2.1 Patient samples
Clinical data were obtained from 132 children (under 16 years old) with NBL diagnosed pathologically as neuroblastoma at the First Affiliated Hospital of Sun Yat-sen University from January 1, 2014, to December 30, 2019. Clinical data were applied to analyze the relationship between the diagnostic stage and METTL1 expression levels. Paraffin-embedded specimens were used for immunohistochemistry (IHC) analysis of METTL1 expression levels. Ethical approval for the study with human subjects was obtained from the Institutional Review Board of the First Affiliated Hospital of Sun Yat-sen University, and written consent was obtained from each patient.
2.2 Experimental Animals
The BALB/c-nu female mice were purchased from GemPharmatech Co., Ltd (Jiangsu, China). All animal care and experimental protocols were approved by the Institutional Ethics Committee for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University. The study complied with all relevant ethical regulations regarding Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Mice were euthanized when their tumor size and overall health status met the institutional euthanasia criteria.
2.3 Cell lines and cultures
Human embryonic kidney 293T (HEK 293T) cells were obtained from Prof. Shuibin Lin's laboratory (Guangzhou, China). Human NBL KELLY cells were obtained from Tongpai Biotechnology Co Ltd (Shanghai, China). Human NBL BE2C cells were from Procell Life Science & Technology Ltd (Wuhan, China). 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA), and 1% GlutaMAX (Gibco, USA). KELLY cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, Australia) and 1% penicillin-streptomycin (Gibco, USA), and 1% GlutaMAX (Gibco, USA). SK-N-BE(2)C (BE2C) cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, USA) supplemented with 10% FBS (Gibco, Australia), 1% penicillin-streptomycin (Gibco, USA), 1× GlutaMAX (Gibco, USA), and 1% MEM non-essential amino acid solution (Gibco, USA). Cells were cultured in a 5% CO2 cell culture incubator (Thermo Scientific, USA) at 37°C.
2.4 Knockdown of METTL1 in NBL cells
Lentiviral vectors expressing pLKO.1 shRNA as a negative control and two shRNA constructs targeting METTL1 were supplied by KeyGEN BioTECH (Nanjing, China). For lentivirus production, the lentiviral shRNA constructs were co-transfected into HEK 293T cells using Lipofectamine 2000 (Invitrogen, USA) with a packaging plasmid (pCMV-ΔR8.9) and an envelope plasmid (pCMV-VSVG). 48 h later, the viruses were collected and infected with Polybrene (Solarbio, China) (4 µg/ml for KELLY cells and 8 µg/ml for BE2C cells). Stably infected cells were screened with puromycin (Solarbio, China) (2 µg/ml for KEELY cells and 4 µg/ml for BE2C cells) for 24 hours. Small interfering RNAs (siRNAs) targeting the 3'-UTR of METTL1 were used to knockdown METTL1 with Lipofectamine 2000 (Invitrogen, USA). siRNA sequences are listed in Table S1.
2.5 Cell proliferation and migration assays
For the cell proliferation assay, 1000 cells were grown in each well of a 96-well plate with 100 µL of fresh medium. Cell viabilities were measured every 24 h for five days using Cell Counting Kit-8 (Dojindo, Japan). For the migration assay, 7.5×104 cells in 200 µL of serum-free medium were added to the upper chamber of the transwell insert (Corning Falcon, USA) and placed in receiving wells containing 700 µL of cell culture medium supplemented with 10% fetal bovine serum. Migrated cells were stained with 0.5% crystal violet and counted after 24 hours.
2.6 Cell apoptosis assays
According to the manufacturer's instructions, the cell apoptosis assay was performed with Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, China). The percentage of positive cells was detected by CytoFLEX (Beckman Coulter, USA).
2.7 Subcutaneous implantation in a mouse model
Four- to six-week-old BALB/c-nu female mice were randomly divided into shNC and shMETTL1-1 groups (n = 5). NBL cells were resuspended by mixing equal amounts of phosphate-buffered saline (PBS, Gibco, USA) and Matrigel (Corning, China). 7× 106 NBL cells in 100 µl of the PBS-Matrigel mixture were injected into the back of the mice. The length (a) and width (b) of the tumors were measured every two days with calipers, and the tumor volume (V) was calculated using the formula V = ab2/2. Fifteen days after injection, the mice were humanely killed, and the harvested tumors were used for further analysis.
2.8 Immunohistochemical (IHC) staining
IHC was performed with an IHC kit (Agilent, USA) and primary antibodies (anti-METTL1, Proteintech, 1:2000 dilution; anti-Ki67, Proteintech, 1:8000 dilution) to detect the described protein expression. To assessing the level of METTL1 expression, histochemistry score (H-score) is generating by the following formula: H Score = summation (1 + i)×pi, where i is the intensity score and pi is the percent of the cells with that intensity. The intensity score was categorized as 0 (absent), 1 (weak), 2 (moderate) and 3 (strong). The percent of the cells was scored as 0, 1, 2, and 3 for < 5%, 5–25%, 25–50%, 50–75%, and > 75%, respectively. Tissues with H-score ≧ 3 were considered as high METTL1 expression group, and tissues with H-score < 3 were classified as low METTL1 expression group.
2.9 RNA isolation and quantitative analysis
According to the manufacturer's instructions, total RNAs were isolated with AG RNAex Pro RNA Reagent (AG, China). For reverse transcription-polymerase chain reaction (RT-PCR), cDNA was synthesized in a 20uL reaction system using HiScript III RT SuperMix for qPCR Kit (Vazyme, China). cDNA samples were then diluted at 1:20 and used for qPCR. Real-time quantitative PCR assays (qPCR) were performed on a StepOnePlus™ real-time PCR system (Thermo Scientific, USA) with TB Green™ Premix Ex Taq™ II (Takara, Japan). Each sample was repeated three times. Results were calculated by the 2−ΔΔCt method using β-ACTIN as an internal control. The primer sequences used in this study are listed in Table S1.
2.10 Northern blot, Northwestern blot, and Western blot
As previously reported, Northern blot and western blot assays were performed[26, 35]. Briefly, for Northern blot, 2 µg of total RNA was separated by electrophoresis on a 15% TBE-UREA gel, and then the RNAs were transferred to a positively charged nylon membrane. Afterward, the nylon membranes were cross-linked with ultraviolet (UV) light. The indicated tRNAs and U6 snRNA were blotted with Digoxigenin-labeled probes. After transfer and cross-linking, nylon membranes were blotted with primary antibody (anti- m7G, MBL International, USA) for Northwestern blotting at 4°C overnight. Anti-digoxigenin or anti- m7G antibody signals were detected according to the previously described Western blot protocol[26, 35]. Probe sequences are listed in Table S1.
2.11 Polysome profiling
Polysome profiling was performed as previously described[36]. Briefly, NBL cells were incubated with 100 µg/ml cycloheximide for 15 min at 37°C. After immediate cold PBS wash, the cells were lysed with multimeric cell extraction buffer (50 mM MOPS, 15 mM MgCl2, 150 mM NaCl, 100 µg/ml cycloheximide, 0.5% Triton X-100, 1 mg/ml heparin, 200 U/ml RNase inhibitor, 2 mM Phenylmethylsulfonyl Fluoride (PMSF), and 1 mM benzamidine) for 10 minutes on ice and centrifuged at 13,000g for 10 min at 4°C. The OD values of the samples were measured and adjusted to be equal. Then 1 ml of cytoplasmic extract was layered onto 11 ml of a 10%-50% sucrose gradient, followed by centrifugation at 36,000 rpm for 3 h at 4°C. Separated samples were fractionated at 0.75 ml/min by a BR-188 density gradient fractionation system (Brandel, USA) and monitored at an absorbance of 254 nm.
2.12 Puromycin intake assay
NBL cells were transfected with METTL1 siRNA and negative control oligos (siNC). After 48 hours, cells were incubated with puromycin (final concentration of 1µM) for 30 min at 37°C. After incubation, the cells were lysed to extract proteins, and the levels of puromycin were detected by Western blot with an anti-puromycin antibody (MABE343, Millipore). The siRNA sequences are listed in Table S1.
2.13 tRNA m7G reduction and cleavage sequencing (TRAC-seq)
TRAC-seq was performed as previously described[37]. Small RNAs were isolated from total RNAs using the Quick-RNA™ Microprep Kit (Zymo Research, USA) according to the manufacturer's instructions, followed by recombinant wild-type and D135S AlkB protein treatment. Half of the AlkB-treated RNAs were used as input for the construction of the library. Next, the remaining AlkB-treated RNAs were treated with 0.2 M NaBH4 for 30 min on ice in the dark. The RNAs were then dark-treated with aniline acetate solution (H2O: glacial acetic acid: aniline, 7:3:1) for 2 h at room temperature in the dark to induce the site-specific cleavage. After the cleavage, the RNAs were purified using the Oligo Clean & Concentrator™ kit (Zymo Research, USA). Finally, the RNA samples were applied for cDNA library construction using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs, USA) and used for high-throughput sequencing on Illumina Nextseq 500. The TRAC-seq data were analyzed as previously described[37]. Briefly, for tRNA m7G analysis, joint and low-quality sequence data were filtered with Trim-Galore. The filtered data were mapped to human mature tRNA sequences using Bowtie, and the read depth at each site and the number of reads starting from that position were calculated using Bedtools. The cleavage rate at the site was defined as the ratio between the number of reads starting and the read depth at the site. The cleavage score for the site was then calculated as:
Cleavage score (i) =\(\frac{l\text{o}\text{g} 2 \left(\text{C}\text{l}\text{e}\text{a}\text{v}\text{a}\text{g}\text{e} \text{r}\text{a}\text{t}\text{e}{ }_{\text{t}\text{r}\text{e}\text{a}\text{t}}\right)}{\text{l}\text{o}\text{g} 2 \left(\text{C}\text{l}\text{e}\text{a}\text{v}\text{a}\text{g}\text{e} \text{r}\text{a}\text{t}\text{i}\text{o}{ }_{\text{n}\text{o}\text{n}-\text{t}\text{r}\text{e}\text{a}\text{t}}\right)}\)
Sites with a cleavage score > 3 and a cleavage rate > 0.1 were considered as candidate m7G sites. To analyze tRNA expression, we extracted sequences containing tRNA genes and 100 bp upstream and downstream of tRNA genes as precursor tRNA genes. The predicted introns were deleted for the mature tRNA sequences, and ''CCA'' was added to the 3' end. During the mapping process with Bowtie2, tRNA reads were calculated and normalized for further analysis.
2.14 Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq)
RNC-seq was performed as previously described[38]. Briefly, cells were pretreated with 100 µg/ml cycloheximide and incubated for 15 min at 37°C. After washing twice with pre-cooled PBS, 1 ml of cell lysate was incubated with 1 ml of ribosomal buffer (RB buffer) containing 1% TritonX-100 [20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200 mM KCl, 100 µg/ml cycloheximide and 2 mM dithiothreitol] for 30 min on ice. The cell lysate was then centrifuged at 16,200g for 10 min at 4°C. 10% of the extract was used as input control. The remaining extract was layered into 11.5 ml sucrose buffer (30% sucrose in RB buffer), and the RNC pellet was collected by centrifugation at 32,000 rpm for 5 h at 4°C. Next, RNA was isolated from the input and RNC samples for sequencing. The isolated RNA was subjected to cDNA library construction and sequencing using the BGISEQ-500 platform (BGI-Shenzhen, China). Gene expression levels were normalized to FPKM. Translational efficiencies were calculated as: TE = (FPKM in polyribosome-seq) / (FPKM in input RNA-seq).
2.15 Gene ontology and pathway analysis
Gene ontology and pathway analysis of TE-down mRNAs identified in RNC-seq data were performed using ToppGene Webtool (https://toppgene.cchmc.org/enrichment.jsp). Benjamini-Hochberg adjusted P values < 0.05 for ontology terms, and pathways were classified as significantly enriched.
2.16 Statistics Analysis
Quantitative data are shown as mean ± SEM. P values in all cases are represented as: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. For statistical analysis, Student’s t-test, one-way ANOVA, or Mann-Whitney U test were used unless otherwise stated. Event-free survival was analyzed using the log-rank test. Statistical analyses were performed using GraphPad Prism version 8 or SPSS version 25.