1. Sample collection
Globally, 32 frozen NPC biopsies and healthy juxta-positioned tissues were collected from NPC patient cohorts that underwent surgical intervention at The First Affiliated Hospital of Bengbu Medical College. Patients were not treated prior to surgery. All procedures involving human samples in this study were conducted according to the Helsinki declaration and accepted by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College. Informed consent was collected from all individuals.
2. Cell culture and transfection
H.Sapiens NPC HNE-1, CNE-2Z, 5-8F and healthy nasopharyngeal epithelial cell line NP69 were from XiangYa School of Medicine of Central South University [Hunan,China]. All NPC cell lines were grown within RPMI-1640 medium [Gibco™, USA], augmented by 10 % fetal bovine serum [FBS, Gibco™, USA] and incubated at 37°C / 5% CO2. NP69 cell cultures were grown within keratinocyte serum-free medium [Gibco™, Thermo Fisher Scientific, China]. Small interfering RNA against CRNDE (si-CRNDE) and relevant control (si-NC), miR-497-5p mimics and relevant controls (miR-NC), together with miR-497-5p inhibitor and relevant control (miR-inhibitor NC) were all procured from Sangon™ [Shanghai, China]. All transfections employed Lipofectamine 2000® [Thermo Scientific™, China].
3. Quantitative reverse-transcription polymerase chain reaction
Total RNA was collected from CNE-2Z / HNE-1 cells using TRIzol® [Ambion™, TX, USA] and reverse transcribed into cDNA through EasyScript® One-step gDNA Removal and cDNA Synthesis SuperMix kit [TransGen™, China]. Consequently, ABI StepOnePlus® [Bio-Rad™, USA] was utilized for 40 cycles of amplification at 95 °C for 30 s, denaturation at 95 °C for 5 s, and extension at 65 °C for 30 s. GAPDH and U6 served as normalization controls of mRNAs and miRNAs, respectively. The - 2ΔCT method was used to calculate relative expression for all investigated miRNAs / transcripts. Primer sequences for RT-qPCR were as follows:
CRNDE (Forward,5‘-GGAAAAATCAAAGTGCTCGAGTGG-3’;Reverse,5‘-TCTTCTGCGTGACAACTGAGGA-3’),
miR-545-5p (Forward,5‘-CGCGCGTCAGTAAATGTTTATT-3’;Reverse,5‘-AGTGCAGGGTCCGAGGTATT-3’),
CCND2 (Forward,5‘-TTTAAGTTTGCCATGTACCCAC-3’;Reverse,5‘-ACGTCTGTGTTGGTGATCTTAG-3’).
4. EdU assay
Cells of each group of CNE-2Z and HNE-1 in logarithmic growth phase were seeded into 6-well plates (1.5×104 cells/well). The cells in each group were labeled with EdU detection solution and consequently fix-treated using 4 % paraformaldehyde at ambient temperature for 15 minutes. Following wash-steps using PBS, 200 µL / well click reaction solution was introduced, and plates placed into incubation for 30 minutes in darkness at ambient temperature. Following another PBS wash-step, 1 mL Hoechst 33342 was added to each well for DNA staining. Following a final PBS wash-step, wells were visualized by laser confocal microscopy.
5. CCK-8 assay
HNE-1 cells and CNE-2Z cells, with good growth status post-transfection, were inoculated in 96-well plates (5000 cells / well) .A 10 μL CCK-8 aliquot was introduced into all wells at 24-, 48-, 72- and 96-hours, respectively, and placed into incubation for 120 minutes. Absorbance (OD) values at 450 nm were measured for individual wells upon a microplate reader, and a proliferation curve was prepared.
6. Transwell assay
In the migration experiment, Materigel gel was not paved. In the invasion experiment, Materigel and Opti-MEM® I Reduced-Serum Medium were diluted at a ratio of 1 : 8, with 50 μL of each chamber spread at the base of the upper-chambers and incubated for 1h to render it semi-solidified. CNE-2Z and HNE-1 cells were inoculated into the upper-chambers within serum-free RPMI-1640 medium, and 600 μL RPMI-1640 medium carrying 10 % FBS was introduced into the lower wells. For the migration experiment, cells were grown for 24 hours. For the invasion experiment, cells were grown for 48 hours. The upper-chambers were consequently removed, fix-treated using 4 % paraformaldehyde for 15 minutes, dyed using crystal violet for 10 minutes, subjected to PBS wash-step, followed by careful removal of excess dye using a cotton swab, and finally observed / counted. The number of cells in five randomly chosen fields was calculated under the microscope, and all experiments were repeated three times.
7. Wound-healing assay
Cells were labeled at the bottom of the six-well plate pre-inoculation. 24 hours post-transfection, once cultures obtained 80 % confluence, cells were lined - perpendicular to the bottom - with a 100 μL sterile pipettor tip/gun. Linear changes at 0 h and 24 h were observed, and the cellular migrative ability was detected. The calculation used was:
Wound healing rate (%) = [ (0-h scratch width – 24-h scratch width) / 0-h scratch width ] × 100 %.
8. Flow cytometric analyses
In order to assess apoptosis, cultures were collected and resuspended into cell suspension, centrifuged, supernatant discarded, and pre-cooled at 4 °C with D-Hanks (pH=7.2~7.4) washing cell precipitation. Consequently, 1 × binding buffer was-step was performed on cultures cells to precipitate once, followed by centrifugation and cell collection. Consequently, 200 μL 1 × binding buffer was used for resuspending cell precipitation. A total of 2μL Annexin V-APC and PI staining were added, with cultures left at room temperature in darkness for 20–60 minutes. According to cell volume, 200-300μL of 1 × binding buffer were added, followed by on-line detection.
In order to detect cell cycle, samples were harvested / fixed overnight using 70 % ethanol, pre-cooled at 4 °C. Fixative was then removed and cells washed using PBS at 4 °C once. Cell staining solution was prepared by adding 0.5 mL propidium iodide staining solution to each cell sample, slowly and fully resuspending cell precipitations, followed by 37°C in darkness for 30 min and temporary storage at 4°C or in an ice bath. Following staining, flow cytometry was used to complete the detection.
9. Dual-luciferase reporter assay
All 3 ′-UTR CRNDE / CCND2 sequences were amplified using polymerase chain reaction (PCR) and ligated with GV272 vector to construct wild-type CRNDE reporter vector (CRNDE-wt) and mutant-type CRNDE (CRNDE-mut), wild-type CCND2 reporter vector (CCND2-wt) and mutant-type CCND2 (CCND2-mut) luciferase reporter plasmids, respectively. MiR-545-5p mimics together with corresponding negative control miR-NC were co-transfected with the two recombinant plasmids into CNE-2Z cells using lipofectamine® 3000 [Invitrogen™, USA]. Luciferase activities for individual study-groups were evaluated in order to analyze possible binding of CRNDE with miR-545-5p, and CCND2 with miR-545-5p.
10. Western blotting
Protein content from all tissue / cell line samples were extracted and quantified through BCA kit® [Beyotime™, China]. Following electrophoresis, samples were transferred onto PVDF membranes [Biosharp™, China], milk-blocked and consequently hybridized with a variety of primary antibodies [1:1,000 dilution, Proteintech Group, INC.™] and incubated overnight (4°C). This was followed by placing in incubation with in second antibodies [1:1,000 dilution, Proteintech Group, INC.™] at room temperature, membrane scanning and observation of band shifts.
11. Xenograft tumor Model
BALB /c nude murines (female) were segregated into two cohort-groups, sh-CRNDE NC (sh-NC) and sh-CRNDE. The tumor cells of each experimental group in the logarithmic growth phase were counted by blood cell counting plate, and finally suspended with required volume of D-Hanks or PBS. Following preparation of tumor cells (5.00 E +06 cells / murine),a disposable sterile syringe was utilized to aspirate absorb cells and consequently inject 200 µL into each nude murine. At 24-days following subcutaneous injection, each experimental animal was injected with 2% pentobarbital sodium (0.5mL) euthanasia, and cervical dislocation confirmed death. Selected tumor samples were fix-treated with 4% paraformaldehyde for immunohistochemical analyses. All other samples were kept at − 80 °C for Western blot assays.
12. Immunohistochemistry
The transplanted tumor was fixed with formalin for 48 hours, soaked in 4 % paraffin, sliced, dewaxed and hydrated. High pressure antigen repair consisted of 3 % hydrogen peroxide being introduced to tissue samples, followed by incubation at room temperature for 20 minutes. Following sample segment washing, drying, ki-67-treatment and 37 °C incubation for 60 minutes, a secondary antibody was added, followed by incubation at 37 °C for 20 minutes. Following further wash/dry-steps, DAB coloring agent was added, with monitoring of color time under a microscope for any positive staining stop colorations. Once coloration step was stopped, samples were treated with hematoxylin staining for one minute, followed by 1 % hydrochloric acid treatment (a few seconds).Finally, samples were dehydrated, rendered transparent, and sealed for image capturing.
13. Statistical analysis
SPSS21.0® software was employed for analyzing experimental datasets, represented as mean ± standard deviation [SPSS, Inc™, USA]. One-/ two-way variance analyses were employed for comparative analyses. LSD-t test was employed for group comparisons. A p-value of <0.05 conferred significance.