FTO promotes liver inflammation by suppressing m6A mRNA methylation of IL-17RA

Figures

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  Figure 1. Dissecting the role of m6A mRNA methylation in the progression of HCC by MeRIP-seq. (A) Enriched RNA motifs from the m6A MeRIP-seq data within each sample. (B) PCA analysis of all samples. (C) Heatmap of m6A methylation levels of all samples. (D) KEGG analysis of differentially m6A modified genes. (E) Heatmap of differentially m6A modified genes related to the cytokine-cytokine receptor interaction. (F) m6A mRNA methylation level of IL-17RA between normal and tumor-adjacent liver tissues. (G) Western blot for the expression of IL-17RA in normal liver and tumor-adjacent liver tissues of patients. (H) Quantifications of IL-17RA expression in different tissues by Western blot. **p < 0.01.

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  Figure 2. FTO functions as a main modulator for the decrease of m6A mRNA methylation of IL-17RA. (A) Western blot for the expression of FTO, ALKBH5, METTL3, and METTL14 in normal liver and tumor-adjacent liver tissues of patients. (B-E) Quantifications of FTO, ALKBH5, METTL14, and METTL3 expression in different tissues by Western blot. All experiments were repeated at least three times and expressed as the means ± SEM. ns, not significant; *p < 0.05; **p < 0.01.

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  Figure 3. Regulation of FTO interferes protein level of IL-17RA in vitro. (A-D). Relative FTO or IL-17RA mRNA level in Huh7 cells after FTO knockdown or overexpression. (E-H). Relative FTO or IL-17RA protein level in Huh7 cells after FTO knockdown or overexpression. (I). Relative IL-17RA mRNA level in Huh7 cells after IL-17RA_WT or IL-17RA_MUT overexpression. (J). IL-17RA protein level in Huh7 cells after IL-17RA_WT or IL-17RA_MUT overexpression. (K). Relative m6A enrichment of IL-17RA mRNA in Huh7 cells after FTO overexpression by m6A-RIP-qPCR. All experiments were repeated at least three times and expressed as the means ± SEM. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 4. FTO promotes liver damage in murine NAFLD model. (A) Schematic of the in vivo experimental setup for construction of murine NAFLD model to ascertain FTO function. (B) Representative images of liver tissues in groups with different treatment by macroscopy and light microscopy (H&E staining and Oil red O staining). (C) NAFLD Activity Score (NAS), including inflammation, steatosis, ballooning, and total NAS in all groups with different treatment. (D) Quantitative analysis of Oil red O intensity in all groups with different treatment. (E) Relative serum TG in all groups with different treatment. (F) Relative TG level in liver tissues of all groups with different treatment. (G) Body weight of mice in all groups with different treatment. (H, I). Relative serum AST, and ALT in all groups with different treatment. (J) Liver weight of mice in all groups with different treatment. All experiments were repeated at least three times and expressed as the means ± SEM. n = 5; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Figure 5. FTO facilitates liver inflammation via induction of IL-17RA in murine NAFLD model. (A-C). Relative serum IL-17A, IL-1α, and IL-1β in all groups with different treatment. (D, E). Relative FTO, and IL-17RA protein level in the control, HFD, and HFD + Rhein groups. (F, G). Relative FTO, and IL-17RA protein level in the HFD + AAV-NC, and HFD + AAV-oeFTO groups. (H, I). Relative m6A enrichment of IL-17RA mRNA in liver tissues of different groups by m6A-RIP-qPCR. All experiments were repeated at least three times and expressed as the means ± SEM. n = 5; *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 6. FTO promotes liver damage in murine chronic liver injury model. (A) Schematic of the in vivo experimental setup for construction of murine chronic liver injury model to ascertain FTO function. (B) Representative images of liver tissues staining for H&E and Sirius red in groups with different treatment by light microscopy. (C) Inflammation score in all groups with different treatment. (D) Quantitative analysis of Sirius red positive area in all groups with different treatment. (E) Relative hydroxyproline level in liver tissues of all groups with different treatment. (F) Relative serum TG in all groups with different treatment. (G) Relative TG level in liver tissues of all groups with different treatment. (H) Body weight of mice in all groups with different treatment. (I, J). Relative serum AST, and ALT in all groups with different treatment. (K) Liver weight of mice in all groups with different treatment. All experiments were repeated at least three times and expressed as the means ± SEM. n = 5; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

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  Figure 7. FTO facilitates liver inflammation via induction of IL-17RA in murine chronic liver injury model. (A-C). Relative serum IL-17A, IL-1α, and IL-1β in all groups with different treatment. (D, E). Relative FTO, and IL-17RA protein level in the control, DEN + CCl4, DEN + CCl4 + AAV-NC, and DEN + CCl4 + AAV-oeFTO groups. (F). Relative m6A enrichment of IL-17RA mRNA in liver tissues of different groups by m6A-RIP-qPCR. All experiments were repeated at least three times and expressed as the means ± SEM. n = 5; *p < 0.05; **p < 0.01; ***p < 0.001.