Processing of TCGA and GEPIA2 data
We characterized the expression profile of CCA RNA-seq datasets downloaded from TCGA-Cholangio carcinoma (CHOL) dataset and then the differential expression of eight m6A methylation-related genes (FTO, HNRNPA2B1, HNRNPC, METTL3, WTAP, YTHDC1, YTHDC2, and YTHDC2) between CCA and normal control samples were evaluated using R package and plotted into heatmap using R package.
The GEPIA2 database contained of 36 tumor tissues of CHOL and 9 adjacent tissues samples. We analyzed the differential expression of METTL3 and AKR1B10 between the CCA and adjacent tissues.
Tissue microarray immunohistochemistry (IHC)
A CCA tissue microarray (No. LVC1202) was generated from 60 cancer tissues and paired pericarcinomas that purchased from Boster Biological Technology co.ltd. IHC staining for METTL3 and AKR1B10 were performed using the above microarray tissue blocks of CCA. Briefly, paraffin-embedded tissues were made into 6 μm sections following deparaffinization and hydration. Sections were repaired by high-pressure following incubated with 0.33% H2O2 in methanol to block endogenous peroxidases and incubated with 10% normal horse serum in TTBS to block non-specific binding. Next, sections were incubated with anti-METTL3 (1:100, Proteintech, 15073-I-AP) or anti-AKR1B10 (1:500, Abcam, ab192865) overnight at 4 °C and then incubated with horse anti-mouse biotinylated antibody. Finally, sections were with colored by chromogen of DAB and counterstained with hematoxylin. Pictures were captured and exported using NDP.view 2.0. IHC staining results of METTL3 were assigned 1–3 scores and AKR1B10 were assigned 0–3 scores based on staining intensity of positive cells and percentage of positive cells. The section with strong staining intensity and diffuse of positive cells was assigned 3 score; strong staining intensity and focal distribution of positive cells was assigned 2 score; weak- medium staining intensity of positive cells was assigned 1 score; no staining or non-specific staining was assigned 0 score. METTL3 scores of 1 and 2 were categorized as low expression group and 3 as high expression group. AKR1B10 scores of 0 and 1 were categorized as low expression group, scores of 2 and 3 as high expression group.
Cell culture, lentivirus construction and transfection
Human liver bile duct carcinoma cell RBE and HCC9810 were purchased from Procell (China) and were maintained in RPMI 1640 with L-Glutamine (CORNING, China) containing 10% FBS (GIBCO, China) and 1% penicillin/ streptomycin (GIBCO, China) at 37 °C and 5% CO2.
To construct METTL3 stably overexpressed stable RBE cell line, the full-length of METTL3 was inserted into the lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-3 × FLAG-WRPE (OE-METTL3 group) and then harvested-lentiviruses were infected with RBE cells using polybrene (hexadimethrine bromide, Sigma 107689-100MG). Blank lentiviral vector was served as negative control (Vector group).
To transient knockdown expression of METTL3 in HCC981 cells and AKR1B10 in RBE cells, we used small interfering RNA (siRNA) method. The synthesized sequence of siRNA targeted METTL3 (siMETTL3) or AKR1B10 (siAKR1B10) by GenePharma (Shanghai, China) were shown in Additional file 1: Table S1.
Besides, for stable knockdown of METTL3 expression used in animal study, lentiviruses vector pLKO.1 puro containing METTL3 shRNA (shMETTL3) and non-targeting scrambled shRNA (shNC) were purchased from GenePharma (Suzhou, China).
According to the manufacturer’s instructions, 5 μL of siMETTL3, siAKR1B10, shMETTL3 and shNC were diluted in 45 μL OPTI-MEM and then mixed with 10 μL Lipofectamine 2000 reagent pre-diluted with 45 μL OPTI-MEM. The mixture was added into cells and cultured for 24 h before further efficiency verification experiments.
TRIzol (Invitrogen Life Technologies) was applied for isolating total RNA from RBE cells and HCCC-9810 cells. Quality qualified RNA reverse-transcription into cDNA was carried out using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and then mRNA expression of METTL3, AKR1B10, and GAPDH were measured by QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) with FastStart Universal SYBR Green Master mix (Takara, China) according to the product’s protocol. Relative expressions of genes were normalized to GAPDH using 2−ΔΔCq method. The primers were listed in Additional file 1: Table S1.
Total protein concentration was measured by the BCA protein assay kit (Thermo scientific, USA). Then, 20 μg proteins were resolved by 10% SDS-PAGE and transferred onto PVDF membranes following blocking for nonspecific binding with 5% nonfat milk at 25 °C for 2 h. The membranes were incubated with anti-METTL3 (1:2000, Proteintech, 15,073-I-AP), anti-AKR1B10 (1:1000, Abcam, ab192865), and anti-GAPDH (1:1000, Proteint, 60004-1-Lg) at 4 °C overnight. After that, membranes were incubated with Goat Anti-Mouse IgG H&L (HRP) (1: 10,000, Abcam, ab205719) at 25 °C for 1 h. Immunore-activity was imaged by Bio-Rad ChemiDoc XRS system and quantified by Image J.
One hundred microliter cells with a density of 1 × 105 cells/well were seeded in 96-well plates and cultured for 24 h. Then, at each indicated times (0 h, 24 h, 48 h, 72 h, 96 h), 10 μL CCK-8 solution (Dojindo, Japan) was added and was incubated for another 1 h at 37 °C. The optical density was read at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific).
Cell apoptosis detection using TUNEL
Cell apoptosis was detected by TUNLE assay using a One Step TUNEL apoptosis kit (red Tunnelyte™ CY3 fluorescence detection) (C1089, Beyotime, China) according to the product instruction. Briefly, adherent CCA cells were washed with PBS and then fixed in immunostaining fixative solution (P0098, Beyotime, China) followed by permeabilized in immunostaining strong permeable solution (P0097, Beyotime, China). After that, cells were incubated with TUNEL solution for 1 h and sealed with anti-fluorescence quenching sealing tablets. Lastly, cells were photographed on a fluorescence microscope.
Cell migration and invasion were accessed by using a Transwell assay. The Transwell chamber was coated with 0.8 μm Matrigel (354480, BioCoat) for cell invasion assay, otherwise for migration assay. CAA cells were seeded into the upper chamber containing serum-free medium and complete medium was added to the lower chamber as a chemoattractant. The cells were cultured 24 h at 37 °C. The migrated cells to the lower chamber was photographed and calculated in three randomly fields under an inverted light microscope. The invaded-cells arriving at the lower chamber were fixed in 10% formaldehyde for 15 min and stained with 0.1% crystal violet for 10 min, and finally photographed in three randomly fields.
Glucose uptake and lactate production assay
Relative glucose uptake required by tumor cells was measured by Glucose Uptake Fluorometric Assay Kit (MAK084, Sigma-Aldrich, USA), and relative lactate production was measured by Lactic Acid Content Assay Kit (D799851-0050, Sangon, China) according to the technical bulletin provided by manufacturer.
Subcutaneous xenograft tumor model
A total of twelve 4-week-old female BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Company and were randomly divided into two groups: ShMETTL3 group (n = 6) and Vector group (n = 6). Mice were single housed at room temperature (21–26 °C) on a nature light cycle for one week before experiments to adapt laboratory environment. All mice were provided free access to diet and water. HCCC-9810 cells with METTL3 knockdown were digested by trypsin and made into single cell suspension. Next, 2 × 106 HCCC-9810 cells were subcutaneously injected into axillary of mice. After injection, mice were continued to be raised normally for 3 weeks. Tumor volume (length × width × width × 0.5) was measured every 3 days using caliper. Mice were euthanized using CO2 inhalation after the last measurement, tumor was collected and weighted.
RNA sequencing was performed at Yingbio Technology (Shanghai, China) using an Illumina HiSeq 2500. For differentially expressed genes (DEGs) identification, the thresholds was Log2fold change (FC) > 1 or < − 1 and false discovery rate (FDR) < 0.05. Gene onology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were carried out for the DEGs using R package. Transcriptome sequencing was repeated in three replicates.
RNA immunoprecipitation (RIP) and m6A RIP qPCR (MeRIP-qPCR)
Total RNA was extracted from RBE cells-overexpressed METTL3 or not, and then isolated mRNA was purified by Dynabeads mRNA Purification Kit (Invitrogen, USA) according to the manufacturer’s instructions. Next, purified mRNA was fragmented by RNA Fragmentation Reagent (Invitrogen, USA) before immunoprecipitation. After that, the anti-m6A antibodies or anti-METTL3 were conjugated to protein magnetic beads for immunoprecipitation, anti-immunoglobulin G (IgG) was served as negative control. Finally, RNA was eluted from RNA–protein immunocomplexes followed by RT-qPCR analysis.
Actinomycin D assay
RBE cells overexpressed METTL3 or NC were seeded in a 12-well plate and cultured for 24 h. Then, 5 μg/mL actinomycin D were added into cells and cultured another 0 h, 3 h, 6 h, 9 h, and 12 h following cell collection. Once collection, the total RNA was extracted from these RBE cells used for RT-qPCR as described above.
Dual-luciferase reporter assays
For m6A reporter assays, the wild-type of AKR1B10 sequence and the mutated at m6A motif 1 (mut-1, m6A was replaced by G), at m6A motif 2 (mut-2, m6A was replaced by G), and at m6A motif 3 (mut-3, m6A was replaced by C) were inserted into XhoI/NotI site of the psiCHECK-2 luciferase reporter vector. Then, these recombinant plasmids were transfected into RBE cells overexpressed METTL3 and NC using Lipofectamine 2000 reagent as described above.
Data analysis was performed by GraphPad Prism 9.0 and data were presented as mean ± SD. Kolmogorov–Smirnov test was used for evaluating the data normality, and Levene test was used for evaluating homogeneity of the variance of data. One-way ANOVA with Tukey test for three groups and t test for two groups were utilized when the data was normality and homogeneous. The chi-square test was used to evaluate the correlation between molecular expression and clinical data. P value less than 0.05 was considered significant.