Patients and tissue samples
Tissue samples were collected from 22 patients with primary neuroblastoma and 9 patients with gangliocytoma treated in Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine from January 2012 to March 2019. All tumor samples from the patients enrolled in the study were surgically removed. The study protocol has been approved by the ethics committee of Xinhua Hospital, and the written informed consent of the participants’ parents or guardians has been obtained.
Data Mining From public databases
The Oncomine (https://www.oncomine.org/) and Tumor Immunity Estimation Resource (TIMER, https://cistrome.shinyapps.io/timer/) database mining tools were used to analyze the expression of MCM6 in different tumors and normal control tissues. Besides, comprehensive information about neuroblastoma-related clinical and prognosis, factors are obtained from R2: Microarray Analysis and Visualization Platform (https://r2.amc.nl) and selected for analysis. We selected three publicly available datasets to analyze tumor prognostic factors, including Kocak (GEO: GSE45547)(13),SEQC (GEO༚GSE49710༉(14) and Oberthuer (ArrayExpress༚E-TABM-38) (15). All Kaplan-Meier analyses were performed online, and the optimal p value and the cutoff value used to separate the high expression group and the low expression group were selected by the median.
Cell culture and transfection
Human neuroblastoma cell lines (SK-N-BE(2), SK-N-AS, SH-SY5Y) were obtained from ATCC (Manassas, USA), SK-N-SH, IMR-32 were from Chinese Academy of Sciences Cell Bank (Shanghai, China). All of the cells were routinely maintained in a 1:1 mixture of Eagle’s Minimum Essential Medium and Ham’s nutrient mixture F-12 Medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37 °C with 5% CO2. The human siRNA MCM6 and the non-targeting siRNA (sequences was in Table S1) were purchased from Riobio Biotechnology (Guangzhou, China). The cells were transfected with siRNA at a final concentration of 60 nM for 24 h performing in Lipofectamine™ RNAimax (Thermo Fisher Scientific, USA). To be specific, cultured the cells in a 6-well plate and performed transfection when the cell density was about 30%. In 200 ul Opti MEM I medium, mixed 0.12 nmol siRNA with 8 ul transfection reagent and incubated for 5 min, added to one well of a 6-well plate, and then add 2 ml complete medium. After 24 h of incubation, the medium was replaced with a standard medium and the cells were ready for further experiments.
Lentivirus-mediated silence for MCM6
The lentivirus containing shRNA interference oligonucleotide sequence (Table S1) and scrambled control shRNA was constructed and packaged by GeneChem (Shanghai, China). The SK-N-BE(2) cells were infected according to GeneChem's manufacturer's protocol. 72 hours after infection, 2ug/ml puromycin (Cat. #ST551, Beyotime, China) was used to screen cells transfected with stable lentivirus. Puromycin-resistant cells were collected 3 days after the addition of puromycin to obtain cells stably transfected with lentivirus.
Total RNA extraction and quantitative reverse transcription polymerase chain reaction
TRIzol reagent (Invitrogen, USA) was used to extract total RNA from cells. Reverse transcription of the extracted RNA was performed using the PrimeScript™ RT Master Mix (Cat. RR036A, TAKARA, Japan). Then, qRT-PCR was performed on the Applied Biosystems QuantStudio 3 real-time quantitative PCR instrument (Appliedbiosystems, Thermofisher Scientific). The primer sequences in this study are: 5'- GAT AAG ATG GAC GTG CGG GA-3' (forward) and 5'- GGC GTT CAG AGT AGC CTT CA-3' (reverse) of MCM6; 5'- ACC CAG AAG ACT GTG GAT GG-3' (forward) and 5'- TCT AGA CGG CAG GTC AGG TC-3' (reverse) for GAPDH. GAPDH was used as a standardization control, and the ΔΔCT method was used to calculate relative mRNA expression.
RNA-seq and data analysis
The Oligo(dT) magnetic beads are used to enrich the mRNA with polyA structure in the total RNA, and the RNA is interrupted to a fragment of about 300 bp in length by means of ion interruption. Using RNA as template, the first strand of cDNA was synthesized with 6-base random primer and reverse transcriptase, and the first strand of cDNA was used as template to construct the second strand of cDNA synthesis library. After that, PCR amplification was used for library fragment enrichment, and then library selection was conducted according to the fragment size, with the library size at 450 bp. The quality of the library was inspected by the Agilent 2100 Bioanalyzer, and then the total concentration of the library and the effective concentration of the library were detected. According to the effective concentration of the library and the amount of data required by the library, the libraries containing different Index sequences (each sample plus a different Index, and finally the off-machine data of each sample are distinguished according to the Index) are mixed in proportion. The mixed library is uniformly diluted to 2 nM, and the single-stranded library is formed by alkali denaturation. After RNA extraction, purification, and library building of the samples, the Next-Generation Sequencing (NGS) is used to perform paired-end (PE) sequencing on these libraries based on the Illumina sequencing platform. The raw sequencing reads can be obtained in the Gene Expression Omnibus (GEO) database with accession number GSE159637.
Analyses were performed in R version 3.5.2. First, filter the raw data, and compare the filtered high-quality sequence (Clean Data) to the reference genome of the species. According to the comparison results, the expression of each gene is calculated. On this basis, further analysis of expression differences, enrichment analysis and cluster analysis were performed on the samples. Compare the Reads on the pair to splice and restore the transcript sequence.
Whole-cell lysates were harvested for protein analysis. Lysed cells with 8M urea lysis buffer supplemented with protease inhibitors. Separate proteins by SDS-PAGE gel electrophoresis and transfer to PVDF membrane (Millipore, Sigma Aldrich, USA). The membrane was blocked with 5% BSA at room temperature for 1 hour, and then incubated with the appropriate antibody overnight at 4 °C. After washing the blots 3 times with TBST, the membranes were incubated with appropriate HRP-conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature, and washed again with TBST. The protein bands were visualized by the Bio-Rad ChemiDoc XRS imaging system. Primary antibody to MCM6 (Cat. ab201683, 1:1000) and β-actin (Cat. #ab8227, 1:1000) were purchased from Abcam. Cyclin D1(Cat. E3P5S, 1:1000), CDK4 (Cat. D9G3E, 1:1000) were purchased from Cell Singling Technology (CST).
Cell viability assay
Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8) (Yeasen, Shanghai, China). Cells at a density of 3 × 103/well were seeded into 96-well plates and cultured for 4 days. CCK-8 determination was performed every 24 h from the time the cells adhered. Add 100 ul of phenol red-free 1640 medium containing 10% CCK8 solution to each well and incubate at 37 °C for 2 h. The absorbance value (OD) of each well was measured at 450 nm and 630 nm.
Colony formation assay
Cells were plated in 6-well culture plates at 100 cells/well. After incubation for 2 weeks at 37 °C, the cells were washed twice with PBS and stained with 0.1% crystal violet solution. The number of colonies containing ≥ 50 cells was counted under a microscope. The colony formation efficiency was calculated as (number of colonies/number of cells inoculated) × 100%.
For the wound-healing assay, cells were grown to confluence in a six-well plate. Artificial wound tracks were created by scraping the confluent cell monolayers with a pipette tip. The cells were fed with serum-free medium. The ability of the cells to migrate into the wound area was assessed every 24 h after scratching.
Cell migration and invasive assays
Cell motility was assessed by cell migration and invasion assays using transwell chambers with or without Matrigel (Corning). Approximately 1 × 106 cells in medium without FBS were seeded on upper transwell chambers with or without Matrigel and incubated at 37 °C for 48 h. Medium containing 10% FBS was put in the lower chamber. The invasive cells attached to the lower surface of the membrane insert were fixed, stained using crystal violet (Beyotime, China) and quantified.
EdU incorporation assay by flow cytometry
For the EdU (5-Ethynyl − 2’- deoxyuridine) incorporation assay, proliferating cells were examined using the Cell-Light EdU Apollo488 In Vitro Flow Cytometry Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. Cells at a density of 1 × 106/well were seeded into 6-well plates and incubated with 50 mM EdU for 3 h. Next, harvested cells and fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and staining with Apollo fluorescent dyes. The fluorescence signal at 488 nm was collected by flow cytometer, and the proportion of positive signals labeled with EdU was analyzed by FlowJo software (FlowJo, LLC).
Cell Cycle Analysis
The cells in logarithmic growth phase were harvested and seeded into 6-well plates (1 × 106/well) and transfected with si-MCM6 or siRNA control. After 48 h, the cells were collected for flow cytometry. This experiment was repeated three times.
The subcutaneous model is used for animal research. In the subcutaneous model, 1 × 10 6 SK-N-BE(2) cells were suspended in 0.1 ml PBS medium and transfected with MCM6 shRNA, lentiviral vector and negative control (scrambled) vector. Ten 4-6-week-old male BALB/c nude mice, were obtained from Shanghai Jihui Laboratory Animal Care Co.,Ltd. (Shanghai, China), and randomly divided into two groups (5 mice/group): the LV-shMCM6 group and LV-shNC group. The 2 kinds of stably transfected cells were injected into the armpits of 5 male BALB/c nude mice separately. The mice were maintained in a barrier facility on a rack filtered by HEPA and fed an autoclaved rodent diet. After 45 days, the mice were sacrificed by cutting off necks after anesthesia, and the tumor tissue was surgically removed, weighed and stained with hematoxylin and eosin. All animal handling and procedures have been approved by the Animal Care and Use Committee.
GraphPad Prism 8 (GraphPad Software, Inc. La Jolla, USA) and SPSS version 25.0 software (SPSS, Chicago, IL, USA) for Windows was applied for statistical analysis. The qualitative data were compared with Fisher's exact test or Pearson's chi-square test, and the quantitative data were compared with Student's t test or analysis of variance. The correlations were analyzed by Spearman. Results were expressed as mean ± standard error of the mean (SEM). p < 0.05 was considered a statistically significant difference. Significance was expressed as: *p < 0.05, **p < 0.01, and ***p < 0.001.