Melanoma stem cells and non-stem cells were sorted in our laboratory previously from cell line MDA-MB-435  or A375 . Melanoma stem cells and non-stem cells were cultured in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (EGF) (Beyotime Biotechnology, China), 10 ng/mL basic fibroblast growth factor (bFGF) (Beyotime Biotechnology), 5 mg/mL of insulin (Beyotime Biotechnology), and 2% of B-27 (Sigma, USA) at 37℃ in a humidified atmosphere with 5% CO2.
Quantification of mRNA with real-time PCR
Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2×ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme) and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was included for normalization. The 2-(△△Ct) method was used to calculate the relative fold change of mRNA expression . PCR was conducted by maintaining the reaction at 95℃ for 30 s, and then alternating for 40 cycles between 95℃ for 5 s and 60℃ for 30 s.
Western blot analysis
Proteins (about 50 μg/protein) were separated using 12% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with triethanolamine-buffered saline solution (TBS) containing 5% skim milk. Subsequently, the membrane was incubated overnight with a primary antibody, followed by incubation with the alkaline phosphatase-conjugated secondary antibody (Roche, Switzerland) for 2 h at room temperature. After rinses, the membrane was detected with BCIP/NBT substrate (Sangon Biotech, China). All antibodies were purchased from Proteintech Group (USA) at catalog number 67445-1-Ig (hnRNP A2B1), 15057-1-AP (TPPP3), 11310-1-AP (EIF3H), 25136-1-AP (DAPK1), A12757 (SYT7), 66969-1-Ig (DOCK2) and 26015-1-AP (RNF128).
Total RNAs were extracted using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) according to the manufacturer’s manual. After electrophoresis of 30 μg RNAs on a 2% agarose gel, the RNAs were transferred to a nylon membrane (Amersham Biosciences, Sweden) for 1 h, followed by ultraviolet cross-linking. The membrane was prehybridized in prehybridization solution (Roche, Switzerland) for 30 min. Subsequently, the membrane was hybridized with a DIG-labeled probe (Table S2) at 420C overnight. The membrane was rinsed and then blocked in a blocking solution (Roche) for 1 h at room temperature. After incubation with the antibody (10 μl) against DIG-labeled alkaline phosphatase (Roche) for 2 h at room temperature, the signals of the membrane were detected with the substrate BCIP/NBT solution (Roche).
RNA immunoprecipitation (RIP) assay
Cells were treated with paraformaldehyde for 10 min at room temperature. After washes with ice-cold phosphate buffer saline (PBS), the cells were incubated with PBS containing 0.125 M glycine for 5 min and then with hypotonic buffer [10 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (HEPES), 1.5 mM MgCl2, 10 mM KCl, 0.4% Nonidet P-40, pH 7.9] on ice for 15 min, followed by centrifugation at 3,000 ×g for 7 min. The pellet was resuspended in sonication buffer [10% sodium dodecyl sulfate (SDS), 0.5 M ethylene diamine tetraacetic acid (EDTA), 1 M Tris-HCl, pH 8.0] and then subjected to ultrasonication. The sample was centrifuged at 12,000 ×g for 20 s and the supernatant was incubated with antibody-coupled Protein G magnetic beads (70μl) (Bio-Rad Laboratories, USA) at 4℃ overnight. The beads were washed with PBS. Subsequently the RNAs were extracted using RNA Isolation Kit (Ambion, USA).
RNA-seq and data analysis
The extracted RNAs were subjected to RNA-seq using an Illumina Hiseq 2500 system by Novogene Corporation (China). Briefly, the rRNAs were removed by ribo-zerotm kit (Epicentre, France). Subsequently fragmentation buffer was added to break the RNA into short segments of 250-300bp, followed by the synthesis of cDNAs with random hexamers. After purification with AMPure XP beads, the double-stranded cDNAs were added with A tails and the connection of sequencing joints. The cDNA library was enriched by PCR. Sequencing was performed. After assembly of RNA-seq data, the raw data was processed to remove the sequences of adapter, ploy-N and low-quality reads. The clean reads were aligned to the genome reference consortium human reference 38 (hg38) by BWA (Burrows Wheeler Aligner) and IGV (Integrative Genomics Viewer). Based on the counts of reads, the gene expression profile was obtained.
Kyoto encyclopedia of genes and genomes (KEGG) analysis
The coding sequences of transcripts were extracted and used as queries to search the protein sequences collected in the GO (gene ontology) database with the blast E-value of less than 1×10-5. The best hit GO identities were assigned to the transcripts. The p-values were corrected for false discovery rate. Deduced genes with homologues in other organisms were used to map to conserved biological pathways.
Semi-quantitative reverse transcription (RT)-PCR
Total RNAs were extracted from cells using a cell/tissue genomic DNA extraction kit (Generay Biotech, China) and then quantified by NanoDrop ND-1000 spectrophotometer. The complementary DNA was synthesized using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, USA) following the manufacturer’s instructions. Subsequently PCR was conducted with sequence-specific primers (Table S3). β-tubulin was used as a loading control.
Cell viability analysis
Cell viability was evaluated using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, USA). Cells were seeded onto a 96-well plate. Thirty-six hours later, the plate was incubated with 20 μL of MTS reagent for 1 h at 37℃. Subsequently the absorbance was recorded at 490 nm. All experiments were repeated three times.
Analysis of caspase 3/7 activity
Caspase-Glo 3/7 assay (Promega) was used to evaluate the activity of caspase 3/7 according to the manufacturer’s protocol. Cells at a density of 1×104/well were plated onto a 96-well plate. Subsequently 50 mL of caspase-Glo 3/7 reagent (Promega) was added to each well. After incubation in the dark at room temperature for 1 h, the luminescence of cells was measured.
Cell cycle analysis
Cell cycle analysis was conducted with flow cytometry. Cells were fixed in ice-cold ethanol overnight. Then the cells were incubated with DNase-free RNase A (20 mg/mL) for 30 min. After centrifugation at 500×g for 5 min, the cells were stained with propidium iodide (PI, 50 mg/mL). The fluorescence intensity of 1×104 cells was measured with a flow cytometer at an excitation wavelength of 488 nm.
Silencing and overexpression of gene in cells
To silence the gene expression in cancer stem cells, RNA interference (RNAi) assay was conducted using gene-specific siRNA (Table S4). The melanoma stem cells (1×105) were transfected with 50 nM of siRNA using Lipofectamine 2000 (Invitrogen, USA). All the siRNAs were synthesized by Shanghai GenePharma Co., Ltd. At different time after transfection, the cells were harvested for later use.
To overexpress a gene in cancer stem cells, the gene was amplified using PCR with sequence-specific primers (Table S5), followed by cloning into pcDNA3.1 (+) vector. The recombinant plasmid was transfected into melanoma stem cells using Lipofectamine 2000 (Invitrogen). At different time after transfection, the cells were collected for later use.
To overexpress hnRNP A2B1 in the hnRNP A2B1-silenced melanoma stem cells, hnRNP A2B1 was mutated at a nucleotide (position 32 A→T) to prevent the recognition by hnRNP A2B1-siRNA. Briefly hnRNP A2B1 was amplified by hnRNP A2B1 primers 1 and 4 (Table S5), and hnRNP A2B1 primers 2 and 3 (Table S5), respectively. Then the amplified products were subjected to PCR with hnRNP A2B1 primers 1 and 2 (Table S5), followed by cloning into pcDNA3.1 (+) vector.
Tumorigenicity in nude mice
Melanoma stem cells were transfected with hnRNP A2B1-shRNA (short hairpin RNA) (5’-AGGAACAGTTCCGTAAGCTCTTTAT-3’) to stably silence the expression of hnRNP A2B1. ShRNA was designed with invitrogen BLOCK-iT™ RNAi Designer (http://rnaidesigner.thermofisher.com). As a control, the sequence of hnRNP A2B1-shRNA was randomly scrambled, generating hnRNP A2B1-shRNA-scrambled (5’-CCGGGCGCGATAGCGCTAATAGCGA-3’). ShRNA was cloned into lentiviral vector pLent-U6-GFP-Puro (Vigene Bioscience, USA), followed by transfection into 293T cells using Lipofectamine 2000 reagent (Life Technologies, USA). At 48 h after transfection, the viral particles were collected to infect melanoma stem cells. Subsequently, the cells were cultured in medium contained 10 µg/ml puromycin for three days. After puromycin screening, only the cells expressing green fluorescence protein (GFP) were selected as stable strains expressing shRNA.
Melanoma stem cells transfected with hnRNP A2B1-shRNA or hnRNP A2B1-shRNA-scrambled were suspended in physiological saline. Matrigel (Becton, Dickinson and Company, USA) was added to the cell suspension at the final concentration of 33%. Subsequently, 100 μL of the cell suspension was subcutaneously injected into each BALB/c mouse to induce tumor growth. The tumor volume was measured every week. Six weeks later, the mice were sacrificed. The tumor sizes and tumor weights were determined. All procedures conducted on mice in this study were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC). All the methods were carried out in accordance with the approved guidelines.
Tumor tissue was cut into 5 μm pieces and mounted onto a slide. The slide was dewaxed and hydrated in 100%, 95% and 80% ethanol for 5 min, respectively. Subsequently the primary antibody was incubated with the slide. After washing with PBS, the slide was incubated with the secondary antibody for 10 min at room temperature. Streptavidin peroxidase was added to the slide, followed by incubation for 10 min at room temperature. Then 3-amino-9-ethylcarbazole (AEC) buffer and AEC chromogen (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were mixed and added to the slide. The slide was incubated for 10 min at room temperature. The proteins and the nucleus were labeled with diaminobenzidine (DAB) (Sigma, USA) or 4’,6-diamidino-2- phenylindole (DAPI), respectively.
The numerical data were analyzed by one-way analysis of variance (ANOVA). The differences between different treatments were analyzed by Student’s t test. All data were presented as mean ± standard deviation.