) were injected subcutaneously into the nude mice. The mice were euthanized to separate the tumor xenografts, the weight and volume of tumor were measured every week for 28 days. Volume (mm3) = 1/2 × width2 (mm2) × length (mm). For metastasis mouse model, HOXC10-silenced A375 cells (200 µL, 2.0 × 106/mL) were injected into the caudal vein of nude mice. After 28 days, the mice were euthanized to obtain the lung tissues. HE staining was performed to estimate the number of metastatic tumors in the lungs.
Lung tissues of mice were embedded in paraffin to obtain paraffin sections. The paraffin sections (5 μm) were stained using HE staining kit (Solarbio, Beijing, China). The number of metastases in lung tissues was observed under an Olympus microscope.
All assays were carried out for 3 times. Statistical analysis was done through SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) or GraphPad Prism 7.0 software (San Diego, CA, USA, USA). The data were showed as the mean ± SD. Statistical differences were carried out through the Student’s t-test or one-way ANOVA. p < 0.05 was supposed as statistically significant.