Clinical tissue specimens
During surgery, PDAC adenoma and normal tissues were received from Jiangsu Province People's Hospital, which is affiliated with Nanjing Medical University. The pathological data corroborated the diagnosis of PDAC and adenoma. Tissues were flash-frozen in liquid nitrogen and kept at -80°C before detection.
Cell culture and transfection
PDAC cell lines were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (100U/mL) in a 5 % CO2 incubator at 37°C. The Lip3000 reagent (Invitrogen, USA) was used to transfect the short hairpin RNA plasmid (Supplementary Table S1), overexpression vector, and negative control.
Establishment of resistant cell lines
CFPAC-1 and Colo357 cells were cultured in medium supplemented with increasingly higher concentrations of gemcitabine (Sigma-Aldrich, USA) for more than a month until the cells could proliferate in the presence of 2000 nM gemcitabine, at which point they were dubbed CFPAC-GM and Colo357-GM, respectively.
Quantitative real‑time polymerase chain reaction
NucleoZOL reagent was utilized to extract total RNA (Macherey-nagel, Germany). HiScript III RT SuperMix was used to create cDNA for qPCR (Vazyme, China). The ChamQ SYBR qPCR Master Mix (High ROX Premixed) was used to conduct the amplification reaction (Vazyme, China). GAPDH or 18s were used to normalize gene expression. In the Supplementary Table S2, the primers are listed.
Western blotting
Protein concentration was evaluated after collecting and lysing cells. The protein was separated and transferred to a PVDF membrane before being incubated for 15 minutes at room temperature with QuickBlockTM Blocking Buffer for Western Blot (Beyotime, China). The membrane was treated with primary antibodies overnight at 4°C. The membranes were then treated for 1 hour at room temperature with secondary antibody (1:10 000, Jackson ImmunoResearch). A chemiluminescence gel imaging device was used to observe protein expression (Thermo Fisher FL1500, USA).
Immunoprecipitation (IP) and IP with mass spectrometry (IP/MS)
Total proteins were extracted from cells and treated overnight at 4°C with suitable primary antibodies. The next day, Dynabeads protein G was added and incubated at room temperature for 1 hour. Mass spectrometry was used to analyze the extracted immunoprecipitates.
Measurement of m6 A modification
The EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) was used to measure m6A levels in whole RNA (P-9005-48). In brief, 200ng of RNA was incubated with capture antibody for 60 minutes before being exposed to detection antibody and enhancer solution. Finally, the samples were incubated for 10 minutes in developer solution. At a wavelength of 450nm, absorbance was measured.
Immunohistochemistry (IHC)
IHC was used to examine protein expression in paraffin-embedded tissues. Tissue slides were treated with xylene and 100 percent ethanol, followed by decreased ethanol concentrations. After antigen retrieval, slides were blocked and stained with primary antibody, followed by secondary antibody incubation using the standard avidin biotinylated peroxidase complex process. Hematoxylin was used as a counterstain, and images were captured using an upright microscope equipment (Nikon, JAPAN).
Cell proliferation assays
PDAC cells were plated in 96-well plates at a density of 6000 cells per well for the Cell Counting Kit-8 (CCK8) test. Cell proliferation was measured at 0, 24, 48, 72, and 96 hours after cell adhesion; CCK-8 (Vazyme) was added, and the plate was incubated at 37 °C and 5 percent CO2 for 1 hour. A microplate reader was used to measure absorbance at 450 nm (TECAN). 1,000 cells were planted and cultured in 6-cm2 cell plates for two weeks for the clonogenic test. The cells were stained with 0.1 percent crystal violet after being fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA). Using a light microscope, colonies were counted until they became visible.
Flow cytometry analysis
The cells were stained with propidium iodide (PI) for cell cycle analysis using a cell cycle staining kit (Beyotime Biotechnology, China) and counted using a FACSCalibur flow cytometer according to the manufacturer's procedure.
RNA stability assay
CFPAC-GM and Colo357-GM cells were planted in 6-well plates for 24 hours before being treated with 5g/mL actinomycin D (MCE, USA) at the 0, 2, 4, and 6 hour intervals. The total RNA was then extracted using NucleoZOL and analyzed using qPCR. The mRNA expression for each group was quantified and normalized using GAPDH at each time point.
Methylated RNA immunoprecipitation sequencing (MeRIP-seq)
MeRIP-seq was conducted in accordance with a previously reported protocol with minor modifications [22]. Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. The cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.
MeRIP-qPCR
Individual gene M6A alterations were identified using the MeRIP-qPCR assay. To summarize, poly(A) RNA was isolated from 50 g of total RNA using the DynabeadsTM mRNA Purification Kit (61,006, Invitrogen), and one-tenth of the RNA was preserved as the input control. PierceTM Protein A/G Magnetic Beads (88,803, Thermo Scientific) were prewashed and incubated for 2 hours at 4 °C with rotation with 5 g of anti-m6A antibody (202,003, Synaptic Systems) or rabbit IgG. Following three washes, the antibody attached beads were combined with pure poly(A) RNA and one immunoprecipitation buffer containing RNase inhibitors. After proteinase K digestion, the methylated mRNAs were precipitated using 5 mg of glycogen and one-tenth volume of 3 M sodium acetate in a 2.5 volume of 100 percent ethanol overnight at 80 °C. qPCR was used to calculate further enrichment, and the corresponding m6A enrichment in each sample was calculated by normalizing to the input.
RNA pull-down assays
The MEGAscript T7 Transcription Kit was used to first transcribe the RNA (AM1334, Thermo Scientific). The amplified RNA was then desthiobiotin-labeled at the ends using the Pierce RNA 3′ End Desthiobiotinylation Kit (20, 163, Thermo Scientific). Finally, the Pierce Magnetic RNA-Protein Pull-Down Kit was used to perform RNA pull-down experiments (20,164, Thermo Scientific). A mixture of up to 50 pmol of biotinylated RNAs, 2 mg of protein lysates, and 50 l of streptavidin beads was used. The streptavidin beads were boiled after incubation and three washing before being utilized in the immunoblotting experiment.
Luciferase reporter assay
Genechem created luciferase reporters comprising the wild type (WT) and mutated-type (Mut) sequences of the NEDD4 exonic region (Chr15: 55924457-55951517). (Shanghai, China). Tsingke created luciferase reporters containing the WT and Mut sequences of the FTO/YTHDF2 promotor (Nanjing, China). In a 24-well plate, cells were seeded and transfected with the WT/Mut reporters. The Luciferase test was carried out according to the manufacturer's instructions using the Luciferase Kit (Promega). BERTHOL chemiluminescence measuring device was used to assess luciferase activity (Centro XS LB 960).
Animal studies
The animal research were carried out in compliance with the animal management committee of Nanjing Medical University's institutional ethical requirements for animal experimentation. Subcutaneously, 5× 106 cells were injected into the axilla of female athymic BALB/C nude mice (4 weeks old, 18-20 g). Every week, the tumor's width (W) and length (L) were measured, and the volume (V) was determined using the formula V = (W2 L)/2.
Statistical analysis
All data and error bars are shown as means standard deviations from at least three separate studies. A two-tailed Student's t-test was used to assess all differences between two independent groups. The log-rank test was used to compare survival curves created using the Kaplan-Meier technique. Univariate and multivariate Cox regression models were used to examine survival data. The Pearson's Chi-square test was used to evaluate the relationship between two independent groups. The ROC curve was created using SPSS software, and the data was evaluated using the two-tailed t test. The P values shown (*P < 0.05 , **P < 0.01, ***P < 0.001 and ****P < 0.0001) were judged statistically significant.