Patients and clinical specimens
A total of 80 NPC specimens and normal nasopharyngeal epithelial tissues were collected from the patients of the Affiliated Hospital of Southwest Medical University. The tissues acquired by surgical operation were immediately frozen in liquid nitrogen for subsequent experiments. All participants signed written informed consent, and the research plan was approved by the Medical Ethics Committee of the Affiliated Hospital of Southwest Medical University. Human NPC cell lines (CNE-1, CNE-2, C666-1, 5-8F, and HONE-1) and the normal nasopharyngeal epithelial cell line NP69 were obtained from ATCC. Human NPC cell lines were cultured in RPMI 1640 (Gibco; Grand Island, NY, USA) supplemented with 10% FBS (Gibco), while NP69 was maintained in Keratinocyte-SFM medium (Gibco).
Quantitative real-time polymerase chain reaction
qRT-PCR with TaqMan microRNA assays (Applied Biosystems) was used to measure the expression levels of miR-221 from tissues and various transfected cells, while qRT-PCR with SYBR Green assays (Takara, Japan) was used to quantify the expression levels of FBXW11. U6 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the relative expression levels of miR-221 or FBXW11 in samples, respectively. The primer sequences were as follows:
miR-221, F-5’-TCTACGTCGTAAGTCGACTGACGATGCTAAGTGCAAGC-3’, R-5’-TACGCTGACGTGCGCGTAGCTGACGATGATCCCTC -3’; FBXW11, F-5’-GTCCTGCGAATGCGATGACGTGA-3’, R-5’-CGAGTGAGTGACGTAGTAGCAG-3’; GAPDH, F-5’-AAAGCGTAGACTGACGTAGGAAA-3’, R-5’‑TGAACCCTGAGCTGACGTACA-3’; U6, F-5’-CTCAGTAGAACCTGACGATGATA-3’; R-5’‑AAGCGTAGACGATGTGTC-3’.
Cell transfection
Firstly, cDNA of FBXW11 gene was cloned into pcDNA3.1 vectors to construct the plasmid pcDNA-FBXW11. And that, miR-221 mimics (5’-GCTCGCGTGCTGCTCTCGCGCGTATGCGCC-3’), miR-negative control (NC) mimic (5’-UUCGCGCCGAGUUCGCGAAUUGUTT-3’), miR-221 inhibitors (5’-GGCCTCGCGACGTATGAGCGAGCGAGC-3’), miR-NC inhibitor (5’-CAGCGCGUUUUCGAGACGAGCGAA-3’), Wnt1 siRNAs (siRNA1: 5’-GCGGCGCUCGAGCGCUCUUUGCGATT-3’; 5’-GCTGAGTGCGCGTATUCGCGAGGTT-3’), and siRNA control (5’-UUGAGCGAGAGACGCGUCGAGAGTT-3’) were provided by Novogene (Beijing, China). Lipofectamine 2000 (Invitrogen) was used for cell transfection according to the protocol of manufacturer.
The abovementioned miRNAs (50 nM) were transfected into C666-1 and 5-8F cell lines with a density of 105/well in 6-well plates (Corning; NY, USA) by Lipofectamine 2000 reagent (Invitrogen), and the cells were collected for analysis 48 h after transfection.
MTT assay
Cell suspensions were seeded in 96-well plates (Corning) and incubated overnight, before being transfected with 50 nM miR-221 mimic or inhibitor, miR-NC mimic or inhibitor, pcDNA-FBXW11 or pcDNA-3.1, and shFBXW11 or negative control. The cell viability was determined by MTT (Sigma; Louis, MO, USA) staining. Live cells were incubating with 20 µl of MTT (5 g/L, Sigma). The supernatant from each well was then aspirated, and 150 µl of dimethyl sulfoxide (DMSO, Sigma) was added to each well to dissolve the MTT methoxypyrimidine crystal to obtain the absorbance, which was measured by a microplate reader at 570 nm (Molecular Devices; Shanghai, China).
Flow cytometry analysis
Cell apoptosis was analyzed by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (BD, San Jose, CA, USA). Briefly, C666-1 and 5-8F cells were seeded into 6-well plates at a density of 1x105 cells/well. After transfection, cells were collected and apoptosis was observed. The apoptotic cells were labeled with FITC-Annexin V and PI and kept dark at 25℃ for 30 min. The data were collected and analyzed by a cytoFLEX LX flow cytometer from Beckman-Counter Electronics (Jiangsu, China).
Cell cycle analysis
C666-1 and 5-8F cells were transfected with miR-221 or NC for 24 h. The cells were then collected by trypsinization and washed with PBS that had been precooled on ice. The cells were fixed in 70% methanol and incubated at 4℃ for 1 h. The cells were then centrifuged and incubated with RNase at 37℃ for 30 min. The cells were stained with propidium iodide for 1 h, and the cell cycle was analyzed using a FACScan flow cytometer; the data were presented using CellQuest software.
Western blot assay
The assay was performed according to previous methods, and described as below. Proteins were isolated from cultured cell using the Protein isolation kit (Tiangen), and the protein concentrations were determined using the Pierce BCA assay (Thermo Scientific; CA, USA). SDS-PAGE gel was used to separate the protein samples (20 µg), after which, the gel was electrophoretically transferred to nitrocellulose membranes (Bio-Rad; Hercules, CA, USA). The membranes were incubated with 10% skimmed milk (Sigma-Aldrich; St. Louis, MO, USA) for blocking, before the addition of the primary antibody and incubation overnight at 4℃. The following primary antibodies were used in Western blot, including FBXW11, Ki67, CDK4, Cyclin-D1, CDK2, Cyclin-E, β-actin, PTEN, phosphor-PI3K (p-PI3K), total-PI3K (t-PI3K), phospho-Akt (p-Akt), and total-Akt (t-Akt), β-catenin, c-myc and GAPDH, and all primary antibodies were purchased by Cell signaling Technology company (Beverly, MA, USA). Peroxidase-conjugated anti-IgG (1:10,000; purchased from Santa Cruz Biotechnology, Inc.) was used as the secondary antibody. The exposure detection of blots was carried out with an enhanced chemiluminescent kit (BD; San Jose, CA, USA), and the corresponding gray value was calculated by Image J software for statistical analysis.
Luciferase reporter assay
A luciferase reporter assay was performed to confirm whether FBXW11 was a direct target of miR-221. Subsequently, miR-221-overexpressing 5-8F cells were seeded in a 48-well plate, and the plasmids psiCHECK-3’UTR-WT (the wild-type 3’UTR fragment of FBXW11, including conserved binding sites for miR-221) and psiCHECK-3’UTR-MUT (the mutant 3’UTR fragment of FBXW11, in which the mutations occur in the conserved binding sites for miR-221) were transfected using Lipofectamine 2000 (Thermo Scientific). Following incubation for 48h, cells were collected and determined by a dual luciferase assay kit (Thermo Scientific).
Statistical analysis
GraphPad Prism 5 software (GraphPad Software Inc.; San Diego, CA, USA) was used for all statistical analyses. Values are presented as mean ± standard deviation (SD), and significance was determined by two-tailed Student’s paired t-test or one way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P-values < 0.01 were taken to indicate statistical significance.