Cell culture, reagents and clinical samples
NP69, C666-1, HNE-1, CNE-1, and 293T cells were maintained in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (FBS, Invitrogen, California) and 100 units/ml penicillin/streptomycin. Primary antibodies against actin and HK2 were purchased from Santa Cruz Biotechnology. A total of five nasopharyngeal carcinoma and paired normal adjacent tissues at least 5 cm away from the tumor sites were acquired from each patient by biopsy prior to any treatment. All human patient samples were collected with informed consents from the patients, and the experiments were approved by the ethics committee of Shanghai Jiaotong University.
Transfections
microRNA-206 inhibitor(5’-ccacaugcuucuuuauauuccaua-3’), microRNA-206 mimics(5’-uggaauguaaggaaguguguaa-3’), and scrambled siRNAs(5’-ugggcguauagacguguuacac-3’) were ordered from GenePharma (Shanghai, China). For the transient transfection, the cells were plated at 70% confluency and transfected with the indicated RNA molecules at a final concentration of 40 nM with the PEI reagent.
Quantitative real-time PCR
Total RNA was extracted from cells with TRIzol reagent as described in the manufacturer’s instructions. Reverse transcription of microRNA and mRNA was performed with the TaqMan qPCR miRNA assay kit. RT-qPCR detection of miR-206 was done with SYBR Green qPCR Supermix. U6 was used as an internal loading control.
Western blot analysis
The Cells were lysed by RIPA buffer. Total protein (30 g) was resolved using 8% SDS-PAGE and then transferred into nitrocellulose (NC) membrane. After blocking with 5% non-fat dried milk for 1 h, the NC membrane was incubated with the primary antibody at 4ºC overnight. After rinsing three times for 10 min each, the membrane was incubated with the secondary antibody for 1 h at room temperature. After washing with TBST three times, the signals were detected with the enhanced chemiluminescence (ECL) kit.
Dual-luciferase reporter assay
Dual-luciferase reporter assay was used to confirm whether miR-206 downregulates Glut1 via binding to the 3´ UTR region. HEK293T cells were seeded in a 48-well plate at 70-80% confluency. After co-transfection with either 20 nM of miR-206 mimics or scrambled controls and 40 ng of pGL-Glut1-3´UTR-wt or pGL-Glut1-3´UTR-mut, cells were harvested to measure the luciferase activity with Dual Luciferase Assay Kit (Promega, Madison, WI). Experiments of luciferase activity assay were performed independently in triplicates.
Cell proliferation assay
Cells were plated at a density of 5000 cells/well in 96-well plates and transfected with the indicated RNA molecules. After culturing in DMEM without serum for 6 h, the cells were allowed to recover in the culture medium for 48 h. After incubation for 48 h, the absorbance at 450 nm was measured with the Cell Counting kit-8 assay kit (CCK8) (DOJINDO. Tabaru, Japan). The cell survival rate was calculated using the equation:
survival rate (%) = (Asample – Ablank)/(Acontrol – Ablank) x 100.
Measurement of glucose uptake and extracellular lactate secretion
The miR-206 inhibitor or mimics were transfected into nasopharyngeal carcinoma cells in serum-free DMEM. After incubation for 48 h, the medium was collected. Lactate production was determined using the lactate assay kit (Sigma-Aldrich). Glucose uptake rate was determined using a colorimetric assay kit (Glucose Uptake Colorimetric Assay Kit, BioVision).
Statistical analysis
Statistical analyses were carried out with GraphPad Prism software. Data are presented as the mean ± SD. Results were considered to be statistically significant if p<0.05.