Cell lines

293 T and HK-2 cells were obtained from Stem Cell Bank, Chinese Academy of Sciences and maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Coring, USA) supplemented with 10% fetal bovine serum (FBS, Coring, USA). G401 cells (provided by Professor Zhengyan Zhao, Children’s Hospital, Zhejiang University, Hangzhou, China) were cultured in McCoy’s 5 A Medium (Procell, China) supplemented with 10% FBS. All the medium contains 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell lines were cultured at 37 °C in 5% CO2 incubator.

Construction of plasmids

For expression of GSTM2, we amplified from cDNA using primers GSTM2 –R1-5.1 and GSTM2–BamH1-3.1 (Table 1) and then cloned into pCDH-MSCV-MCS-EF1α-GFP + Puro expression vector (SBI system Bioscience, USA) by 2X MultiF Seamless Assembly Mix Kit (Abclonal, China). All DNA fragments for cloning purpose were amplified by chain polymerase reaction (PCR) with the high-fidelity Phusion enzyme (New England Biolabs, USA). All constructive plasmids were verified by DNA sequencing.

Table 1 Primers used in this studyLentivirus package and infection

Lentivirus was packaged in 293 T cells as previously described. Briefly, the lentiviral vector containing inserts of interest and three packaging system plasmid (Rev,Gag and VSVG) were mixed and transfected into 293 T cells with EndoFectin-Lenti reagent (GeneCopoeia, USA), and then the virus containing culture medium was collected 48 h after transfection and spun for 10 min at 3000 r.p.m. Lentivirus infection was carried out in 6-well plates by mixing 500 μl virus supernatant and 500 μl regular culture medium containing 0.8 μl polybrene (10 mg/ml).

YTHDF1 KO by CRISPR/Cas9 approach

DF1 KO in G401 cells was performed as previously described (Peng et al. 2023). Briefly, the lentiviral vector carrying Cas9 and dual gRNAs were introduced into G401 cells by infection. At 3 days after infection, cells were subject to puromycin selection. Then 2 weeks later, individual colonies were manually collected and expanded in 24-well plates. KO clones were characterized by western blot.

Cell growth assay by CCK-8

CCK-8 assay was conducted to check the effect of DF1 KO or GSTM2 expression on cell growth. Briefly, G401 cells were seeded into 96-well plates at a density of 1 × 104 cells/well, and the measurement was performed at 24, 48, 72 h after seeding. The absorbance at 450 nm was determined using a microplate reader (TECAN, Spark, Switzerland).

Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated by Direct-zol RNA Miniprep Kits (Zymo, USA). Real-time PCR was performed using SYBR Green kit (Vazyme, Q221) and specific primers on a Bio-Rad CFX Opus96 platform (Bio-Rad, Hercules, USA). All primers used are listed in Table 1. Delta-delta Ct values were used to determine their relative expression.

Cell migration and invasion assay

Cell migration and invasion assays were determined using 24-well transwell inserts or Matrigel transwell inserts from BD Biosciences (San Jose, CA, USA), respectively as previously described with modification. Briefly, 3 × 104 cells in 100 μl serum-free medium were added to the upper compartment of the chamber, while the lower compartment was filled with 600 μL of regular culture medium, then the cells were incubated at 37 °C for 36 h. Cells remaining on inside membrane surface in the upper chambers were removed with a cotton swab, whereas the cells on the lower surface were fixed in methanol for 10 min at room temperature, washed with PBS and stained with 0.5% crystal violet and counted as described previously (Gupta et al. 2014). The invasion assay was done by the same procedure with transwell inserts pre-coated with matrigel (Corning® BioCoat™, CLS354480, USA), and 5 × 104 tumor cells were added to the upper chamber.

Colony formation assay

For the colony formation assay, cells were seeded in 6-well plates. After 14 days, the cells were fixed with 4% PFA (Solarbio, Bejing) and then stained with 0.5% crystal violet. The number of colonies was countered for five representative fields. The experiments were repeated for three times.

Western blot

Cells were harvested, and proteins were extracted and quantified as previously described with some modification (Sachdeva et al. 2011). Briefly, cells were harvested and the cell pellets were washed with ice-cold phosphate buffered saline (PBS). Next, the cells were lysed with RIPA buffer (Fdbio, China) and denatured with 5xSDS sample buffer or directly lysed by home-made 2xSDS sample buffer. Protein samples were separated using 10% or 12% SDS-PAGE before transferring to PVDF membrane. Membrane was then blocked by 5% BS in TBST at room temperature for 1 h and then incubated with primary antibodies overnight at 4 °C. After washed six times with TBST, the membrane was incubated with a secondary antibody labeled with either IRDye 800 CW or IRDye 680. Finally, signal intensity was determined using the Amersham Typhoon NIR System.

Quantitative proteomics analysis

The 4D-label-free-based quantitative proteomic analysis of G401 DF1 KO cells and vector controls cells was carried out by Jingjie PTM Biolabs Inc. (Hangzhou, China). Cell samples were sonicated, lysed, and centrifuged to obtain the supernatant and its protein concentration was measured. The tryptic peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile/in water), directly loaded onto a home-made reversed-phase analytical column (25-cm length, 75/100 μm i.d.). Peptides were separated with a gradient from 6 to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 450 nL/min on a nanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with a MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle. The dynamic exclusion was set to 30 s.

The MS/MS data were analyzed using the MaxQuant search engine (version 1.6.15.0). Tandem mass spectra were matched against the human SwissProt database (20,422 entries), combined with a reverse decoy database. Trypsin/P was selected as the cleavage enzyme, allowing for up to two missed cleavages. The precursor ion mass tolerance was set to 20 ppm for the initial search and 5 ppm for the main search, while the fragment ion mass tolerance was set to 0.02 Da. Carbamidomethylation of cysteine was specified as a fixed modification, and acetylation at the protein N-terminus and oxidation of methionine were set as variable modifications. The false discovery rate (FDR) for protein and peptide identification was set to 1%.Then, Pearson's correlation coefficient, principal component analysis (PCA), and relative standard deviation were used to evaluate the repeatability of samples from each group. Differential proteins were obtained after the qualification of samples, whose differences in relative quantification in two groups were compared by T-test, and the corresponding p-value was calculated. In addition, with a criterion of p-value ≤ 0.05, the protein ratio > 1.5 was regarded as up-regulation, while the protein ratio < 1/1.5 as down-regulation.

Assessment of cytosolic ROS

Cells were seeded at 3 × 105 cells per well in 6-well plates. Next day, cells were incubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA; 10 mM; Beyotime, S0033S) for 30 min at 37 °C in the dark. The fluorescence-labeled cells were visualized by an inverted fluorescence microscopy (Leica, DMi8, Germany), and number of fluorescent cells were analyzed by ImageJ software (11.27.42).

In silico analysis of the binding of YTHDF1 to GSTM2 m6 A site

The potential m6 A sites were predicted using the online tools SRAMP (http://www.cuilab.cn/sramp). CLIP-seq data of GSTM2 were extracted from the POSTAR3 database (http://postar.ncrnalab.org) (Zhao et al. 2022).

RNA immunoprecipitation (RIP) assay

G401 cells were grown in 10 cm dish to 90% confluency, and then washed twice with ice-cold PBS and scraped off in 1 mL PBS. Next the cells were centrifuged and re-suspended in 1 mL RIP lysis buffer (150 mM KCl, 25 mM Tris–HCl (pH 7.4), 5 mM EDTA, 0.5% NP-40, with 100 U/ml RNase inhibitor and protease inhibitor cocktail). After that, 5 μg DF1 antibody or negative control rabbit IgG (Boytime, China) was added to the cell lysates and incubated overnight at 4℃ with rotation. Next day, protein A/G PLUS-Agarose Beads (Santa Cruz, USA) were added to IP reaction and continued for another 2 h incubation. Beads were washed 4 times with NP-40 lysis buffer and then treated with proteinase K to remove proteins. Then interested RNAs were purified by TRIzol methods (ThermoFisher Scientific) and detected by qRT-PCR.

m6 A RNA immunoprecipitation (MeRIP) assay

The MeRIP assay was performed as previously described. Total RNA was isolated from a 10 cm dish G401 cells and dissolved in 50 µl RNase-free water, of which 5 μl was saved as RNA input and the remaining 45 µl RNA was added to 500 µl pulldown lysis buffer containing RNase inhibitor. The total RNA was mixed with mouse IgG or m6 A antibody. The tubes were then rotated overnight at 4 °C, followed by incubation with protein A/G beads under the same condition overnight. Finally, m6 A bound RNA was extracted with TRIzol, followed by RT-PCR as described above.

RNA stability assay

To measure the stability of GSTM2 mRNA, 2 μg/mL of actinomycin D (Selleck, USA) was used to treat G401 cells. The procedure of isolating total RNA for qPCR analysis was as described above. Finally, the mRNA level at the indicated time points was calculated and normalized using U6.

Statistical analysis

All data are expressed as the mean ± SD. Statistical analyses were performed with GraphPad Prism 10.1 software. The differences between two groups of data were analyzed by unpaired Student’s t test, and differences among three or more groups were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA followed by a post hoc Tukey test. A value of P < 0.05 was considered statistically significant.