Mettl14 KO and WT mouse single cell RNA sequencing

Without intervention, a considerable proportion of metabolic fatty liver disease (MAFLD) patients will progress from simple steatosis to metabolic steatohepatitis, liver fibrosis and even hepatocellular carcinoma. However, the molecular mechanisms that control progressive MAFLD have yet to be fully determined. Here, the N6-methyladenosine (m6A) methyltransferase METTL14 hepatocyte conditional knockout mouse model was created. In vivo and in vitro experiments were performed to identify the mechanism of MAFLD progression caused by low METTL14 expression in hepatocytes. In details, METTL14 downregulation decreased GLS2 by affecting the translation efficiency mediated by YTHDF1 in a m6A-depedent manner, which might help to form an oxidative stress microenvironment. Considering that the oxidative stress microenvironment of liver tissue is the basic feature of MAFLD-related liver tissue and the key factor in fostering the progression of inflammation, non-parenchymal cells of one KO and one WT mouse liver were used for single cell RNA sequencing (scRNA-seq). It was further revealed that METTL14/GLS2 downregulation recruited Cx3cr1+Ccr2+ monocyte-derived macrophages (Mo-macs). Further experiments revealed that CX3CR1 can activate the transcription of S100A4 via CX3CR1/MyD88/NF-κB signaling pathway in Cx3cr1+Ccr2+ Mo-macs. Restoration of METTL14 or GLS2, or blocking the above pathway such as inhibiting MyD88 could ameliorate liver injuries and fibrosis. Taken together, these findings indicate potential therapies for the treatment of MAFLD progression.

若无干预措施，相当比例的代谢相关脂肪性肝病(metabolic fatty liver disease, MAFLD)患者会从单纯性脂肪变性进展为代谢性脂肪性肝炎、肝纤维化，甚至肝细胞癌。然而，调控MAFLD进展的分子机制尚未完全阐明。本研究构建了肝细胞条件性敲除N6-甲基腺嘌呤(m6A)甲基转移酶METTL14的小鼠模型，通过体内外实验探究了肝细胞中METTL14低表达诱发MAFLD进展的分子机制。具体而言，METTL14表达下调通过以m6A依赖的方式影响YTHDF1介导的翻译效率，降低了GLS2的表达，这可能促进氧化应激微环境的形成。鉴于肝脏组织的氧化应激微环境是MAFLD相关肝脏组织的核心特征，也是推动炎症进展的关键因素，本研究分别对1只敲除小鼠和1只野生型小鼠的肝脏非实质细胞进行了单细胞RNA测序(single cell RNA sequencing, scRNA-seq)，进一步分析发现，METTL14/GLS2表达下调招募了Cx3cr1+ Ccr2+单核细胞源性巨噬细胞(monocyte-derived macrophages, Mo-macs)。后续实验证实，在Cx3cr1+ Ccr2+ Mo-macs中，CX3CR1可通过CX3CR1/MyD88/NF-κB信号通路激活S100A4的转录。恢复METTL14或GLS2的表达，或是阻断上述通路（如抑制MyD88），均可改善肝脏损伤与肝纤维化。综上，本研究结果为MAFLD进展的临床治疗提供了潜在的干预策略与治疗靶点。