Fig 1: Spliceosome components and splicing factors are altered in hepatocellular carcinoma (HCC): (A) fold-change of spliceosome components and splicing factors expression in HCC tissue versus non-tumour adjacent tissue (NTAT) in Retrospective-1 cohort. Data are presented as mean ± standard error of the mean (SEM); (B) bubble plot of the expression pattern of significantly dysregulated spliceosome components and splicing factors in seven validation cohorts. The y-axis indicates the spliceosome component and splicing factors altered in Retrospective-1. Bubbles indicate the expression pattern. The bubble size indicates the p-value; (C) VIP score analysis showing the spliceosome components and splicing factors with higher discriminatory capacity in Retrospective-1; (D) spliceosomal components more frequently found among the five elements (Top 5) with more discriminatory capacity in all the studied cohorts by VIP score analysis; (E) ROC curve analysis constructed with the expression levels of EIF4A3, ESRP2, SRPK1 and RBM3 to discriminate between tumour and non-tumour samples in all the studied cohorts; (F) transcriptomic and genomic alteration landscape of EIF4A3, ESRP2, SRPK1 and RBM3 in the TCGA cohort, and the clinical features of the patients. The asterisks (*p < .05; **p < .01; ***p < .001) indicate statistically significant differences
Fig 2: Pharmacologic inhibition or overexpression of EIF4A3 alter aggressiveness of liver cancer cells: (A) cell proliferation was determined in EIF4A3-IN-1 and vehicle-treated HepG2, Hep3B and SNU-387 cells by the Alamar Blue assay at 24, 48 and 72 h; (B) number of colonies formed in EIF4A3-IN-1 treated cells compared to vehicle-treated cells. Representative images of colonies formed after 10 days are depicted; (C) mean tumoursphere size of in EIF4A3-IN-1 treated cells compared to vehicle-treated cells. Representative images of tumourspheres formed after 10 days are depicted; (D) cell proliferation was determined in pEIF4A3-transfected HepG2 cells in comparison with mock cells by the Alamar Blue assay at 24, 48 and 72 h; (E) number of colonies formed in pEIF4A3-transfected cells compared to mock cells. Representative images of colonies formed after 10 days are depicted; (F) mean tumoursphere size of in pEIF4A3-transfected cells compared to mock cells. Representative images of tumourspheres formed after 10 days are depicted. Data are presented as mean ± standard error of the mean (SEM) from n = 3–5 independent experiments. Asterisks (*p < .05; ***p < .001) indicate statistically significant differences versus scramble-treated controls. Dashes (#p < .05; ##p < .01; ###p < .001) indicate statistically significant differences versus vehicle-treated controls
Fig 3: EIF4A3 silencing exerts its inhibitory actions by modulating FGFR4 splicing: (A) validation of EIF4A3 and FGFR4 expression in rescue experiments. EIF4A3 was silenced alone or in combination with FGFR4 overexpression in HepG2 and Hep3B cells, and the expression of both genes was validated in all the experimental conditions by qPCR; (B) cell proliferation determined in siEIF4A3‐treated, pFGFR4‐transfected and siEIF4A3‐treated/pFGFR4‐transfected, compared with scramble‐treated/mock HepG2 and Hep3B cells by Alamar Blue assay at 24, 48 and 72 h; (C) number of colonies formed in siEIF4A3‐treated, pFGFR4‐transfected and siEIF4A3‐treated/pFGFR4‐transfected, compared with scramble‐treated/mock HepG2 and Hep3B cells. Representative images of colonies formed after 10 days are depicted; (D) mean tumoursphere size of siEIF4A3‐treated, pFGFR4‐transfected and siEIF4A3‐treated/pFGFR4‐transfected, compared with scramble‐treated/mock HepG2 and Hep3B cells. Representative images of tumourspheres formed after 10 days are depicted; (E) number of colonies formed in siEIF4A3‐treated, BLU‐treated and siEIF4A3‐treated/BLU‐treated HepG2 and Hep3B cells compared to scramble‐ treated/control cells. Representative images of colonies formed after 10 days are depicted. Data are presented as mean ± standard error of the mean (SEM) from n = 3–5 independent experiments. Asterisks (*p < .05; **p < .01; ***p < .001; ****p < .0001) indicate statistically significant differences versus scramble‐treated or mock controls, whereas dashes (## p < .01; ### p < .001) indicate statistically significant differences versus vehicle‐treated controls
Fig 4: EIF4A3 associates with the expression and splicing of key hepatocellular carcinoma (HCC)‐related genes: (A) GSEA analysis performed by GenePattern in Reactome using the TCGA cohort classified by EIF4A3 expression levels in low and high EIF4A3 groups; (B) differentially expressed genes (DEGs) in EIF4A3‐silenced HepG2 cells obtained from RNAseq data (FDR < .05); (C) splicing event types in EIF4A3‐silenced HepG2 cells obtained from RNAseq data (FDR < .05); (D) the Venn diagram of DEGs and differentially spliced genes (DSGs) in EIF4A3‐silenced HepG2 cells (FDR < .05); (E) STRING analysis of the 55 genes with differential expression and splicing pattern in EIF4A3‐silenced HepG2 cells (FDR < .05)
Fig 5: EIF4A3 silencing decreases aggressiveness of hepatocellular carcinoma (HCC) cells: proliferation of EIF4A3‐silenced with siEIF4A3#1 (A) and siEIF4A3#2 (B) compared to scramble‐treated cell lines (HepG2, Hep3B and SNU‐387) at 24, 48 and 72 h determined by the Alamar Blue assay; (C) migration of EIF4A3‐silenced compared to scramble‐treated cells. Representative images of cell migration after 24 h are depicted; (D) number of colonies formed in EIF4A3‐silenced compared to scramble‐treated cells. Representative images of colonies formed after 10 days are depicted; (E) mean tumoursphere size of EIF4A3‐silenced compared to scramble‐treated cells. Representative images of tumourspheres formed after 10 days are depicted; (F) mRNA expression levels of key tumour markers genes in EIF4A3‐silenced versus scramble‐treated cells; (G) Validation of EIF4A3 expression by qPCR after in vivo silencing in xenograft models; (H) growth rate of tumours in Hep3B‐induced xenograft tumours in nude mice (n = 5) before and after in vivo EIF4A3‐silencing (indicated by the arrow). Representative images of scramble‐ and siEIF4A3‐treated tumours are depicted; (I) final tumour weight of scramble‐ and siEIF4A3‐treated tumours. Data are presented as mean ± standard error of the mean (SEM) from n = 3–5 independent experiments. The asterisks (*p < .05; **p < .01; ***p < .001; ****p < .0001) indicate statistically significant differences
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