Cell lines
The nasopharyngeal epithelial cell line, NP-69 and NPC cell lines (CNE-2, CNE-1, SUNE-1 and C666-1) were cultured with RPMI-1640 containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA). C666-1 and SUNE-1 cells are not misidentification and contamination of human cell lines (ExPASy: SIB Bioinformatics Resource Portal, https://www.expasy.org/). All cell lines were obtained from Nanjing KeyGen Biotech Co., Ltd (Nanjing, China) and were cultured in humid chamber at 5% CO2, 37°C.
Cell transfection
MiR-454-3p mimic and negative control (miR-NC), shRNA negative control (sh-Ctrl) and shRNA against LINC00839 (sh-LINC00839 #1 and sh-LINC00839 #2) were synthesized by GenePharma (Shanghai, China). The pcDNA3.1 control plasmid (pc-vector) and plasmid for mediating LINC00839 overexpression (pc-LINC00839) were purchased from GenePharma. pcDNA3.1 plasmid or miRNA mimic was transfected into C666-1 and SUNE-1 cells using Lipofectamine 3000 (Thermo Fisher Scientific). To stably obtain sh-LINC00839 stable transfected cell, lentivirus carrying sh-LINC00839 or sh-Ctrl packaged in HEK-293T cell and secreted into culture medium. SUNE-1 cells were infected by lentivirus using polybrene (Sigma, Shanghai, China) and selected by 1.5 μg/mL puromycin (Sigma) for 14 days.
Quantitative real‐time PCR analysis (qPCR)
Total RNAs were isolated using a TRIZOL kit (Thermo Fisher Scientific). 1 μg of RNA was used for cDNA synthesis with a reverse transcriptase PCR kit (Thermo Fisher Scientific). qPCR was conducted using a SYBR Green qPCR Master Mix kit (TAKARA). The relative expression of miR-454-3p was measured use an All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, Montgomery, USA). The primer sequences are shown below. miR-454-3p forward primer: 5’-ACCCTATCAATATTGTCTCTGC-3’, Reverse primer: 5’-GCGAGCACAGAATTAATACGAC-3’; U6 forward primer: 5’-CGCGCGTCGTGAAGCGTTC-3’, Reverse primer: 5’-GTGCAGGGTCCGAGGT-3’; ZO-1 forward primer: 5’-GCCGCTAAGAGCACAGCAA-3’, Reverse primer: 5’-TCCCCACTCTGAAAATGAGGA-3’; Vimentin forward primer: 5’-GACGCCATCAACACCGAGTT-3’, Reverse primer: 5’-CTTTGTCGTTGGTTAGCTGGT-3; GAPDH forward primer: 5’-TGTGGGCATCAATGGATTTGG-3’, Reverse primer: 5’-ACACCATGTATTCCGGGTCAAT-3’. U6 was used for miR-454-3p normalization and GAPDH for mRNA normalization. The relative expression of RNA was calculated using the 2-△△Ct method.
Cell proliferation
miR-454-3p mimic or sh-LINC00839 transfected SUNE-1 or C666-1 cells were seeded into 96 well culture plates (1×104). At 24, 48, 72 or 96 hours, 20 μl of MTT (5 mg/ml) were added into plates. Following the 4 hours incubation, supernatant in each well was gently removed and 200 μl of dimethyl sulfoxide (DMSO) was added. The absorbance was measured at 450 nm.
Clone-formation assay
SUNE1 or C666-1 cells (1000 cells/well) were seeded into six-well plates. After 14 days, cell colonies in plates were fixed using methanol and stained by 0.1% crystal violet (Sigma).
Migration assay
After transfection, SUNE1 or C666-1 cells were seeded into 6-well plates to incubate until 80% confluence. Then, the scratch was generated with a 200 µL pipette tip and cultured for 24 hours. The healing distance was captured by a microscope. The percentage of migration = (width at 0 hour − width at 24 hours)/width at 0 hour × 100%.
Cell invasion
The membrane in Transwell chamber (8 μm pore size, Corning Costar, NY, USA) was coated with 40 μl of Matrigel (BD). After transfection, 200 μl of SUNE1 or C666-1 cells were plated into the upper chamber and 600 μl of medium was added into the lower chamber. After 24 hours, the invading cells on the lower surface of membrane were stained by 0.1% crystal violet and calculated.
Immunoblotting
Total proteins were extracted using RIPA lysis buffer (Biyuntian, Nanjing, China). 35 μg of proteins were separated using 10% SDS-PAGE and transferred onto PVDF membrane (Millipore, Braunschweig, Germany). After blocking with 5% skim milk, membrane was incubated with c-Met, E-cadherin, N-cadherin or GAPDH antibody (1:1000, Santa Cruz Biotechnology, CA, USA) at 4°C overnight. After washed with TBST for three times, membrane was incubated with HRP-linked secondary antibody (1:10000, Biyuntian) for 2 hours. The bands were assessed using an ECL kit (Millipore).
Ago2-RNA immunoprecipitation (RIP) assay
After transfection of miR-454-3p, C666-1 and SUNE1 cell were collected, and cell lysate was prepared. Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore) was utilized to measure the mRNA level of LINC00839 from the samples bound to the Ago2 or IgG antibody. After washing, the retrieved RNAs were subjected to qPCR analysis.
Luciferase reporter assay
The LINC00839 3′‐UTR containing putative wild type binding sites for miR-454-3p was inserted into pGL3 luciferase reporter (Promega, Madison, WI, USA) to generate pGL3-LINC00839-wt. The mutated miR-454-3p-binding sequence was constructed using a QuikChange Site‐Directed Mutagenesis Kit (Stratagene, USA) to construct pGL3-LINC00839-mut. C666-1 and SUNE1 cells were co-transfected with miR-454-3p mimic plus pGL3-LINC00839-wt or miR-454-3p plus pGL3-LINC00839-mut. After 48 hours, the luciferase activity was measured by a luciferase reporter assay kit (Promega). The c-Met 3′‐UTR containing putative wild type binding sites or mutated binding sequence for miR-454-3p was inserted into pGL3 luciferase reporter to generate pGL3-c-Met-wt or pGL3-c-Met-mut, respectively. SUNE-1 and C666-1 cells were co-transfected with miR-454-3p mimic plus pGL3-c-Met-wt or miR-454-3p plus pGL3-c-Met-mut. After 48 hours, the luciferase activity was measured by a luciferase reporter assay kit (Promega).
Tumor growth in vivo
sh-Ctrl or sh-LINC00839 stable transfected SUNE-1 (100 μL, 2×106) cells were subcutaneously inoculated into BALB/c nude mice (n=3 in each group). The width and length of tumor were recorded every week. Tumor volume = length × width2 × 0.5. After five weeks, nude mice were sacrificed. Tumor tissues was subjected for immunohistochemical staining assay. The animal experiment was in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Weifang Hospital of traditional Chinese Medicine.
Statistical analysis
Data are presented as Mean±SD. Statistical differences were assessed by Student's t-test or one-way ANOVA analysis followed by Dunnett's test. P value less than 0.05 was considered as statistical difference.