Fig 1: Wnt signalling pathway is active in PN but not in MES GBM.(a) Gene Set Enrichment Analysis (GSEA) of TCGA data set was performed for the expression of gene components of the canonical Wnt pathway in clinical PN and MES GBMs. Wnt signalling pathway is active in PN GBMs, but not in MES GBMs. (b) Hierarchical cluster analysis on expression data of the Wnt pathway genes differentially expressed between PN and MES spheres. (c) IB analysis of Wnt pathway components including TCF4, FZD6, APC, and activated ß-catenin (ABC) in indicated PN and MES spheres. (d) TopFlash/FopFlash assays that quantify relative Wnt signalling activity in indicated PN and MES spheres. Values in bar graphs were normalized to those in MES 83. (e) Indicated PN and MES spheres were shown expressing GFP under the control of a 7 × TCF/LEF optimal promoter cassette (7 × TOP) and constitutively expressed nuclear mCherry. PN 84 and MES 1123 were treated with Wnt3a (200 ng ml-1), indomethacin (Indo; 20 µM), or a control (vehicle). Scale bar, 100 µm. Bar graph, quantification of fluorescent signal of GFP (TOP-GFP) versus mCherry (PGK-H2BmCherry). The percentage of GFP- and mCherry-positive spheres was determined by FACS. (f) Cell proliferation (upper panels) and sphere formation (lower panels) assays for PN 84 expressing control or specific shRNAs of Wnt3a, Wnt4, and Wnt5a, respectively. Error bars (s.d.) represent the data of triplicate samples for each cell line. *P<0.05, **P<0.01, paired two-way Student's t-test. Data in a–f are representative from three independent experiments with similar results.
Fig 2: METTL3-enhanced m6A of APC mRNA and subsequent binding of YTHDF2 suppresses APC expression.a RIP analyses of KYSE180 cells were performed with an anti-YTHDF1 or anti-YTHDF2, or anti-YTHDF3 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. ***p = 0.0006 (left), **p = 0.0092 (right) based on two-tailed Student’s t-test. b KYSE180 cells were transfected with or without a vector expressing METTL3 shRNA. RIP analyses were performed with an anti-YTHDF2 antibody followed by qPCR analyses with primers against APC mRNA. Data represent the means ± SD of triplicate samples. ***p = 1.85E - 05 (left), **p = 0.0023 (middle) and 0.0056 (right) based on two-tailed Student’s t-test. c KYSE450 cells were transfected with or without a vector expressing YTHDF2 shRNA. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. ***p = 0.0006, 0.0009, 5.58E - 05 and 0.0007 (left to right) based on two-tailed Student’s t-test. d KYSE450 cells were transfected with or without YTHDF2 shRNA. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. e KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. The relative mRNA expression levels of APC were measured using quantitative PCR. Data represent the means ± SD of triplicate samples. ***p = 9.33E - 04 (left) and 2.99E - 05 (right), *p = 0.031 based on two-tailed Student’s t-test. f KYSE180 cells were transfected with or without a YTHDF2 siRNA or combination of YTHDF1–3 siRNAs. Immunoblotting analyses were performed with the indicated antibodies for three times with similar results. g KYSE180 cells expressing luciferase reporter genes fused with or without the wild-type (WT) or mutated m6A nucleotides from APC genes were transfected with or without a vector expressing YTHDF2 shRNA. The relative luciferase activity after normalization to the shControl group is shown. Data represent the means ± SD of triplicate samples. ***p = 3.94E - 06 based on two-tailed Student’s t-test. ns, not significant. Source data are provided as a Source Data file.
Fig 3: miR-20b and miR-125b augment Wnt signalling through targeting FZD6.(a) Top panel: IB of activated ß-catenin (ABC) and TCF4 in miR-transduced 293T cells. Lower: cells were then treated with PBS (control), Wnt3a (200 ng ml-1) and BIO (2 µM), respectively. Firefly luciferase activities were normalized to Renilla luciferase activities. (b) Top: targeting sites of miR-20b and miR-125b (underlined, seed sequence) in mRNA of FZD6 gene. Lower: qRT–PCR for expression of FZD6 and APC in PN 157 (left) and PN 84 (right) expressing miR-20b ZIP, miR-125 ZIP or a non-targeting control miRZIP. ß-Actin, an internal control. (c) Effects of miR-20b or miR-125b on the activities of indicated reporter genes containing WT or mutations at the miR-binding sites. Luciferase activities were normalized as in a. (d) IB for FZD6 and APC in miR-transduced MES 83 and 1123, or miRZIP-transduced PN 157 and 84. (e) Levels of ß-catenin in cytoplasm (C) and cell nucleus (N) were examined in miR-transduced MES 83 or miR ZIP-transduced PN 84. GAPDH, control for cytoplasmic protein; Histone H3, control for nuclear protein. (f) Subcellular localization of ß-catenin in glioma spheres. Yellow arrows, stained ß-catenin. Scale bar, 20 mm. (g) Knockdown of FZD6 by specific shRNAs (#1/#2 targeting ORF of FZD6 mRNA and shRNA #3 targeting 3'-untranslated region (UTR) of FZD6 mRNA) inhibited growth of MES 83 and 1123 brain tumour xenografts. Re-expression of FZD6, but not a vector control, rescues shFZD6 #3-inhibited MES 83 tumour growth (n=5). Top panels: IB. Tumour areas in both experiments were quantified (Supplementary Fig. 6e). Scale bar, 1.0 mm. (h) sphere formation of MES 83 expressing control shRNA or shRNAs of FZD6, with or without re-expression of FZD6, or a control (right). (i) Cell proliferation (left) and sphere formation (right) for PN 157 expressing miR-20b ZIP, miR-125b ZIP, or a control with or without FZD6 knockdown. shR, shRNAs; C, control shRNA; shF, shRNA#1 for FZD6. Error bars (s.d.) represent the data of triplicate samples for each cell line or tumours from five individual mice in each group. *P<0.05, **P<0.01, paired two-way Student's t-test. Data are representative from three independent experiments with similar results.
Fig 4: METTL3 reduces APC expression and promotes ß-catenin downstream gene expression, aerobic glycolysis, ESCC cell proliferation and tumour development.a Immunoblotting analyses of KYSE180 cells with or without METTL3 shRNA expression or combined METTL3 shRNA and APC shRNA expression were performed with the indicated antibodies for three times with similar results. b, c Glucose consumption (b) and lactate production (c) of KYSE180 cells with or without METTL3 shRNA expression, or combined METTL3 shRNA and APC shRNA expression were determined. Data represent the means ± SD of triplicate samples. **p = 0.0093 (b) and p = 0.0062 (c) based on two-tailed Student’s t-test. ns, not significant. d KYSE180 cells with or without METTL3 shRNA expression or combined METTL3 shRNA and APC shRNA expression were cultured for the indicated periods of time and collected for cell counting. Data represent the means ± SD of triplicate samples. **p = 0.0023 based on two-tailed Student’s t-test. ns, not significant. e–i KYSE180 cells with or without METTL3 shRNA expression or combined METTL3 shRNA and APC shRNA expression were subcutaneously injected into the flank regions of nude mice (n = 6). Six weeks later, tumour sizes (e), volumes (f) and weight (g) were measured. Data represent the means ± SD of six mice in each group. **p = 0.0025 (f), *p = 0.0135 (g) based on two-tailed Student’s t-test. h IHC staining of tumour tissues was performed with the indicated antibodies for three times with similar results. Representative images are shown. Scale bar, 100 µm. i The protein expression levels of the indicated proteins were displayed. *p = 0.0269, **p = 0.0033, 0.0069 and 0.0011 (left to right), ***p = 8.35E – 05, 0.0002 and 0.0008 (left to right) based on two-tailed Student’s t-test. ns, not significant. Source data are provided as a Source Data file.
Fig 5: A mechanism underlying METTL3-promoted ESCC development.Overexpressed METTL3 increases the levels of the APC mRNA N6-methyladenosine, which recruits YTHDF to APC mRNA, to downregulate APC expression. Downregulated APC stabilizes ß-catenin expression and promotes ß-catenin-dependent gene expression for aerobic glycolysis, and ESCC cell proliferation and tumour growth.
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