Cell culture and Tissue specimens
HONE1 and SUNE1 cells were cultured in RPMI-1640 (Invitrogen) containing 10% fetal calf serum (FCS; Hyclone, Invitrogen). HONE1 and SUNE1 cells were cultured at 37 ° C in a 5% CO2 incubator. Tissue microarrays including 132 NPC samples were purchased from Outdo BiotechCo., Ltd, Shanghai, China.
In situ hybridization
The expression of miR-4721 in 132 cases of nasopharyngeal carcinoma tissues was examined by in situ hybridization in Bioscience (Guangzhou, China). The positive staining was detected by a diaminobenzidine (DAB) substrate kit (AxyBio, Guangzhou, China).
Transient transfection with FOXA1 or miR-4721 mimics/inhibitor
RiboBio Inc. (Guangzhou, China) and Biosense Technologies (Guangzhou, China) designed and synthesized the mimics and inhibitor of miR-4721 and the plasmid of FOXA1. The sequence of miR-4721 and its control are listed in Supplementary Table S1. The specific experimental steps are the same as before[14].
Western blotting
Follow the steps (https://www.westernblotprotocol.com) for western blotting. The following antibodie were used: rabbit anti-FOXA1 (1:1000; CST, USA), rabbit anti-Nanog (1:1000; CST, USA), rabbit anti-Snail (1:1000; CST, USA), rabbit anti-ZEB2 (1:1000; CST, USA), rabbit anti-E-Cadherin (1:1000; CST, USA), mouse anti-β-actin (1:5000; Beyotime AA128, China). The antibodies are listed in Supplementary Table 3.
Tumor spheroid formation assay
NPC cells (1000 cells/ml) were cultured in serum-free Ham'sF-12 medium (Gibco) supplemented with 20 ng/mL epidermal growth factor (EGF, Invitrogen, Grand Island, NY, USA), B27 (1:50; Gibco), and 20 ng/mL fibroblast growth factor (FGF, Invitrogen). After 72 hours of culture, rumen spheres were counted under a microscope.
Side population assay
1×106 nasopharyngeal carcinoma cells/ml are suspended in DMEM/2% FBS. Then mixed into a single cell suspension, alone or in combination with verapamil (50 mmol/ml; Sigma-Aldrich), and then incubated with Hoechst 33342 dye (5μg/ml; Sigma-Aldrich) at 37°C for 90 minutes. After incubation, the cells were stained with propidium iodide (1μg/ml; Sigma Aldrich). The FACS Aria flow cytometer (BD Biosciences) was used for flow cytometry analysis.
Establishment of subcutaneous xenograft mouse model
To determine the tumor formation ability, a mouse model of subcutaneous xenograft tumors was established. 1×106, 5×105, 1×105, and 5×104 NPC cells were respectively injected into mice (N = 6 per group). According to a previous description (http://bioinf.wehi.edu.au/software/elda/), Extreme limiting dilution analysis were performed to assess tumor-initiating frequencies.
ALDEFLUOR
The ALDEFLUOR assay kit (Stem Cell Technologies) was used to detect the activity of ALDH. Single cells isolated from cell lines or tumor samples were mixed in ALDEFLUOR assay buffer (1.5 µM) containing the ALDH substrate BODIPY amino acetaldehyde (BAAA) and incubated for 1 h at 37°C. Diethylamino benzaldehyde (DEAB), was used as a negative control (10-fold molar excess). BD FACS Diva software V6.1.3 (BD Biosciences) or Flow Jo software (Tree Star) were used to analyze flow cytometry data.
Immunofluorescent staining
According to the previous description, Immunofluorescent staining assays were performed[13]. Store the slides in a dark box at 4°C and observe under a fluorescence microscope.
Immunohistochemical staining
IHC is used to assess the expression level of FOXA1 and Nanog in samples of NPC and its control tissues, which has been previously described[14]. Two pathologists who were blind to the clinical parameters examined the stained tissue sections and scored them separately. The staining scoring criteria are also described[14].13 In order to statistically analyze the expression of FOXA1 and Nanog in NPC tissues in non-cancerous tissues, the staining scores 0-4 and 5-6 are regarded as low expression and high expression, respectively.
Luciferase reporter assay
Luciferase reporter assay was performed to show that miR-4721 directly regulates FOXA1. The WT 3 ’UTR or mutant 3’UTR was cloned into psiCHECK-2 vectors. The WT or mutant 3’UTR vector was co-transfected with miR-4721 mimics/inhibitors or a non-specific control into NPC cells. To investigate whether FOXA1 effects the promoter activity of miR-4721 and Nanog, we constructed the Nanog promoter-luciferase reporter plasmid pGL3-Nanog and the miR-4721 promoter-luciferase reporter plasmid pGL3-miR-4721. Luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The primers sequences used in the luciferase activity reporter assay are listed in Table S2.
CHIP assay
According to the previous description[8], we used was using a ChIP assay kit (Millipore) to perform ChIP assay according to the manufacturer’s instructions. DNA-protein complexes were immunoprecipitated from NPC cells with antibodies anti-FOXA1 or IgG (Cell Signaling Technology, Danvers, MA, USA). The enrichment of the miR-4721 and Nanog promoter region were measured by qRT-PCR analysis and PCR analysis. PCR Primers are listed in Table S2.
EMSA analysis
An EMSA kit (Roche Diagnostics, Basel, Switzerland) was used to detect the binding site of FOXA1 to the promoters of miR-4721 and Nanog according to the manufacturer’s instructions. According to the previous description[8], Biosense Bioscience (Guangzhou, China) completed EMSA analysis. Summary of the sequences of the probes used in the EMSA assay were seen in Table S4.
Statistical analysis
Data analysis were completed by IBM SPSS v20.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prismv5.0 (GraphPad Software, Inc., La Jolla, CA, USA) software. Chi-square test analysis of the relationship between FOXA1 expression level and clinicopathological characteristics. Two independent group was compared by wo-tailed Student’s t-test. The differences between groups for all in vitro analyses were determined by One-way ANOVA. Differences were considered significant if P < 0.05.