HEK293T, human PC (SW1990, BxPC-3, AsPC-1, CFPAC-1 and PANC-1) and normal pancreatic ductal epithelial cells (HPDE6-C7) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The short tandem repeat profiling was used for cell authentication. HEK293T, SW1990, BxPC-3, CFPAC-1 PANC-1 and HPDE6-C7 cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA). AsPC-1 cells were grown in RPMI-1640 (Gibco, USA) supplemented with 10% FBS. All cells were maintained at 37 °C in an atmosphere of 5% CO2. In certain experiments, the administration of specific amino acids (2 μM phenylalanine, 4 μM leucine, 4 μM isoleucine, 1 μM methionine and 4 μM valine) was used to detect an effect on PC.
Alcohol-induced cell model
HPDEC6-7, BxPC-3 and SW1990 cells were cultured as described above. Ethanol (100%; Sigma, USA) was added to the medium to a concentration of 20 mg/100 ml. The cells were cultured continuously for three months, and the medium was replaced every 3 days.
The PC tissues and fragments of the normal pancreas were collected during surgery. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University. Informed consent was obtained from all patients.
The 2,000bp sequence upstream of the KIAA1429 TSS (-2000/0) was obtained from UCSC (http://genome.ucsc.edu/). The 2,000bp sequence and five deletion sequences (-1012/0, -512/0, -262/0, -137/0 and -94/0) were inserted into the PGL3-basic vector.
The full sequences of SLC43A2, YTHDF2, C/EBP β, GATA1, GR-α and TFII-I were cloned into the PCDNA3.1 vector.
The sequence adjacent to the m6A motif in the SLC43A2 3'-UTR was cloned into the pmirGLO vector (WT), and a mutant plasmid (MUT) wad produced by substituting A with T at the m6A site.
Gene knockdown or overexpression
The sequence for stable KIAA1429 knockdown was designed by Hanbio Tech (Shanghai, China) and inserted into the pHBLV-U6-MCS -PGK-PURO plasmid.
Stable KIAA1429 overexpression was produced by GeneChem Tech (Shanghai, China) using the Ubi-MCS-IRES-PURO plasmid.
siRNAs were used for transient knockdown of YTHDF2 or SLC43A2.
The detailed sequences are listed in Supplementary Table 3.
RNA preparation and qRT–PCR
An RNA extraction kit (Accurate Biology, China), a reverse transcription kit (Toyobo, Japan) and SYBR Green (Roche, Switzerland) were used for qRT–PCR. Gene expression was normalized to the level of GAPDH. Supplementary Table 2 contains a complete list of the primers.
The antibodies for western blotting were as follows: β-actin (Boster, China), GAPDH (Sangon Biotech, China), KIAA1429 (CST, USA), SLC43A2 (Sangon Biotech, China), C/EBP β (Santa Cruz, USA) and YTHDF2 (Proteintech, USA).
Cell proliferation and cell metastasis assays
CCK-8 (MedChemExpress) and EdU (Beyotime Biotechnology) experiments were performed to investigate cell growth as described elsewhere.
Transwell assays were used to evaluated the invasive and migratory capacities as described previously.
A total of 800 ng of RNA was loaded onto a Hybond-N+ membrane (Amersham, USA). After UV crosslinking, the membrane was blocked and immunoblotted with an anti-m6A antibody (CST, USA) and a secondary antibody. The signals on the membrane were visualized by enhanced chemiluminescence (Bio-Rad, USA). The samples were stained with methylene blue (MB), and this signal was used for normalization.
An mRNA purification kit (ThermoFisher, USA) was used to separate mRNA from the total RNA. The percentage of methylated mRNA was detected by an EpiQuik™ m6A RNA methylation quantification kit (Epigentek, USA).
RNA-seq, MeRIP-seq and MeRIP-qPCR
Total RNA extraction and mRNA purification were conducted as described above. A NEBNext® Ultra™ RNA library prep kit (NEB, USA) was used to generate the sequencing libraries. The 150 bp paired-end reads were obtained using the Illumina HiSeq 4000 platform.
Me-RIP was performed as described previously . Briefly, purified RNA was fragmented into ~100 nt oligonucleotides. A portion of the RNA served as the input, and the remaining RNA was immunoblotted with an anti- m6A antibody (Synaptic Systems). RNA captured by an anti-m6A antibody was collected on protein A beads (Thermo Fisher, USA). Finally, methylated RNA was used for standard RNA-seq or qRT–PCR. The SLC43A2 primers are listed in Supplementary Table 2.
The RIP experiment was carried out using a RIP kit (BersinBio, China). Briefly, the protein–RNA complexes were extracted using polysome lysis buffer. RNA bound to the target protein was immunoprecipitated with an anti-YTHDF2 antibody (Protein, USA) and pulled down by magnetic beads.
ChIP was performed using a chromatin IP kit (CST, USA). In brief, 1×107 cells were crosslinked with 1% formaldehyde; the reaction was stopped by glycine, and the adducts were collected. After nuclei isolation and chromatin fragmentation, 2% of the digested chromatin was used for the input, and remaining material was coincubated with an anti-C/EBP β antibody (Santa Cruz, USA) or IgG (CST, USA) for 4 h. Finally, the immunoprecipitated chromatin was collected using protein G magnetic beads, purified and quantified by PCR.
Luciferase reporter assay
A luciferase reporter assay was performed using a luciferase system (Promega, USA) following the manufacturer’s protocol.
The cells were incubated with actinomycin D (ActD, 5 μg/ml) for 15, 30, 60, 120 and 180 min. The expression of SLC43A2 mRNA was detected by qRT–PCR.
The cells were transfected with SLC43A2-containing or empty PCDNA 3.1 plasmids. Fresh medium was replaced 48 h after the transfection, and the cells were continuously cultured for 48 h. The medium was collected, and the concentration of phenylalanine was detected by a phenylalanine enzyme linked immunosorbent assay (ELISA) kit (Spbio, China).
In vivo experiments
Six-week-old female C57BL/6 mice were purchased from Charles River (Beijing, China). The KPC1199 cell-derived KPC pancreatic ductal adenocarcinoma mouse model was provided by Dr. Jing Xue from Renji Hospital (Shanghai, China). To generate an orthotopic model, 5×105 KPC1199 cells with stable KIAA1429 knockdown and luciferase expression or the corresponding controls were surgically implanted in the pancreas. To generate a liver metastasis model, 5×105 KPC1199 cells were surgically injected into the spleen. Splenectomy was performed after 5 min. Four weeks later, mice were anaesthetized, injected with luciferin and imaged using a Night-OWL II LB983 imaging system (Berthold Technologies, Germany).
The differences were evaluated using Student’s t test. The correlations between KIAA1429 mRNA and the clinical data were analysed by chi-squared test. Kaplan–Meier plots used for survival analysis were compared using log-rank test. A P value < 0.05 was considered significant.