Cell culture and treatment
The NPC cell line CNE2 and the human immortalized nasopharyngeal epithelial cell line NP69 were accepted from Sun Yat-Sen University (Guangzhou, China) as gifts. The NPC cell lines (CNE1, 5-8F, 6-10B) were gifts from the Xiangya School of Medicine, Central South University. NP69 cells were cultured in serum-free medium. The four human NPC cell lines (CNE-1, CNE-2, 5-8F, 6-10B) were cultured in RPMI 1640 medium with 10% FBS (Gibco). Among them, 5-8F and 6-10B cells were cultured in complete medium with 100 U/mLpenicillin, 100 μg/mL streptomycin (Shanghai Genebase Gen-Tech Co., Ltd., Shanghai, China). All of these cell lines were incubated at 37 ° C in a humidified atmosphere containing 5% CO2 . In the hypoxic experiments, cells were placed in an anoxic model incubator (Billups-Rothenberg) maintained in an atmosphere of 1% O2 , 5% CO2 and 94% N2 .
Statement of Ethics
The experiment was conducted with the true understanding and written consent of each patient who have been performed with the appropriate participants' informed consent in compliance with the Helsinki Declaration. All fresh primary NPC specimens with paired nasopharyngeal epithelial samples were obtained from Nantong University Affiliated Hospital(2019-L065). All patients did not receive any anti-tumor treatment prior to biopsy. All patients were confirmed by histopathology. All experiments conducted were endorsed by the Academic Committee of Nantong University. All animal experiments were ethically approved by the Jiangsu Provincial Laboratory Animal Management Committee and followed the NIH guidelines.
miR-433 mimic/ mimics NC/inhibitor/ inhibitor NC (Shanghai,China) at a final concentration of 30 nM were transfected into cells (1 x 105 / well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Hs-miR-433 mimic: 5′-AUCAUGAUGGGCUCCUCGGUGU-3′. Hs-miR-433 inhibitor: ACACCGAGGAGCCCAUCAUGAU. The same method as mentioned above was used for the PEGFP-shHif-1α expression vector and the PEGFP-shRNA-NC expression vector (GeneChem, China). After 24 hours of transfection, cells were harvested and subjected to further analysis.
Western blot analysis
Western blot analysis was carried out as described previously. The antibodies used were as follows: anti-HIF-1α (Abcam; 1: 2,000 dilution), β-actin (Santa Cruz Biotechnology, CA, USA, 1: 2,000 dilution), Vimentin, E-cadherin, N-cadherin, P65,p-P65 (Cell Signaling Technology, Danvers, MA, USA, 1: 1,000 dilution). Membranes were washed, then incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology; 1: 1,000 dilution).Blots were then developed by enhanced chemiluminescence (ECL; Cell Signaling Technology).
According to the manufacturer's instructions, 1 ml of Trizol (Invitrogen) was added to the tissues and cells, and total RNA was extracted by homogenization on ice. The real-time PCR system was subjected to real-time quantitative PCR (RT-qPCR) and SYBR Green PCR master mix (Thermo Fisher) for miRNA and mRNA analysis. The relative expression of miRNA was normalized against U6 RNA and mRNA expression was normalize using GAPDH, respectively.
Dual Luciferase Reporter Assay
To study the regulation of target genes by microRNAs, bioinformatics methods to predict potential target genes and intervention site sequences of miR-433, and design appropriate microRNA plasmids or intervention fragments, and construct reporter plasmids of target genes. Then, 293 cells (or other cells of interest) were cultured and seeded in a 12-well plate and grown for 24 hours. The reporter plasmid was co-transfected with the miR-433 binding sequence. The harvested cells are added to a specific luciferase substrate, and the luciferase reacts with the substrate to produce fluorescein, and the activity of the luciferase is determined by measuring the intensity of the fluorescence.
Cell viability assay
Cell viability was determined over time via Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Shanghai, China). Cells were dissociated and seeded in 96-well plates (5 × 103 cells/well) and the plates were pre-incubated in an incubator for a suitable time (37 ° C, 5% CO2 ). 10 μl of CCK8 solution was added to each well(100 μl medium), and plates were returned to the incubator for 1-4 hours. The absorbance at 450 nm was measured by using a microplate reader.
Briefly, 5×104 cells were mixed and transfected with serum-free DMEM, placed in the upper chamber of a 24-well Boyden Chamber (Millipore, 8-μm pore size), and 500 μl containing 10% FBS in the lower chamber. Medium. The plates were placed in an anoxic modular incubator (Billups-Rothenberg). After culturing for a suitable period of time, the chamber was taken out, fixed by methanol for 30 minutes, and crystal violet stained for 30 minutes. The upper layer of unmigrated cells was gently wiped off with a cotton swab; and washed 3 times with PBS. All migrated cells were counted using a microscope.
Cells were transfected in 6-well plates for 24 hours under hypoxic conditions. 1 x 106 transfected cells were harvested and fixed in 70% ethanol at 4 °C overnight. Wash cells 3 times with washed phosphate buffered saline (PBS), and the cells were stained in PI (BD Biosciences, Oxford, UK) staining solution in the presence of DNase without DNase in the dark. After incubation for 30 minutes in the dark at room temperature, cells were assayed by flow cytometry using cell quest software (BD FACS Aria).
The expression of EMT markers, ki67 and HIF-1α in NPC was analyzed by immunohistochemistry using DBA Assay Kit (ZSGB, China). Antibody drops were incubated on sections: anti-E-cadherin/N-cadherin/vimentin ( 1:100, CST, USA), anti-ki67 (1:100, Abcam, USA), anti-HIF-1α (1:100, Abcam, USA). Each experimental group was observed under a microscope (Leica, Wetzlar, Germany).
Quantitative analysis of serum HIF-1α expression levels
The concentration of HIF-1α in the serum samples of NPC patients was quantitatively analyzed using an enzyme-linked immunosorbent assay (ELISA) (HIF-1α ELISA kit, CSB-E12112h, CUSABIO) according to the kit instructions. About 100 μL of the standard control sample and the serum sample were added to a microtiter plate coated with HIF-1α antibody, followed by incubation for 2 hours at 37 ° C. Finally, absorbance can be measured at 450 nm and analyzed by the following method: Curve Expert.
Zebrafish transfer model
Nasopharyngeal carcinoma cells were injected into zebrafish, nasopharyngeal carcinoma cells were stained (2 g/ml of DiI, 30 min), 100-500/5 μl suspended in minimal medium, and injected into F1 zebrafish embryo (48h) yolk sac. After 48h, the cell mass transfer in zebrafish was observed by fluorescence microscope and statistical analysis was performed.
All BALB/c at hymic nude mice (5-6 weeks old) were provided by Shanghai Laboratory Animal Center, China, and kept in a specific pathogen-free environment. All mouse experiments followed institutional guidelines. CNE2 cells (1 × 106) in 0.1 ml 1640 medium without fetal bovine serum were subcutaneously injected into the mice. After 24 days, tumors were resected and measured for volumes.
Each experiment was repeated three times. Statistical analysis using Prism6 and SPSS 19.0 software for statistical analysis.The t test was used for comparison between groups. Data are expressed as mean ± standard deviation; P < 0.05 is considered statistically significant.