Samples, cell lines, and plasmids
The 28 human esophageal cancer samples were obtained from abdominal surgery at the First Affiliated Hospital of University of Science and Technology of China, and patients provided informed consent. The pathological condition was determined by an experienced surgical specialist. The comprehensive clinical and pathological information of esophageal cancer patients is shown in SI Appendix, Table S1. The esophageal cancer cell lines KYSE140, KYSE180, KYSE450, KYSE30,and KYSE150 and the human normal esophageal epithelial cell line HEEC were obtained from the Chinese Academy of Cell Resource Center (Shanghai, China) .
Lentiviruses for overexpressing and silencing the expression of FTO, METTL14 and AKT3 were constructed from Hanbio Biotechnology Co., Ltd. Plasmids for the expression of Flag-tagged wild-type (YTHDF1-WT, YTHDF2-WT, YTHDF3-WT, YTHDC1-WT, YTHDC2-WT) were constructed withthep3xFLAG-Myc-CMV vector. Detailed information regarding the primers used for plasmid construction is depicted in SI Appendix, Table S2. For shRNA plasmids used in lentivirus-mediated interference, complementary sense and antisense oligo nucleotides encoding shRNAs targeting YTHDF1 were synthesized, annealed and cloned into the pHBLV-U6-MCS-EF1-mcherry-T2A-PURO vector. The related sequences of shRNAs are shown in Table S2 of the SI Appendix.
Gene expression and survival analysis in esophageal cancer datasets
Kaplan–Meier plotter (http://kmplot.com/analysis/) was used to assess the prognostic value of FTO and METTL14 expression in patients with esophageal cancer. The mRNA expression of FTO, YTHDC1, and YTHDF1 in cancer tissues and matched adjacent normal tissues of esophageal cancer patients was obtained from TCGA (The Cancer Genome Atlas) database. GEPIA2 (http://gepia.cancer-pku.cn/) was used to assess the correlation analysis of FTO and AKT3.
m6A content analysis
The EpiQuik TM m6A RNA Methylation Quantification Kit (Epigentek) was used to analyze the content of m6A in total RNA.
Dot blot assays
mRNA was obtained according to the PolyA Ttract® mRNA Isolation System (Promega) instruction manual. mRNA (50 ng/100 ng) was diluted to 2 µl with DEPC water and placed in a PCR instrument for thermal denaturation at 65 ℃ for 10 minutes. The denatured RNA was evenly dotted onto a positively charged nylon membrane (Beyotime). UV irradiation was placed under the operating platform for 15 minutes, and RNA was fixed to the membrane. The fixed nylon film was washed in 1 x PBST 3 times for 5 minutes each time. Ten milliliters of 5% skim milk sealant was prepared, and the membrane was placed in the sealant and sealed at room temperature for 2 hours. Rabbit m6A primary antibody (active motif) was formulated at a ratio of 1:1000. The nylon membrane was dipped in the primary antibody and incubated overnight at 4 ℃. The nylon film incubated overnight was washed with PBST 3 times for 5 minutes each time. Rat anti-rabbit secondary antibody was prepared according to 1:2000. The nylon membrane was immersed in the secondary antibody and incubated at room temperature for 2 hours. The nylon film was washed 4 times with TBST for 5 minutes each time. The ECL color solution was prepared, and the nylon film was immersed in the color solution for 10 seconds. The nylon film was removed and observed under developer. Then, the nylon film was stained with 0.2% methylene blue dye (pH 5.2, corrected by 0.3 M sodium acetate for pH) for 0.5 hours. Photographs were taken, and the sample load volume of each sample was compared.
According to described protocol, m6A-RT–PCR was conducted . To obtain the m6A pull-down portion, 2 µg RNA was used for immunoprecipitation with m6A antibody in 500 µl IP buffer. m6A RNA was immunoprecipitated with Dynabeads ® Protein A and then eluted twice with elution buffer. m6A IP RNA was recovered by ethanol precipitation. Then, 2 ng of the total RNA and m6A IP RNA were used as templates in qRT–PCR.
In vitro cell assays
Total of 5 × 103 cells per well were seeded onto a 96-well plate and checked every 24 hours (0, 24, 48, 72 and 96 hours), and cell proliferation was measured using CCK-8.
The cell migration test was performed in a 24-well plate with an 8 µm pore size Transwell chamber (Corning). A total of 200 µl of a suspension containing 5 ×104 cells made of RPMI 1640 without FBS was inoculated into the upper part of the chamber. Then,600 µl of RPMI 1640 medium containing 20% FBS was added to the lower part of the chamber. After incubation for 30 hours at 37 ℃ and 5% CO2, the transwell chamber was removed, the culture medium in the well was discarded, the cells were washed twice with PBS, fixed with methanol for 5 minutes, stained with 0.1% crystal violet for 30 minutes, the upper layer of cells was wiped off with a cotton swab, and the cells were washed with PBS 3 times. Five fields of view were randomly taken under the microscope to observe the cells and count them.
Cell invasion assays were performed in a 24-well plate with 8 µm pore size chamber inserts (Corning). A total of 8 ×104 cells were seeded in the upper portion of the invasion chamber with 200 µl of RPMI 1640 without FBS. The lower portion of the chamber contained 600 µl of medium supplemented with 20% FBS and glutamine. After incubation for 36 hours at 37 ℃ and 5% CO2, the noninvading cells were removed from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet for 30 minutes. The cells were then imaged and counted in four separate areas with an inverted microscope.
RNA pull-down assays
RNA was taken from the Megascript® T7 Transcription Kit (Ambion) through the in vitro Transcription Kit, and then the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific) was used to conduct the RNA pull-down experiment. In short, a Pierce RNA 3' End Desthiobiotinylation Kit (Thermo Scientific) was used to biotin the RNA. Then, 50 pmol of biotinylated RNA, 50 µl of streptavidin magnetic beads and 200 µg of cell lysates were incubated at a suitable temperature for a certain period of time, and the supernatant was collected after repeated washing for real-time PCR and Western blotting analysis. Detailed information regarding the primers used for real-time PCR analysis is depicted in SI Appendix, Table S2.
A Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used for RIP according to the instructions . Briefly, approximately 2–4 × 107 KYSE150 cells were lysed before centrifugation, incubated with magnetic beads and coated with antibodies for 4 hours or overnight at 4 ℃. Then, the complexes were washed and incubated with proteinase K. Then, the samples were centrifuged and placed on a magnetic separator, and the supernatants were used to extract RNA with an RNA extraction kit (Bioline). Purified RNA was used for analysis. Detailed information regarding the primers used for PCR analysis is depicted in SI Appendix, Table S2.
m6A-seq and data analysis
Total RNA was isolated from KYSE150 and KYSE30 FTO knockdown (sh-FTO) cells using TRIzol reagent (Tiangen). RNA fragmentation, m6A-seq, and library preparation were performed according to the manufacturer’s instructions . An RNA Library Prep Kit (NEB, USA) was used for library preparation. The m6A-seq data were analyzed according to the manufacturer’s protocols.
Vector and m6A mutation assays
The potential m6A sites of full-length AKT3 transcripts were predicted using an online tool, SRAMP (http://www.cuilab.cn/sramp/). The m6A motif-depleted 3’UTR regions were cloned into pGL3 for luciferase reporter gene analysis. The specific sequences are shown in SI Appendix, Table S2.
To measure the RNA stability of FTO knockdown in KYSE150 cells, actinomycete D (6 μg/ml)-treated control cells and down regulated FTO cells were used to block RNA transcription at 0, 2, 4, 6, and 8 hours. AKT3 mRNA residue was detected by quantitative PCR, and the stability of mRNA was calculated.
Luciferase reporter assays
FTO knockdownKYSE150 cells were transfected with pGL3, pGL3-WT-3’UTR, pGL3-Mut1-3’UTR, or pGL3-Mut2-3’UTR in a 6-well plate. After transfection for 8 hours, each cell line was reseeded into a 96-well plate. After 24 hours of incubation, both firefly and Renilla luciferase activities were measured 24 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega) . Detailed information regarding the primers used for plasmid construction is depicted in SI Appendix, Table S2.
In vivo xenograft model
For the subcutaneous transplanted model, sh-control, sh-FTO, NC-OE and AKT3-OE KYSE150 cells (6 × 106 per mouse, n = 3 for each group) were diluted in 100 μl of PBS + 100 μl Matrigel (BD) and subcutaneously injected into immuno deficient male mice to investigate tumor growth. When the tumor volume in each group reached ~100 mm3, the mice were anesthetized with a small flow of carbon dioxide to make them unconscious. Then, the mice were killed completely by increasing the flow, and by pulling the leg of the mouse to see if there was muscle tension, the mice were judged as dead. Then, the tumors were removed and weighed for use in immunohistochemistry assays and further studies. The equation V=0.5×D×d2 was used to calculate the tumor volume (V: volume, D: longitudinal diameter, d: latitudinal diameter). For the in vivo lung metastasis model, mice were injected with WT (wild-type), sh-FTO, AKT3-OE and sh-FTO+AKT3-OE KYSE150 cells (1 × 106 per mouse, n = 3 for each group). Six weeks after injection, mice were killed, and metastatic lung tumors were analyzed.
Tissue arrays were constructed using 28 pairs of esophageal cancer and paracancerous tissues as well as animal experimental specimens. Paraffin embedding, sectioning, and immunohistochemistry (IHC) were programmed to detect FTO expression in esophageal cancer and paracancerous tissues and Vimentin, E-cadherin, and MMP2 expression in animal specimens. Pictures were taken usinga LEICA DM 4000B microscope.
Microsoft Excel software and GraphPad Prism were used to assess the differences between experimental groups. Statistical significance was analyzed by a two-tailed Student’s t test and one-way ANOVA. p values less than 0.05 were considered to be statistically significant: *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001.