Samples, cell lines, and plasmids
The human ESCCs samples obtained from abdominal surgery, Anhui Provincial Cancer Hospital, West Branch of the First Affiliated Hospital of USTC, and with patients’ informed consent. The pathological condition was determined by experienced surgical specialist. The comprehensive clinical and pathological information of ESCCs patients were shown in SI Appendix, Table S1. ESCCs cell line KYSE140, KYSE180, KYSE450, KYSE30, KYSE150 and human normal esophageal epithelial cell line HEEC were obtained from the Chinese Academy of Cell Resource Center (Shanghai, China) and maintained as previously described [32]. Cells were cultured in DMEM or RPMI 1640 (BI) medium supplemented with 10% fetal bovine serum (PAN), 100 U/ml penicillin, and 100 mg/ml streptomycin (WISENT) in humidified air at 37°C with 5% CO2. All cell lines tested negative for mycoplasma contamination. FTO, METTL14, and AKT3 expressing lentivirus vector were purchased from Hanbio (Shanghai). Plasmids for expression of Flag tagged wild-type (YTHDF1-WT, YTHDF2-WT, YTHDF3-WT, YTHDC1-WT, YTHDC2-WT) were contructed with p3xFLAG-Myc-CMV vector. The detailed information regarding the primers used for plasmid constructs is depicted in SI Appendix, Table S2. For shRNA plasmids used in lentivirus-mediated interference, complementary sense and antisense oligonucleotides encoding shRNAs targeting FTO and were synthesized, annealed and cloned into pHBLV-U6-MCS-EF1-mcherry-T2A-PURO vector. The related sequences of shRNAs were shown in Table S2 of the SI Appendix.
Gene expression and survival analysis in ESCCs cancer datasets
Kaplan–Meier plotter (http://kmplot.com/analysis/) was used to assess the prognostic value of FTO and METTL14 expression in patients with ESCCs cancers. mRNA expression of FTO, YTHDC1, and YTHDF1 in cancer tissues and matched adjacent normal tissues of ESCCs cancers were obtained from TCGA (The Cancer Genome Atlas) database. GEPIA2 (http://gepia.cancer-pku.cn/) was used to assess correlation analysis of FTO and AKT3.
m6A content analysis
The content of m6A in total RNA was analyzed with the EpiQuik TM m6A RNA Methylation Quantification Kit (Epigentek).
Dot-blot assays
The mRNA was obtained according to the PolyATtractR mRNA Isolation Systerms kit (Promega) instruction manual. 50ng/100ng mRNA was diluted to 2 µl with DEPC water and placed in a PCR instrument for thermal denaturation at 65°C for 10 minutes. The denatured RNA was evenly dotted onto the 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 for 3 times, 5 minutes each time. 10 ml 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 ratio of 1 :1000. Dip the nylon membrane in the primary antibody and incubate overnight at 4°C. The nylon film incubated overnight was washed with PBST for 3 times, 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, 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 taken out and observed under the developer. Then the nylon film was stained with 0.2% methylene blue dye (pH5.2, corrected by 0.3 M sodium acetale for pH) for 0.5 hours. Photograph was taken and the sample load volume of each sample was compared.
m6A-RT-PCR
m6A-RT-PCR was conducted according to previously described protocol with a slight modification [33]. Briefly, the total RNA was extracted using Trizol and fragmented by RNA fragmentation reagents (Thermo, AM8740) or not. After saving 50 ng of the total RNA as input, the remaining RNAs (2 µg) were used for m6A-immunoprecipitation with m6A antibody (Synaptic Systems) in 500 µl of IP buffer (150 mM NaCl, 0.1 % NP-40, 10 mM Tris, pH 7.4, 100 U RNase inhibitor) to obtain m6A pull down portion (m6A IP portion). m6A RNAs were immuno-precipitated with Dynabeads ® Protein A (ThermoFisher Scientific) and eluted twice
with elution buffer (5 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.05% SDS, 20 mg/ml Proteinase K). m6A IP RNAs were recovered by ethanol precipitation, and RNA concentration was measured with Qubit® RNA HS Assay Kit (ThermoFisher Scientific). Then 2 ng of the total RNA and m6A IP RNA were used as templates in qRT-PCR, as described above.
In vitro cell proliferation, migration, and invasion assays
Cell proliferation was measured using the CCK-8 according to the manufacturer's instructions, 5 × 103 cells per well were seeded onto a 96-well plate and checked every 24 hours (0, 24, 48, 72 and 96 hours). Triplicated samples were counted.
The cell migration test was performed in a 24-well plate with 8 mm pore size transwell chamber (Corning). 200 µl of a suspension containing 5 ×104 cells made of RPIM 1640 without FBS was Inoculated into the upper part of the chamber. 600 µl of RPIM 1640 medium containing 20% FBS was added to the lower part of the chamber. After incubating for 30 hours at 37°C and 5% CO2, take out the transwell chamber, discard the culture medium in the well, wash twice with PBS, fix with methanol for 5 minutes, stain with 0.1% crystal violet for 30 minutes, wipe off the upper layer of cells with a cotton swab, wash with PBS 3 times, randomly take 5 fields of view under the microscope to observe the cells and count them.
Cell invasion assays were performed in a 24-well plate with 8 mm pore size chamber inserts (Corning). 8 ×104 cells were seeded in the upper portion of the invasion chamber with 200 µl of RPIM1640 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°C and 5% CO2, the non-invading 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 extraction and real-time PCR for gene expression
RNA extraction with Trizol (Invitrogen) and real-time PCR were performed according to the protocol used in our previous study [34]. Primers of targeted genes were depicted in SI Appendix, Table S2.
Colony formation assays
For the colony formation assay, about 500 infected or transfected cells were seeded into each well of a 6-well plate and maintained in a medium containing 10% FBS for 10 days. The colonies were fixed with methanol and stained with 0.1% crystal violet, and the number of clones was counted. Triplicated samples were counted.
Cell apoptosis assays
Cells were harvested and rinsed twice with pre-cooling PBS. The samples were diluted with 150 µl of 1×annexin-binding buffer, then 5 µl of APC-labeled enhanced annexinV and 5 µl (20 µg/ml) of propidium iodide (PI) were added. Then the cells were incubated in the dark for 15 minutes at room temperature. Flow cytometry was conducted on a FACSCalibur instrument (BD).
Wound-healing assay
For wound-healing assay, cells were seeded and cultured until a 90% confluent monolayer was formed. Cells were then scratched by a sterile pipette tip and treated as indicated in the text in the FBS-free medium. Cell migration distances into the scratched area were measured in 10 randomly chosen fields under a microscope.
Western blot assays
Cells were harvested and washed twice with PBS. After adding lysis buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue and 1.25% 2-mercaptoethanol), samples were lysed and heated for 10 minutes at 95°C, followed by centrifugation at 4°C and 13,000 rpm for 30 minutes. Whole-cell proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF). Membranes were blocked with 5% nonfat milk at room temperature for 1 hour or at 4°C overnight and then incubated with the appropriate antibody. Images were captured by the Image Reader. Detailed information regarding the full-length gels is depicted in Supplementary Figures of SI Appendix.
Biotin RPD assays
RPD assays were performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the manufacturer's instructions. KYSE150 cells were transfected with biotinylated AKT3 sense probe and antisense probe (50 µl streptavidin beads were washed once with RPD buffer, then 10 µl of sense probe and 10 µl of antisense probe were added, and incubated for overnight at 4°C). and the cells were incubated at 4°C for 1–3 hours and the total cell lysates were incubated at room temperature for 2 hours. The bead-RNA-protein complexes were washed with 1 × binding-washing buffer four times. The proteins were precipitated and diluted in protein lysis buffer. Finally, the retrieved proteins were measured by real-time PCR and/or western blot analysis. Detailed information regarding the primers used for real-time PCR analysis is depicted in SI Appendix, Table S2.
RNA immunoprecipitation (RIP) assays
RIP was performed with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the instructions provided by the manufacturer [35]. Briefly, approximately 2–4 × 107 KYSE150 cells were lysed in hypotonic buffer supplemented with RNase inhibitor and protease inhibitor before centrifugation. The lysates were incubated with magnetic beads and coated with the indicated antibodies for 4 hours or overnight at 4°C. Then, the RNA-protein complexes were washed 6 times and incubated with proteinase K digestion buffer, the bead-bound immunocomplexes were treated with proteinase K for 30 minutes at 55°C. Samples were centrifuged and placed on a magnetic separator, and supernatants were used to extract RNA by Kit (Bioline). Purified RNAs were then subjected to PCR analysis and normalized to the input. Detailed information regarding the primers used for PCR analysis is depicted in SI Appendix, Table S2.
m6A sequencing (m6A-seq) and data analysis
The total polyadenylated 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 [36]. RNA Library Prep Kit (NEB, USA) was used for library preparation. Each experiment was conducted with two biological replicates. m6A-seq data were analyzed according to protocols. Significant peaks with FDR < 0.05 were annotated to RefSeq database (hg19). Sequence motifs were identified by using Homer. Gene expression was calculated by Cufflinks using sequencing reads from input samples. Cuffdiff was used to find DE genes.
Vector and m6A mutation assays
The potential m6A sites were predicted using an online tool, SRAMP (http://www.cuilab.cn/sramp/). Full-length AKT3 transcripts, the AKT3 CDS region, the AKT3 three prime untranslated region (3’UTR), and the m6A motif depleted CDS or 3’UTR regions were cloned into pcDNA3.1 for the RNA pull down assay. The specific sequences are shown in SI Appendix, Table S2.
RNA stability
To measure RNA stability of FTO knockdown to KYSE150 cells, actinomycetes D (6 µg/ml) treated control cells and down-regulated FTO cells to block RNA transcription at 0, 2, 4, 6, 8 hours, respectively. AKT3 mRNA residue was detected by quantitative PCR and the stability of mRNA was calculated.
Luciferase reporter assays
Luciferase assay was performed using reporter lysis buffer (Promega) and luciferase assay reagent according to the manufacturer’s instructions. Briefly, FTO knockdown-KYSE150 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 re-seeded into a 96-well plate. After 24 hours incubation, both firefly and Renilla luciferase activities were measured 24 hours after transfection using the
Dual-Luciferase Reporter Assay System (Promega) and a Promega GloMax 20/20
luminometer. The relative firefly luciferase activities of the UTR construct and pathway reporter
constructs were analyzed as previously reported [37]. Detailed information regarding the primers
used for plasmid construction is depicted in SI Appendix, Table S2.
In vivo xenografts model
Four-week-old male BALB/c nude mice were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. The nude mice were kept in the SPF Animal Laboratory of China University of Science and Technology. Animal experiments were conducted in accordance with the National Guidelines for the Health Use of Laboratory Animals. Animal research was approved by the Biomedical Ethics Committee of China University of Science and Technology. The procedures for mouse experiments were implemented in accordance with the Regulations on the Administration of Laboratory Animals approved by the State Council.
For 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 immunodeficient male mice to investigate tumor growth. When tumor volume, in each group, reached ~ 100 mm3, all the mice were killed, and tumors were removed and weighed for use in immunohistochemistry assays and further studies. The tumor volume was calculated using the equation V = 0.5×D×d2 (V: volume, D: longitudinal diameter, d: latitudinal diameter). For in vivo lung metastasis model, mice were injected with WT (wide-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.
Immunohistochemistry assays
The protein expression levels of Vimentin, E-cadherin, and MMP2 were determined by immunohistochemistry (IHC). IHC staining was performed on 4-mm sections of paraffin-embedded tissue samples. Briefly, the slides were incubated with the appropriate antibody at 4°C overnight. The subsequent steps were performed using the GTVision III Detection System/Mo&Rb (GeneTech). Detailed information regarding the antibody is depicted in SI Appendix, Table S2.
Statistical analysis
Statistical analysis was carried out using Microsoft Excel software and GraphPad Prism to assess 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.