PURPOSE: Radiation therapy (RT), vital for treatment of primary liver cancer, comes with unavoidable liver injury, which limits its implementation. N6-methyladenosine (m6A) methylation is involved in many molecular functions. However, its role in radiation-induced liver diseases (RILD) remains unknown. Here, we intend to investigate the role of m6A methylation in RILD. METHODS AND MATERIALS: Methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and RNA transcriptome sequencing (RNA-seq) were used to reveal the methylation pattern of human hepatic stellate cells (HSCs) exposed to irradiation. C3H/HeN mice and STING-deficient mice underwent X-ray irradiation of 24 Gy in three fractions. The m6A methylation of HMGB1 transcript was validated using MeRIP, RIP, luciferase assays and mRNA decay assay. RESULTS: Human HSCs showed significant differences in methylation patterns after 8 Gy of X-ray irradiation. Irradiation recruited AlkB Homolog 5 (ALKBH5) to demethylate m6A residues in the 3' untranslated region (UTR) of High-Mobility Group Box 1 (HMGB1), which resulted in activation of STING-IRF3 signaling. Changes in transcription of the 3'UTR of HMGB1 occurred after knockdown of ALKBH5, which were eliminated after m6A residue mutation. Strikingly, ALKBH5 deficiency or HMGB1 silencing both attenuated type I interferon production and decreased hepatocyte apoptosis. In vivo depletion of ALKBH5 abolished upregulation of HMGB1-mediated STING signaling and decreased liver inflammation, which was consistent with STING-/- mice treated with irradiation. Notably, YTHDF2, a m6A reader protein, directly bound to HMGB1 m6A-modified sites and promoted its degradation. CONCLUSIONS: ALKBH5-dependent HMGB1 expression mediates STING-IRF3 innate immune response in RILD.
PURPOSE: Radiation therapy (RT), vital for treatment of primary liver cancer, comes with unavoidable liver injury, which limits its implementation. N6-methyladenosine (m6A) methylation is involved in many molecular functions. However, its role in radiation-induced liver diseases (RILD) remains unknown. Here, we intend to investigate the role of m6A methylation in RILD. METHODS AND MATERIALS: Methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and RNA transcriptome sequencing (RNA-seq) were used to reveal the methylation pattern of human hepatic stellate cells (HSCs) exposed to irradiation. C3H/HeN mice and STING-deficient mice underwent X-ray irradiation of 24 Gy in three fractions. The m6A methylation of HMGB1 transcript was validated using MeRIP, RIP, luciferase assays and mRNA decay assay. RESULTS:Human HSCs showed significant differences in methylation patterns after 8 Gy of X-ray irradiation. Irradiation recruited AlkB Homolog 5 (ALKBH5) to demethylate m6A residues in the 3' untranslated region (UTR) of High-Mobility Group Box 1 (HMGB1), which resulted in activation of STING-IRF3 signaling. Changes in transcription of the 3'UTR of HMGB1 occurred after knockdown of ALKBH5, which were eliminated after m6A residue mutation. Strikingly, ALKBH5deficiency or HMGB1 silencing both attenuated type I interferon production and decreased hepatocyte apoptosis. In vivo depletion of ALKBH5 abolished upregulation of HMGB1-mediated STING signaling and decreased liver inflammation, which was consistent with STING-/- mice treated with irradiation. Notably, YTHDF2, a m6A reader protein, directly bound to HMGB1m6A-modified sites and promoted its degradation. CONCLUSIONS:ALKBH5-dependent HMGB1 expression mediates STING-IRF3 innate immune response in RILD.