MOE Joint International Research Laboratory of Animal Health & Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China; Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China.
Division of Nutritional Sciences, Cornell University, Ithaca, New York.
Cell Mol Gastroenterol Hepatol. 2021;12(3):839-856. doi: 10.1016/j.jcmgh.2021.05.007. Epub 2021 May 13.
Transforming growth factor β1 (TGF-β1) secreted from activated Kupffer cells (KC) promotes the progression of nonalcoholic steatohepatitis (NASH) to liver fibrosis. N6-methyladenosine (mA) RNA modification participates in various cell stress responses, yet it remains unknown whether it plays a role in TGF-β1 upregulation in activated KCs.
Western blot, dot blot, and liquid chromatography with tandem mass spectrometry were used to determine the expression of mA methyltransferase, METTL3, and METTL14, as well as global mA modification. RNA-sequencing and mA-seq were employed to screen differentially expressed genes and responsive mA peaks. Nuclear factor κB (NF-κB)-mediated METTL3/METTL14 transactivation were validated with chromatin immunoprecipitation polymerase chain reaction and dual-luciferase reporter system, and the role of mA in TGF-β1 upregulation was further verified in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice.
Serum lipopolysaccharide (LPS) concentration is increased in high-fat diet-induced NASH rats. TGF-β1 upregulation is closely associated with METTL3/METTL14 upregulation and global mA hypermethylation, in both NASH rat liver and LPS-activated KCs. LPS-responsive mA peaks are identified on the 5' untranslated region (UTR) of TGF-β1 messenger RNA (mRNA). NF-κB directly transactivates METTL3 and METTL14 genes. LPS-stimulated TGF-β1 expression is abolished in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. Mutation of mA sites on the 5'UTR of TGF-β1 mRNA blocks LPS-induced increase of luciferase reporter activity.
NF-κB acts as transcription factor to transactivate METTL3/METTL14 genes upon LPS challenge, leading to global RNA mA hypermethylation. Increased mA on the 5'UTR of TGF-β1 mRNA results in mA-dependent translation of TGF-β1 mRNA in a cap-independent manner. We identify a novel role of mA modification in TGF-β1 upregulation, which helps to shed light on the molecular mechanism of NASH progression.
活化的枯否细胞（Kupffer cells，KC）分泌的转化生长因子 β1（TGF-β1）促进非酒精性脂肪性肝炎（NASH）向肝纤维化进展。N6-甲基腺苷（m6A）RNA 修饰参与各种细胞应激反应，但尚不清楚其是否在活化的 KC 中 TGF-β1 的上调中发挥作用。
使用 Western blot、点印迹和液相色谱-串联质谱法测定 m6A 甲基转移酶 METTL3 和 METTL14 的表达以及全局 m6A 修饰。使用 RNA 测序和 m6A 测序筛选差异表达基因和响应 m6A 峰。核因子 κB（NF-κB）介导的 METTL3/METTL14 反式激活通过染色质免疫沉淀聚合酶链反应和双荧光素酶报告系统进行验证，并在 METTL3/METTL14 缺陷型 KC 和骨髓谱系细胞特异性 METTL14 敲除小鼠中进一步验证 m6A 在 TGF-β1 上调中的作用。
高脂饮食诱导的 NASH 大鼠血清脂多糖（LPS）浓度升高。TGF-β1 的上调与 NASH 大鼠肝和 LPS 激活的 KC 中 METTL3/METTL14 的上调和全局 m6A 高甲基化密切相关。TGF-β1 信使 RNA（mRNA）的 5'非翻译区（UTR）上鉴定出 LPS 响应的 m6A 峰。NF-κB 直接反式激活 METTL3 和 METTL14 基因。在 METTL3/METTL14 缺陷型 KC 和骨髓谱系细胞特异性 METTL14 敲除小鼠中，LPS 刺激的 TGF-β1 表达被消除。TGF-β1 mRNA 5'UTR 上 m6A 位点的突变阻止了 LPS 诱导的荧光素酶报告活性的增加。
NF-κB 作为转录因子，在 LPS 刺激下反式激活 METTL3/METTL14 基因，导致全局 RNA m6A 高甲基化。TGF-β1 mRNA 5'UTR 上增加的 m6A 以帽非依赖性方式导致 TGF-β1 mRNA 的 m6A 依赖性翻译。我们发现了 m6A 修饰在 TGF-β1 上调中的新作用，这有助于揭示 NASH 进展的分子机制。
