Clinical data were obtained from 132 children (under 16 years old) with NBL diagnosed pathologically as neuroblastoma (with INSS stage4S cases removed) 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. This project was approved by the Medical Ethical committee for Clinical Research and Animal Trails of the First Affiliated Hospital of Sun Yat-sen University (Application ID:486). Baseline information of NBL sample were listed in Supplementary Table 1.
The NBL datasets (GSE62564, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62564) from GEO database were used for the univariate and multivariate cox hazard analysis of risk factors of NBL and the analysis of association between the expression of METTL1 and NBL patients’ prognosis. Baesline information of GSE62564 were listed in Supplementary Table 2.
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. This project was approved by the Medical Ethical committee for Clinical Research and Animal Trails of the First Affiliated Hospital of Sun Yat-sen University (Application ID:486).
Cell lines and cultures
Human embryonic kidney 293 T (HEK 293 T) 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 SK-N-BE (2) C (BE2C) cells were from Procell Life Science & Technology Ltd. (Wuhan, China). 293 T 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). 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.
Knockdown of METTL1 in NBL cells
Lentiviral vectors expressing pLKO.1 Short hairpin RNA (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 293 T cells using Lipofectamine 2000 (Invitrogen, USA) with a packaging plasmid pCMV-ΔR8.9 and an envelope plasmid pCMV-VSVG. 48 hours 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 RNA (siRNA) targeting the 3′ untranslated regions (3′ UTR) of METTL1 were used to knockdown METTL1 with Lipofectamine 2000 (Invitrogen, USA). siRNA sequences are listed in Supplementary Table 3.
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 hours 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% FBS. Migrated cells were stained with 0.5% crystal violet and counted after 24 hours.
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).
Subcutaneous implantation in a mouse model
Four to six-week-old BALB/c-nu female mice were randomly divided into two groups: shRNA targeting green fluorescent protein (shGFP) (N = 5) and shRNA targeting METTL1–1 (shM1–1) (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.
Immunohistochemical (IHC) staining
IHC was performed with an IHC kit (Agilent, USA) and anti-METTL1 antibody (Proteintech, 1:2000 dilution) and anti-Ki67 antibody (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 (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, 3 and 4 for < 5, 5 to 25%, 25 to 50%, 50 to 75%, and > 75%, respectively. Tissues with H-score≧6 (the median H-score) were considered as high METTL1 expression group, and tissues with H-score < 6 were classified as low METTL1 expression group. The antibodies used in this study are listed in Supplementary Table 4.
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 Real-time quantitative PCR assays (qPCR). 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 primer as an internal control. The primer sequences used in this study are listed in Supplementary Table 3.
Northern blot, northwestern blot, and Western blot
As previously reported, northern blot and western blot assays were performed [26, 34]. Briefly, for northern blot, 2 μg of total RNA was separated by electrophoresis on a 15% TBE-UREA gel containing tris base, boric acid, Ethylene Diamine Tetraacetic Acid (EDTA) and urea. Then the RNAs were transferred to a positively charged nylon membrane. Afterward, the nylon membranes were cross-linked with ultraviolet light. The indicated tRNAs and U6 small nuclear RNA (snRNA) were blotted with Digoxigenin-labeled probes. After transfer and cross-linking, nylon membranes were blotted with primary antibody, anti-m7G antibody (MBL International, 1:1000 dilution), for Northwestern blotting at 4 °C overnight. Anti-Digoxigenin-AP (Roche 1:15000 dilution), or anti-m7G antibody signals were detected according to the previously described Western blot protocol [26, 34]. Probe sequences are listed in Supplementary Table 3. The antibodies used in this study are listed in Supplementary Table 4.
Polysome profiling was performed as previously described . Briefly, NBL cells were incubated with 100 μg/ml cycloheximide for 15 minutes at 37 °C. After immediate cold PBS wash, the cells were lysed with multimeric cell extraction buffer containing 50 mM 3- (N-morpholino) propanesulfonic acid (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 13,000 g for 10 minutes at 4 °C. The optical density (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 hours at 4 °C. Separated samples were fractionated at 0.75 ml/minutes by a BR-188 density gradient fractionation system (Brandel, USA) and monitored at an absorbance of 254 nm.
Puromycin intake assay
NBL cells were transfected with siRNA targeting METTL1 (siM1) and siRNA targeting negative control oligos (siNC). After 48 hours, cells were incubated by using puromycin with final concentration of 1 μM for 30 minutes 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 (Millipore, 1:2000 dilution). The siRNA sequences are listed in Supplementary Table 3. The antibodies used in this study are listed in Supplementary Table 4.
tRNA m7G reduction and cleavage sequencing (TRAC-seq)
TRAC-seq was performed as previously described . 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 minutes 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 . 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. Chemical treatment (NaBH4/aniline-treated) in TRAC-Seq resulted in cleavage of tRNA at m7G site, which were detected by sequencing. Therefore, the principle of tRNA m7G site recognition is founded on cleavage sites. In order to obtain the global m7G cleavage sites in tRNAs, the cleavage scores were determined by comparing the ratio of reads starting at the specific site to reads passing through that site in treated and non-treated (control samples without NaBH4/aniline treatment) samples. Then, the cleavage ratio of site i is defined as the ratio between the number of reads starting and the read depth at the site i. And the cleavage score of the site was then calculated as:
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.
Ribosome nascent-chain complex-bound mRNA sequencing (RNC-seq)
RNC-seq was performed as previously described . Briefly, cells were pretreated with 100 μg/ml cycloheximide and incubated for 15 minutes 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 minutes on ice. The cell lysate was then centrifuged at 16,200 g for 10 minutes 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 (Fragments per kilobase per million). Translational efficiencies were calculated as: TE = (FPKM in polyribosome-seq) / (FPKM in input RNA-seq).
Gene ontology and pathway analysis
Gene ontology and pathway analysis of TE-down (Down-regulated Translational efficiencies) 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.
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 R studio, GraphPad Prism version 8 and SPSS version 25.