Immunohistochemistry
Resected NSCLC samples from Hamamatsu University School of Medicine and Seirei Mikatahara General Hospital were collected and named as the HUSM cohort. Tissue microarray (TMA) sections were analyzed using immunohistochemistry (IHC) as previously described [39]. Cores of insufficient quality or quantity were excluded from analysis. Antibodies for ALKBH5 (HPA007196, Atlas Antibodies, Stockholm, Sweden) and FTO (Ab124892, Abcam, Cambridge, UK) were diluted at 1:400, whereas those specific for EGFR E746-A750 deletion (#2085, D6B6, Cell Signaling Technology [CST], Danvers, MA, USA) and EGFR L858R mutant (#3197, 43B2, CST) were diluted at 1:100, followed by incubation at room temperature for 0.5 h. Protein expression levels were then assessed using the H-score, which was calculated by multiplying the percentage of stained tumor area (0–100%) by the staining intensity (scored on a scale of 0–3) to yield a value ranging from 0 to 300.
Analysis Of Publicly Available Datasets
We used the lung cancer database in the Kaplan–Meier plotter (http://kmplot.com/analysis/index.php? p = service&cancer = lung) to analyze the association between prognosis and ALKBH5 and FTO mRNA expression in NSCLC cohorts. Data were downloaded on December 10, 2020. Kaplan–Meier curves for overall survival (OS) were generated and stratified according to the median expression of each mRNA. To assess the mRNA expression of ALKBH5 and FTO, data from the Cancer Genome Atlas (TCGA) (NSCLC, Provisional) were downloaded from cBioPortal (http://www.cbioportal.org/) on November 11, 2019. Expression data were obtained in the form of RNA-seq by Expectation Maximization (RSEM).
Immunofluorescence Analysis
Cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with 5% bovine serum albumin in PBS (−) at room temperature for 1 h, the cells were probed with primary antibodies against ALKBH5 (HPA007196, Atlas Antibodies) and then incubated with a Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (#A-11010, Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with ProLong® Gold Antifade Reagent with DAPI (#8961, CST), after which the cells were imaged via fluorescence microscopy using z-stack image reconstructions (BZ-9000; Keyence, Osaka, Japan).
Cell Lines And Transient Knockdown With Sirna
The human lung cancer cell lines H1299, H460, H2087, A549, ABC1, and H358 and the human immortalized cell lines BEAS2B and HEK293 were obtained from Health Science Research Resources Bank (Osaka, Japan) or the American Type Culture Collection (Manassas, VA, USA). PC3 and PC9 lung cancer cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and RIKEN BioResource Center (Tsukuba, Japan), respectively, whereas ACC-LC176 cells were a kind gift from Dr. Takashi Takahashi (Nagoya University). RERF-LC–MS, HLC-1, and LC-2/ad were kind gifts from Dr. Toshiro Niki (Tokyo University). Lung cancer cell lines were cultured in RPMI1640 medium (R8758, Thermo Fisher Scientific), whereas HEK293 cells were cultured in DMEM (D5796 MERCK, Darmstadt, Germany) containing 10% (vol./vol.) fetal bovine serum (FBS), 100 IU/mL penicillin G, and 100 µg/mL streptomycin. LHC9 (12680013, Thermo Fisher Scientific) was also used as a medium for BEAS2B cells. Cells were maintained in a 5% CO2 and 95% air incubator at 37℃. Silencer Select Pre-designed siRNA for ALKBH5 (siALKBH5: s29743, s29744, s29745, Invitrogen), FTO (siFTO: s28147, s28148, s28149, Invitrogen), IGF2BP1(siIGF2BP1: s20916, s20917, Invitrogen, Carlsbad, CA, USA), IGF2BP2 (siIGF2BP2: s20922, s20923, Invitrogen), IGF2BP3(siIGF2BP3: s20919, s20920, Invitrogen), and Silencer Select Negative control (siNC: 4390843, Invitrogen) were purchased for transient knockdown. More than two different sequences were used for one target gene to minimize off-target effects. Cells were cultured for 24 h before transfection, after which they were transfected with 15 nM of final siRNA concentrations using Opti-MEM (31985070, Gibco, Dublin, Ireland) and Lipofectamine® 2000 (11668019, ThermoFisher). The cells were then used for further assays at 48–96 h after transfection. When no siRNA sample number was available, siRNA no. 1 (#1) and siRNA no. 3 (#3) were pooled for ALKBH5 unless otherwise specified. siIGF2BP1, siIGF2BP2, and siIGF2BP3 were pooled for all transfections.
Generation Of Retro-x Tet-on Inducible Cell Lines Overexpressing Alkbh5
The retroviral plasmid pRetroX-TetOne puro (634307, Clontech, Mountain View, CA, USA) was amplified using NEB Stable competent Escherichia coli (high efficiency) (C3040H, NEW ENGLAND BioLabs, Ipswich, MA, USA). The full-length ALKBH5 sequence (NM_017758), which was confirmed using Sanger sequencing, was subcloned into pRetroX-TetOne puro vector using EcoRI and BglII restriction sites (pRetroX-TetOne puro-ALKBH5). Retroviral supernatants were produced using the GP2-293 packaging cell line (Clontech), in which pRetroX-TetOne puro empty vector or pRetroX-TetOne puro-ALKBH5 were each cotransfected with the envelope vector VSV-G using Xfect transfection reagent (Clontech). BEAS2B, HEK293, PC9, and A549 cells were transfected for 24 h using 4 µg/mL polybrene (H9268, Sigma-Aldrich, St. Louis, MO, USA). Puromycin selection (0.5–1.5 µg/mL) began 48 h after transfection and lasted for 3 days until all non-infected cells had died. In subsequent experiments, Retro-X cells were induced with 0.1–100 ng/mL of doxycycline (DOX) diluted in culture media upon cell seeding for 24–96 h. Cells transfected with pRetroX-TetOne puro-ALKBH5 with 100 ng/mL DOX were designated as ALKBH5-overexpressed (OE) cells, whereas those infected without DOX were designated as negative control (NC) unless otherwise noted.
Rna Isolation And Quantitative-polymerase Chain Reaction (Qpcr)
Total RNA was extracted using the RNeasy Plus Mini Kit (#74136, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions, with the total RNA concentration calculated using Nanodrop (NanoDrop1000, Thermo Fisher Scientific). cDNA was synthesized from 1 µg of total RNA using the ReverTra Ace qPCR RT Master Mix (FSQ-201, TOYOBO) according to the manufacturer’s instructions. qPCR reactions were performed on a Step One Plus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) using the THUNDERBIRD qPCR Mix (QPS-201, TOYOBO, Osaka, Japan). The relative RNA expression levels were calculated using the ΔΔCt method, with the levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. All amplicons were confirmed as a single product using agarose gel visualization and/or melting curve analysis. The applied primer sequences are listed in Additional file 7: Table S1.
Protein Isolation And Western Blotting
Protein isolation and western blotting
Total protein lysates were extracted from whole cells using 1× sodium dodecyl sulfate (SDS) sample buffer. The Pierce BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scientific) was used to determine the protein concentration. All proteins were separated using SDS-polyacrylamide gel electrophoresis and transferred to PVDF Blotting Membrane (P 0.45, A29532146, GE healthcare Life science, Chicago, IL, USA) using the Trans-Blot Turbo Cassette (Bio-Rad, Hercules, CA, USA). Blocking One (03953, Nacalai, Kyoto, Japan) or 5% skimmed milk were used for blocking. Primary antibodies for ALKBH5 (1:1000 dilution, HPA007196; Atlas Antibodies), FTO (1:1000 dilution, Ab124892; Abcam), IGF2BP1 (1:1000 dilution, 22803-1-AP; Proteintech), IGF2BP2 (1:1000 dilution, 11601-1-AP; Proteintech), IGF2BP3 (1:2000 dilution, 14642-1-AP; Proteintech), TIMP3 (1:3000 dilution, Ab39184; Abcam), p21 (1:1000 dilution, A1483; ABclonal, Woburn, MA, USA), E2F1 (1:500 dilution, A2067; ABclonal), CCNG2 (0.2µg/mL, Ab251826; Abcam), p53 (1:200 dilution, Sc-126; SANTA CRUZ BIO TECHNOLOGY, Dallas, TX, USA), and GAPDH (1:1000 dilution, Ab8245; Abcam) were incubated for overnight at 4℃. Secondary antibodies for rabbit (1:20000 dilution, NA9340; GE healthcare Life science) or mouse (1:20000 dilution, NA9310; GE Healthcare Life Science) were incubated at room temperature with 1–5% skimmed milk for 1 h. Enhanced chemiluminescence (Pierce ECL Plus Substrate or West Atto Ultimate Sensitivity Substrate, Thermo Fisher Scientific) was used to visualize the protein bands using ChemiDocTouch (Bio-Rad).
Cell Viability Assay
Cells were seeded into 96-well plates with 3000 cells per well after 48 h of knockdown or overexpression. Cell proliferation was monitored using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Thereafter, the cells were incubated with 10% CCK-8 for 1 h, followed by absorbance assessment at 450 nm in each well via spectrophotometry (Synergy HT, BioTek, Winooski,VT, USA) every 24 h.
Transwell Migration Assay
Cell migration was evaluated using a 24-well plate with cell culture inserts (353097, Falcon, Mexico City, Mexico) containing a filter with 8 µm-diameter pores. Briefly, after serum starvation for 24 h with 0.1% FBS-containing RPMI1640 medium, 1 × 105 cells resuspended in 500 µL of RPMI1640 medium (Gibco) were seeded into the upper chamber, after which RPMI1640 medium containing 10% FBS was placed in the lower compartment of the chamber. After incubation for 16 h, the upper surface of the membrane was wiped with a cotton-tipped applicator to remove non-migrating cells, whereas the migrating cells on the lower surface were fixed with cold methanol and stained with 0.5% crystal violet. Migrating cells were automatically counted in three random microscopic fields using the Hybrid Cell Count software (BZ-Ⅱ Analyzer, Keyence, Osaka, Japan).
Wound-healing Assays
To assess cell migration, 2 × 105 cells were seeded into 6-well plates. Thereafter, cells were incubated in 5% CO2 at 37℃ for 48 h and an additional 24 h with 0.1% FBS-containing RPMI1640 medium. A wound was scratched into the cells using a 200-µL plastic tip and washed with PBS (−). The cells were then incubated in RPMI1640 containing 10% FBS. The relative distance of the scratches was observed under an optical microscope (IX53, Olympus, Tokyo, Japan) at 3–6 time points after wounding and assessed using the Image J software.
Cell Cycle Assay And Apoptosis Assay
Cell Cycle Assay Solution Blue (C549, Dojindo) was used to measure the cell cycle according to the manufacturer’s instructions. Briefly, treated cells were synchronized at the G1 phase through serum starvation with 0.1% FBS-containing medium for 48 h. At 24 h after the release of serum starvation, the treated cells were collected, washed with PBS (−), and incubated with 5 µL cell cycle assay solution for 15 min at 37℃. Thereafter, DNA content was determined based on staining intensity using a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA). The Annexin V-FITC Apoptosis Detection Kit (15342-54 Nacalai) was used to detect apoptosis by measuring annexin V and propidium iodide (PI)-positive cells following the manufacturer’s instructions. Briefly, cells were incubated for 96 h after siRNA transfection. To induce apoptosis, the cells were exposed to either 7.5 µM of gefitinib (078-06561, FUJIFILM) or 10 µM of cisplatin (P4394, Sigma-Aldrich) alone for 48 h after siRNA transfection. The treated cells were collected, washed with PBS (−), and incubated with 5 µL of annexin V-FITC solution and 5 µL of PI solution for 15 min. Thereafter, apoptotic cells were determined using a Gallios flow cytometer. Results were analyzed using the FlowJo software (Becton, Dickinson, Franklin Lakes, NJ, USA), after which the extent of apoptosis and cell cycle distribution were determined.
Rna Stability Assay
Cancer cells were incubated for 48 h after siRNA transfection. Cells were treated with actinomycin D at a final concentration of 5 µg/mL. Total RNA was extracted at 0, 2, 4, and 6 h after adding actinomycin D. The remaining CDKN1A and TIMP3 mRNA was measured through quantitative real-time PCR and normalized to RPL32 mRNA, which has a half-life of 25 h.
Quantitative analysis of global m 6 A levels using liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS)
PolyA-enriched RNA was extracted using PolyATract mRNA isolation systems (#Z5310 Promega, Madison, WI, USA) according to the manufacturer’s instructions. PolyA-enriched RNA concentration was calculated using Qubit 2.0. The polyA-enriched RNA was enzymatically hydrolyzed using 8-OHdG Assay Preparation Reagent Set (292-67801, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan). Technically, 100 ng of polyA-enriched RNA was digested using 5.7 µL of acetic acid buffer and 3 µL of Nuclease P1 included in the 45-µL sample containing nuclease-free water at 37℃ for 30 min, followed by incubation with 6 µL of Tris Buffer and 0.3 µL of alkaline phosphatase at 37℃ for 30 min. After digestion, the sample was centrifuged at 14,000 g and 4℃ for 20 min using a Nanosep 3K Omega centrifugal device (Pall Corporation, Port Washington, NY, USA).
As an internal standard, N6-methyladenosine-d3 (m6A-d3; M275897, Toronto Research Chemicals, Toronto, Canada), which is a stable isotope of N6-methyladenosine labeled with three deuterium atoms on the N6-methyl group, was added to the nucleosides obtained via digestion of polyA-enriched RNA. These nucleosides were separated using an Acquity UPLC HSS T3 column (2.1 mm × 100 mm; Waters, Milford, CT, USA) with 0.1% (vol./vol.) formic acid in water as mobile phase A and methanol as mobile phase B at a flow rate of 200 µL/min in a linear gradient elution of 5–60% B from 0 to 7 min. Standard compounds of adenosine (A; A9251, Sigma-Aldrich), N6-methyladenosine (m6A; A170736, Sigma-Aldrich), and m6A-d3 were used to confirm the nucleoside-to-base ion mass transitions of 268.1–136.4 (A), 282.2–150.2 (m6A), and 285.2–153.2 (m6A-d3). Peak areas of A, m6A, and m6A-d3 in the nucleosides digested from polyA-enriched RNA were calculated using the column retention time of the standard compounds using Analyst 1.6.1 software (AB SCIEX, Foster City, CA, USA). The m6A level was quantified as the ratio of m6A to A or m6A-d3 based on the calibrated concentrations.
Microarray Analysis Of Differentially Expressed Genes
Total RNA was extracted from ALKBH5-knockdown or control PC9 cells 96 h after transfection. RNA samples were used for global gene expression profiling on human Clariom S Assay microarrays (Thermo Fisher Scientific, Wilmington, DE, USA), which include 24351 genes. All microarray analyses were entrusted to Filgen Inc. (Aichi, Japan). A total RNA quality control check was performed using a NanoDrop ND-1000 (Thermo Scientific) and an Agilent 2100 Bioanalyzer. Using the Gene Chip TM WT PLUS Reagent Kit, fragmented and labeled cDNA samples were prepared from 250 ng of total RNA according to the manufacturer’s instructions (Gene Chip TM WT PLUS Reagent Kit User Manual). Thereafter, 100 µL of hybridization solution was prepared using 73 µL of Hybridization Master Mix and 2.3 µg of fragmented and labeled cDNA. The array was incubated using the Gene Chip TM Hybridization Oven 645 at 45℃ for 16 h (60 rpm). The array was cleaned using the Gene Chip TM Fluidics Station 450 and scanned using the Gene Chip TM Scanner 3000 7G according to the manufacturer’s instructions [Gene Chip TM Command Console (AGCC) 4.0 User Manual]. The Microarray Data Analysis Tool version 3.2 (Filgen, Aichi, Japan) was used for data normalization and subsequent processing. Differentially expressed mRNAs were identified using a set cutoff (fold change > 1.5 or < 0.67; P < 0.01). Gene set enrichment analysis (GSEA) was performed to examine the gene set regulated by ALKBH5 knockdown (http:/software.broadinstitute.org/gsea/omdex.jsp) [40]. For analysis, the false discovery rate (FDR) based on gene set permutation was used. Microarray data has been deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) (accession number GSE165453).
Epitranscriptomic Microarray Analysis
Unfragmented total RNA was extracted from ALKBH5-knockdown or control PC9 cells at 96 h after transfection and quantified using the NanoDrop ND-1000. RNA samples were used for global m6A expression profiling on an Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8 × 60 K; Arraystar), which includes 44,122 protein-coding mRNAs and 12,496 long non-coding RNAs. Microarray analyses were entrusted to Arraystar Inc. (Rockville, MD, USA). Sample preparation and microarray hybridization were performed based on Arraystar’s standard protocols. Briefly, total RNAs were immunoprecipitated with an anti-m6A antibody (Synaptic Systems, 202003). The “immunoprecipitated (IP)” and “supernatant (Sup)” RNAs were labeled with Cy5 and Cy3, respectively, as cRNAs in separate reactions using the Arraystar Super RNA Labeling Kit. The cRNAs were combined and hybridized onto Arraystar Human mRNA&lncRNA Epitranscriptomic Microarray (8 × 60 K, Arraystar). After washing the slides, the arrays were scanned in two-color channels using an Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Raw intensities of IP (Cy5-labeled) and Sup (Cy3-labeled) were normalized with an average of log2-scaled Spike-in RNA intensities. The “m6A methylation level” was calculated to determine the percentage of modification based on the IP (Cy5-labeled) and Sup (Cy3-labeled) normalized intensities. “m6A quantity” was calculated to determine the amount of m6A methylation based on the IP (Cy5-labeled) normalized intensities. Differentially m6A-methylated RNAs between both comparison groups were identified by filtering with a fold change of > 1.5 or < 0.67 (P < 0.01) through the unpaired t-test. Microarray data had been deposited in the GEO at the NCBI (accession number GSE165454).
Qpcr For Methylated Rna Immunoprecipitation (Merip) With Ma Antibody
ALKBH5-knockdown or control lung cancer cells were used for methylated RNA immunoprecipitation assay. The Magna MeRIP m6A kit (catalog no.17-10499, Millipore, Burlington, MA, USA) was used according to the manufacturer’s protocol. Briefly, the polyA-enriched RNA was fragmented into 100–200 nucleotides incubated with RNA fragmentation buffer for 55 s (CS220011, Millipore). The size of polyA-enriched RNA fragments was optimized using the Agilent 4200 TapeStation (Agilent technologies, Santa Clara, CA, USA). We used 0.5 µg of fragmented polyA-enriched RNA as input control and 5 µg of fragmented polyA-enriched RNA for m6A mRNA immunoprecipitation, followed by incubation with m6A antibody (MABE1006, Millipore)- or mouse IgG-conjugated Protein A/G Magnetic Beads in 500 µL 1× IP buffer supplemented with RNase inhibitors at 4℃ overnight. Methylated RNAs were immunoprecipitated with beads, eluted via competition with free m6A, and purified using the RNeasy kit (Qiagen). Moreover, modification of m6A toward particular genes was determined using qPCR analysis with specific primers [primers for the positive control region (stop codon, EEF1A1+) or NC region (exon 5, EEF1A1−) of human EEF1A1 was included in the Magna MeRIP m6A kit]. To design primers for MeRIP qPCR, m6A sites of specific genes were predicted using the sequence-based RNA adenosine methylation site predictor algorithm (http://www.cuilab.cn/sramp). We focused on the potential m6A sites in the 3′ UTRs near the stop codon and designed primers to ensure that the target sequence were present in these sites with a limited length of 120 nt. Self-designed primers for MeRIP qPCR are listed in Additional file 7: Table S1.
Statistical analysis
Discrete variables were expressed as numbers (percentages), whereas continuous variables were expressed as means ± standard deviations (SDs) unless otherwise specified. The Mann–Whitney U test was used to compare continuous individual samples, whereas Student’s t-test was applied to compare continuous experimental data. Fisher’s exact test for independence was used to compare categorical data between groups. The Wilcoxon matched-pairs signed-rank test was used to compare two corresponding groups. Spearman’s correlation coefficient was used for correlation analysis. Kaplan–Meier curves with log-rank tests were used to analyze survival. Accordingly, OS was defined as the duration from baseline to the date of death, whereas recurrence-free survival (RFS) was defined as the duration from baseline to the recurrence date. Univariate and multivariate Cox proportional hazards models were applied to generate hazard ratios (HRs) for death while adjusting for other potential confounding factors. Cell proliferation and RNA stability assays were analyzed using two-way analysis of variance. Statistical analyses were performed using GraphPad Prism Version 8 (GraphPad Software, San Diego, CA, USA) and EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), with P values of < 0.05 indicating statistical significance.