Fig. 1
FGFR4 inhibitor treatment elevates EZH2 expression by activating non-canonical NF-kB signaling in HCC. A Western bolt detected the expression of EZH2 and FGFR1-4 in human hepatic cell line THLE-2 and human hepatoma cell lines HepG2, SMMC-7721 and MHCC97L. B Dose responses of zebrafish KRASG12V+ HCC primary tumors treated with Roblitinib for 72 h. Scale bars: 100 μm. Data are presented as mean ± SEM (n = 15, one-way ANOVA with Dunnett’s multiple comparison test, **p < 0.01, ns, no significance). C Clustered heatmaps showed the differentially expressed genes between control group and Roblitinib treatment group (fold change ≥ 2 or ≤  − 2 and p-values < 0.05). HepG2 cells were treated with Roblitinib for 48 h, followed by RNA-Seq analysis. Red indicates high relative expression, and blue indicates low relative expression. D Bubble chart showed the Gene Ontology (GO) biological process and molecular function analysis of differentially expressed genes following Roblitinib treatment in (C). P-values < 0.05 was regarded as statistically significant. E Gene set enrichment analysis (GSEA) of the KEGG pathway showed that HCC cells treated with Roblitinib were enriched for the drug resistance/antagonism. F GSEA of Reactome pathway showed that cells treated with Roblitinib were enriched for transcriptional signatures associated with PRC2-mediated methylation. G-H Western bolt detected the expression of EZH2 after different FGFR inhibitor treatment for 48 h (G) and Roblitinib treatment for different durations (H). I High levels of EZH2 were associated with worse survival of HCC patients. J Gene set enrichment analysis (GSEA) showed that the NF-kB signaling pathway was enriched in the Roblitinib treatment group compared with Control group. K RNA-Seq analysis of the expression level of the indicated NF-kB genes after Roblitinib treatment for 48 h. The abscissa represented the indicated NF-kB genes, while the ordinate represented Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak’s multiple comparison test, ****p < 0.0001, ns, no significance). L Western bolt detected the expression of the indicated NF-kB genes after Roblitinib treatment for 48 h. M Western blot analysis of EZH2 expression in HepG2 cells transfected with empty vector control (Con), HA-tagged NFKB2 (HA-NFKB2) and NFKB2-shRNAs (shNFKB2-1 and 2). N Western blot analysis of EZH2 expression in HepG2 cells after lentiviral transduction with control shRNA (shVector) or 3′ UTR NFKB2-targeting shRNA (shNFKB2) and restored with empty vector control (Con) or HA-tagged NFKB2 (HA-NFKB2), following by Roblitinib treatment for 48 h