Human lung cancer specimens and cell lines
We obtained fresh and paraffin-embedded lung cancer specimens from 72 patients who underwent lung cancer surgery between January 2016 and September 2019 at the Cancer Hospital of Harbin University Medical College, China. Clinical characteristics of patients were retrospectively analyzed. Two pathologists performed blinded histological confirmation by hematoxylin and eosin (H&E) staining. The clinical pathology variables of lung cancer patients are summarized in Additional table S1. We collected all clinical samples with informed consent according to Health Insurance Portability and Accountability Act (HIPAA)-approved protocols. The use of human patient samples was approved by the Harbin University Medical College Ethics Committee.
MRC-9, H1299, H1650, H1703, H1795, H1792, A549, DLD-1, SW480, Panc-1, and MIA PaCa-2 cell lines were purchased from ATCC, USA. Cells were cultured in medium according to the manufacturer’s instructions and grown in a humidified incubator at 5% CO2. All cell lines were authenticated and confirmed negative for mycoplasma contamination by providers.
RNAi and protein overexpression transfection
To knockdown endogenous gene expression, we purchased si-RNAs targeting human LKB1 (sc-35816), ALKBH5 (sc-93856), and CTCF (sc-35124) from Santa Cruz and transiently transfected these siRNAs into lung cancer cells for 48 h using Lipofectamine RNAiMAX (Invitrogen). We ordered LKB1 (#8590) and ALKBH5 (#38073) overexpressing vectors from Addgene and transfected them into human cancer cells using Lipofectamine 2000 (Invitrogen) for 48 h. Si-RNA-A (sc-37007, Santa Cruz) and c-Flag pcDNA3 (Plasmid #20011, Addgene) were served as transfect controls for genes knockdown and overexpression, respectively.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
We extracted total cellular and tissue RNA using TRIzol Reagent (Thermo Fisher Scientific, USA) and used 1 µg total RNA for reverse transcription using the iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instructions. We performed qRT-PCR was performed with 2 × SYBR Green qPCR Master Mix (Bimake, USA) with a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad Inc. USA). We calculated the relative gene expression using the comparative CT method and β-actin RNA sequences as a control. Primer sequences are listed in Additional table S2.
Western blot analysis
We lysed total protein from treated cells using RIPA buffer (Cell Signaling Technology) supplemented with Protease Inhibitor Cocktail (ThermoFisher; 78430). We loaded 20 µg of protein and separated the sample using 12% SDS-PAGE. Western blotting was performed as previously described [23], and the primary antibodies included GAPDH (Santa Cruz, sc-137179), LKB1 (Santa Cruz, sc-32245), ALKBH5 (Proteintech, 16837-1-AP), CTCF (Santa Cruz, sc-271474), METTL3 (Abcam, ab195352), METTL14 (Abcam, ab220030), FTO (Abcam, ab126605), WTAP (Proteintech, 60188-1-Ig), SOX2 (Cell signaling, #3579), SMAD7 (Santa Cruz, sc-11392), and MYC (Cell signaling, #13987). We performed densitometric analyses of band intensity using ImageQuant TL 8.2 image analysis software (GE Healthcare Life Sciences, USA) and GAPDH was as an internal control.
Immunohistochemistry (IHC) staining
We performed IHC analysis of paraffin-embedded lung cancer tissues containing primary tumors and matched normal lung tissues as previously described [24, 25]. In brief, we de-paraffinized and rehydrated human tissue sections to retrieve antigens by microwaving the sections in 10 mM Sodium Citrate (pH 6.0), and then incubating in 1% hydrogen peroxide to suppress endogenous peroxidase activity. After blocking in 2.5% horse serum for 1 h, we applied primary antibodies to the slides at 1:500 (anti-LKB1, METTL3, METTL14, WTAP, and FTO antibodies), 1:200 (anti-ALKBH5 antibody), and 1:1000 (anti-m6A and 5-mC antibody) dilutions and incubated at 4°C overnight. We stained slides with EnVision + Dual Link System-HRP (Dako) for 1 h at room temperature, then washed, counter-stained with hematoxylin, dehydrated, treated with xylene, and mounted the samples. Images of stained cells in four random fields were captured by using an optical microscope (Olympus, Japan). Relative protein expression were evaluated by a Histoscore (H-score) system. This semiquantitative approach was calculated as the product of the percentage of positive cells by the staining intensity (graded as: 0, non-staining; 1, weak; 2, median; or 3, strong using adjacent normal mucosa as the median). Possible scores ranged from 0 to 300. The results were evaluated by two independent pathologists.
Immunofluorescence staining
We performed immunofluorescence staining on cultured A549 cells transfected with siRNA or pcDNA. After treatment, we fixed cells with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton/phosphate-buffered saline (PBS), blocked in 1% bovine serum albumin (BSA), and then incubated with the indicated primary antibody for LKB1 (Santa Cruz, sc-32245), ALKBH5 (Proteintech, 16837-1-AP), m6A (Synaptic Systems, 202111), or YAP (Cell signaling, 4912) overnight at 4°C. The next day, cells were washed and incubated with secondary antibody conjugated to Alexa Fluor 555 (donkey anti-rabbit, Invitrogen) for 1 h at room temperature. We used DAPI (Sigma) staining to label nuclei. Fluorescence was observed under a Leica SP8 confocal laser scanning microscope.
Luciferase reporter assays
For the ALKBH5 promoter reporter assay, we generated serial ALKBH5 promoter reporters by PCR amplification and inserted into pGl3-basic plasmid, as we previously reported [23]. We deleted the CTCF region using the Q5 Site-Directed Mutagenesis Kit (NEB) per the manufacturer’s protocol. Firefly luciferase activity was used to evaluate the effect of m6A modification on SOX2, SMAD7, and MYC activation. We used the pmirGLO Dual-Luciferase miRNA target expression vector from Promega to construct the reporter plasmid, which contained both a firefly luciferase and a Renilla luciferase. The wild-type SOX2, SMAD7, and MYC reporter plasmid was individually cloned by inserting the fragment containing the m6A peak region after the firefly luciferase coding sequence. We constructed mutant three reporter plasmids by replacing the adenosine bases within the m6A consensus sequences with cytosine. All constructs were confirmed by Sanger sequencing. Nucleotide sequences of primers are in Additional table S2. A549 cells grown in 96-well plates were transfected with reporter vectors and SV-40-Renilla-Luc in the presence of Lipofectamine 2000 Reagent (Invitrogen). After a 24-h transfection, we prepared cell extracts with passive lysis buffer. Luminescence was measured with the Dual-Luciferase Reporter Assay System (Promega), according to manufacturer’s instructions. The relative luciferase reporter activities were normalized to that of Renilla. We performed experiments for each vector as biological triplicates with six technical repeats.
Global 5-mC and m6A measurement
We measured total cellular and tissue 5-mC in DNA and m6A in mRNA levels by the EpiQuik m6A RNA Methylation Quantification ELISA kit (Colorimetric) and MethylFlash Methylated DNA 5-mC Quantification Kit (Colorimetric) (Epigentek Group Inc.), respectively. Total genomic DNA and RNA were isolated using the PureLink Genomic DNA Mini Kit and TRIzol Reagent (Thermo Fisher Scientific). We used 200 ng of DNA or RNA for additional global 5-mC and m6A measurement according to the manufacturer’s instructions. Measurements were performed in triplicate.
Bisulfite genome sequencing (BGS)
For DNA methylation analysis, we used BGS method, as previously described [26, 27]. Briefly, 500 ng of genomic DNA was bisulfite converted using the BisulFlash DNA Modification Kit (Epigentek) following the manufacturer's instructions. We amplified the fragment containing CTCF peak region of ALKBH5 promoter using primers listed in Additional table S2. PCR products were purified and cloned into a pCR4 TOPO vector using the TOPO TA Cloning Kit (Thermo Fisher Scientific, Rockford, IL, USA). We isolated and sequenced plasmid DNA from 10 randomly selected clones (Genewiz, Piscataway, NJ, USA).
Methylated DNA immunoprecipitation (MeDIP) analysis
To confirm BGS results, we performed MeDIP using the Methylamp Methylated DNA Capture Kit (EpiGentek) according to manufacturer's instructions. Briefly, we extracted cellular and tissue chromatin DNA and digested to approximately 150–700 bp using a micrococcal nuclease (CST). The fragmented DNA was immunoprecipitated with anti-5-mC (Abcam, ab10805) at room temperature for 2 h. After washing and purifying the DNA, we quantified the methylation status using qPCR. Primers are list in Additional table S2.
m6A-RNA Immunoprecipitation (m6A-RIP)
We extracted total RNA from treated A549 cells or tumor tissues, and incubated with DNase according to the TURBO DNA-free TM Kit (ThermoFisher) protocol to avoid DNA contamination. Then, we chemically fragmented 1 µg/µl RNA into ~ 100nt size and incubated with m6A antibody to immunoprecipitate according to the standard protocol for the EpiMark N6-Methyladenosine Enrichment Kit (NEB). Enrichment of m6A containing mRNA was then analyzed using qRT-PCR. Primers targeting m6A-enriched regions of SOX2, SMAD7, and MYC are listed in Additional table S2.
Cell proliferation and migration assay
For cell colony formation assay, A549 or H1792 cells were transfected with siRNA or pcDNA for 24-h and seeded into 6-well plates (500/well). After 1 week, we fixed formed colonies and stained with 0.1% crystal violet in 20% methanol, and counted colonies consisting of at least 50 cells. We calculated relative cloning numbers and created plots using GraphPad Prism 7.0 (GraphPad Software, Inc.). For the migration assay, we re-suspended 5 × 105 cells in Opti-MEM Reduced Serum Media (Invitrogen) and seeded into the upper chamber of a transwell apparatus (0.8 µm, BD Biosciences), and added complete medium to the bottom chamber to provide chemoattractants for migration. After 24 h, we gently wiped away cells remaining on the upper side of the membrane, and fixed cells that had migrated to the lower side of the membranes with methanol and stained with 1% crystal violet. To count migrated cells, we captured images of stained cells in five random fields by using an optical microscope (Olympus, Japan) and counted samples in triplicate.
Bioinformatics assay from database
The differentially expressed genes (DEGs) of m6A modulators (METTL3, METTL14, WTAP, FTO, and ALKBH5) and readers (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) between KL and K-lung cancer cell lines or tissues from the databases of the Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/), Mouse Tumor Biology (MTB, http://tumor.informatics.jax.org/mtbwi/index.do), and Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle/home). The RPKM values for each tissue and cell line were obtained from the CCLE repository. Then we compared the differentially expressed genes between K and KL-lung cancers in above three databases.
m6A-seq data analysis
Based on the NCBI-GEO database (GEO: GSE76367), we determined the number of m6A-seq fragments mapped to each gene using HT-SEq. We overlapped the core m6A peaks based on functional m6A signal enriched around the stop codon (-5kbp ~ + 5 k bp) of mRNAs and canonical m6A peaks [28, 29]. Then, core m6A peaks were subjected to functional enrichment analysis by Ingenuity Pathway Analysis (IPA) (http://www.qiagen. com/ingenuity) and KEGG pathway (https://www.genome.jp/kegg/).
Statistics
All experiments were repeated at least three times, unless otherwise stated in the figure legend. We performed statistical analyses using the SPSS v20.0 software (SPSS Inc., Chicago, IL), and used Student’s t-test (two-tailed) or one-way ANOVA analysis followed by Tukey’s, Sidak’s, or Bonferroni test to assess statistical significance between or among groups. The relationship between ALKBH5 mRNA expression and DNA methylation, as well as between ALKBH5 and LKB1 mRNA expression, were analyzed by Pearson correlation coefficient. We calculated the survival rate using the log-rank (Mantel–Cox) test. Normality was assumed and variance was compared between or among groups. All numerical data were presented as mean ± standard deviation (SD) and a p-value of < 0.05 was considered significant.