ALKBH5 修饰的 HMGB1-STING 激活通过先天免疫反应导致放射性肝损伤。
ALKBH5-Modified HMGB1-STING Activation Contributes to Radiation Induced Liver Disease via Innate Immune Response.
机构信息
Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, China, P.R. China.
Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, China, P.R. China.
出版信息
Int J Radiat Oncol Biol Phys. 2021 Oct 1;111(2):491-501. doi: 10.1016/j.ijrobp.2021.05.115. Epub 2021 May 24.
PURPOSE
Radiation therapy, which is vital for the 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. Herein, we investigate the role of m6A methylation in RILD.
METHODS AND MATERIALS
Methylated RNA-immunoprecipitation sequencing and RNA transcriptome sequencing were used to reveal the methylation pattern of human hepatic stellate cells (HSCs) exposed to irradiation. C3H/HeN mice and stimulator of interferon genes (STING)-deficient mice underwent x-ray irradiation of 24 Gy in 3 fractions. The m6A methylation of the high-mobility group box 1 (HMGB1) transcript was validated using methylated RNA immunoprecipitation, RNA immunoprecipitation, luciferase assays, and a messenger RNA decay assay.
RESULTS
Human hepatic stellate cells 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 of HMGB1, which resulted in the activation of STING-interferon regulatory factor 3 signaling. Changes in the transcription of the 3' untranslated region of HMGB1 occurred after the 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 the upregulation of HMGB1-mediated STING signaling and decreased liver inflammation, which was consistent with STING mice treated with irradiation. Notably, YTHDF2 (m6A reader protein) directly bound to HMGB1 m6A-modified sites and promoted its degradation.
CONCLUSIONS
ALKBH5-dependent HMGB1 expression mediates STING-interferon regulatory factor 3 innate immune response in RILD.
目的
放射疗法对原发性肝癌的治疗至关重要，但会不可避免地导致肝损伤，从而限制了其应用。N6-甲基腺苷（m6A）甲基化参与了许多分子功能，但它在放射性肝损伤（RILD）中的作用尚不清楚。本研究旨在探讨 m6A 甲基化在 RILD 中的作用。
方法和材料
采用甲基化 RNA 免疫沉淀测序和 RNA 转录组测序技术，揭示了辐照下人肝星状细胞（HSCs）的甲基化模式。采用 3 次 24 Gy X 射线照射 C3H/HeN 小鼠和干扰素基因刺激因子（STING）缺陷型小鼠。采用甲基化 RNA 免疫沉淀、RNA 免疫沉淀、荧光素酶报告基因实验和信使 RNA 衰变实验验证高迁移率族蛋白 1（HMGB1）转录物的 m6A 甲基化。
结果
人肝星状细胞在接受 8 Gy X 射线照射后，其甲基化模式出现显著差异。辐照募集 AlkB 同源物 5（ALKBH5）去甲基化 HMGB1 的 3'非翻译区的 m6A 残基，导致 STING-干扰素调节因子 3 信号的激活。ALKBH5 敲低后，HMGB1 的 3'非翻译区转录发生变化，突变 m6A 残基后消除了这些变化。令人惊讶的是，ALKBH5 缺陷或 HMGB1 沉默均能抑制 I 型干扰素的产生并减少肝细胞凋亡。体内敲除 ALKBH5 可消除 HMGB1 介导的 STING 信号上调，并减少肝脏炎症，这与接受辐照的 STING 小鼠的结果一致。值得注意的是，YTHDF2（m6A 阅读蛋白）直接结合 HMGB1 m6A 修饰位点并促进其降解。
结论
ALKBH5 依赖性 HMGB1 表达介导了 RILD 中的 STING-干扰素调节因子 3 固有免疫反应。
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