The clinical samples used in this research were primary pediatric NB patients histologically diagnosed in Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine from September 2012 to February 2015. Tumor samples from all the patients in the study were surgically removed. All specimens were first quick-frozen in liquid nitrogen and then stored at -80°C for tissue microarray analysis. The research and consent procedure have been validated by the Clinical Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine.
Tissue microarray (TMA) preparation and immunohistochemistry (IHC)
Separate a small part of the tissue specimen and shape it in a customized mold for chip production. Then fix overnight in 4% paraformaldehyde, after that，tissue blocks were embedded in paraffin in a prepared array. Then the sample was sliced (5 μm) and adhered to a poly-L-lysine coated glass slide for immunohistochemical staining, which was performed as previously described [18, 19], using specific antibody against ALDOB (1:100 dilutions, Cat.18065-1-AP, Proteintech Group, Chicago, USA). Without knowing the clinicopathological characteristics of the tumor, the immunoreactivity in tissue sections was observed under three random microscopes, and then evaluated by three pathologists. Differences in scoring were discussed until consensus was reached. The tissue sections were scored under an optical microscope according to the degree of staining (0~3 points were negative staining, light yellow, light brown, dark brown) and the positive range (1~4 points were 0~25%, 26~50%, 51~75%, 76~100%). The two scores are then multiplied to get the final score for comparison. Finally, the intensity of staining was divided into four levels according to the final score: "0" means negative expression (the final score was 0), "1" means weak positive expression (the final scores were 1~4 points), "2" means moderate expression (the final scores were 5~8 points), and "3" means strong expression (the final scores were 6~12 points).
Public database analysis
Four publicly available data sets were selected from the R2: Microarray analysis and Visualization platform (http://r2.amc.NL) : Oberthuer-251-custom-ampexp255, Maris-101-custom-u95a, NRC-283-rma_sketch(bc)-huex10t and Asgharzadeh-249-Custom-huex10Tx, which contained information on the clinical characteristics and prognosis of patients with neuroblastoma, the target genes were obtained and selected for analysis. All Kaplan-Meier analyses were performed online, and the optimal P value and corresponding cutoff value for separating the high-expression group from the low-expression group were selected by the median.
Human neuroblastoma cell lines SK-N-BE (2) and SH-SY5Y were acquired from American Type Culture Collection (Manassas, USA) and maintained in a mixture of Eagle basic medium and Ham nutrient mixture F12 1:1, 10% fetal bovine serum (FBS) , both from Gibco, USA, and cultivated in a 5% CO2 moist incubator at 37°C.
Production and infection of lentivirus
Lentivirus particles expressing shRNA sequence targeting human ALDOB gene (5'-CCTATTGTTGAACCAGAGGTATA-3') were designed and constructed, and shRNA particles expressing nonsense sequences were used as negative control (shNC). Lentivirus particles expressing shALDOB or shNC were added to 6-well plates with SH-SY5Y and SK-N-BE(2) growing to 30%-60% confluency, and the infection complex (MOI) was 20. After 48 hours of transfection, fluorescent microscope (Olympus) was used to detect the transfection efficiency of stably expressed green fluorescent protein (GFP) cells, and 1 g/ml puromycin (Cat. #ST551, Beyotime, China) was used to screen the transfected cells, and stable transfected cell lines were obtained after 48 hours of continuous treatment. After screening, some cell RNA was collected to confirm the ALDOB mRNA level by qPCR, and the extracted protein was detected by Western blot.
RNA extraction, reverse transcription and real-time PCR
Total RNA was extracted from cells by Trizol Reagent (Thermo Fisher Scientific, USA). Reverse transcription was executed using PrimeScript RT kit (TaKaRa Biotech, Shanghai, China), bio-RAD Labs (Hercules, CA) perform the Real-time PCR with SYBR Green PCR Master Mix (TaKaRa Biotech), following the manufacturer’s protocol. The expression of ALDOB mRNA was normalized to GAPDH and used the 2-ΔΔCt method for relative quantitative. We repeated three independent porous experiments for statistical analysis. Primer sequences used in this research are
ALDOB forward: 5'-AGCTATGGCCACCGTAACAG-3',
ALDOB reverse: 5'-GGGCTTTGGTAGAGGGCAAA-3'
GAPDH forward: 5'-TGTGGGCATCAATGGATTTGG-3'
GAPDH reverse: 5'-ACACCATGTATTCCGGGTCAAT-3'
Cell viability assay
Stably transfected SH-SY5Y and SK-N-BE(2) cells were inoculated in 96-well plates at a density of 5×10 3/ well and cultured in 100 microliters of MEM/ F12 containing 10% FBS for 4 days. Cell viability was detected by cell counting kit (CCK-8) (Dojindo Molecular Technologies Inc., Shanghai, China) every 24 hours. The specific procedure was to add 10 microliters of CCK-8 solution to each well, and then to measure the absorbance value (OD) of each well at 450nm and 630nm after incubation at 37℃ for 1 hour.
Colony formation test
The stably transfected SH-SY5Y and SK-N-BE(2) cells were inoculated into a 6-well plate at a density of 1000 cells per well. The cells were cultured in a cell incubator for 14 days, and the liquid was changed every three days, the cell state and the size of the clones was observed under the microscope. When each clone was larger than 20 cells, the culture medium was removed, paraformaldehyde fixed the cell, then crystal violet was used to stain cells. Next the cells were washed with PBS, and photographs were taken.
Determination of wound healing
SH-SY5Y and SK-N-BE(2) cells were cultured in a six-well plate and grown to confluence 90%. Artificial wound marks were created by scraping a single layer of converging cells with a pipette suction head and incubating the cells with a serums free medium for 48 hours. After PBS rinsing, photographed transplanted cells at 0 and 48 hours, and calculated the migration distance during wound healing was.
Cell invasion assay was performed in transwell chamber using Matrigel (BD, Biosciences, CA). 6×10 cells in the matrix medium with no fetal bovine serum were incubated in Transwell upper chamber with matrix gel. In the lower chamber, there was the cell culture medium with 10% FBS. Then cultivated at 37°C for 48 hours. After 48 hours, 4% paraformaldehyde fixed the cells on the submembrane surface of the compartment and then stained with crystal violet.
Western blot analysis
Lyse cells on ice with RIPA buffer which contains PMSF (Cat. # ST506, Beyotime, China) (Cat. # P0013B, Beyotime, China) for 30 minutes, centrifugation at 20,000 g for 15 minutes, collected the supernatant, and used the BCA kit (Thermo Scientific, Rockford) to test the protein concentration. Gel electrophoresis used 10% SDS-PAGE gel and contains the same amount of protein per lane. Protein transfer used PVDF membranes (Millipore, Sigma Aldrich, USA). Then took the 5% BSA TBST (Tris buffered saline, pH 7.6, 0.1% Tween®20) to seal the PVDF membranes at room temperature for 1 hour, followed by primary incubation at 4°C overnight. TBST washed the membrane 3 times for 5 minutes each time, then incubated with TBST and an appropriate secondary antibody at room temperature for 30 minutes. Next, TBST wash the membrane 3 times for 5 minutes each time. Proteins are visualized by electrochemical luminescence (ECL). The primary antibodies of ALDOB (catalog number AB75751) and β-actin (catalog number AB8227) were purchased from Abcam. E-cadherin, Vimentin, N-cadherin, Claudin-1, Slug, and ZEB1 were purchased from Cell Singling Technology (CST). Image J (X64, v. 2.1.4) software for quantitative analysis of imprinting.
The experimental data were showed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism8 software (GraphPad Software, Inc. La Jolla, USA). Between the two groups, we used Student's T-test to estimate the differences, and between the three groups, we used and one-way ANOVA. Kaplan-Meier analysis was used for survival assessment, and survival differences was analyzed by log-rank test. P <0.05 was considered as a statistically significant difference. Significance was displayed as: * P <0.05, ** P <0.01, and *** P <0.001