N6-Methyladenosine Reader YTHDF3-Mediated CEBPA Translation Maintains Genomic Stability and Stem Cells Function to Prevent Liver Injury and Hepatocellular Carcinoma [RIP-seq]. N6-Methyladenosine Reader YTHDF3-Mediated CEBPA Translation Maintains Genomic Stability and Stem Cells Function to Prevent Liver Injury and Hepatocellular Carcinoma [RIP-seq]

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) induced by chronic liver damage is a major cause of cancer mortality, but its precise epigenetic mechanisms are severely under studied. In addition, the role of N6-methyladenine (m6A) reader YTHDF3 in human diseases remains poorly understood. METHODS: Liver injury and hepatocarcinogenesis in mice were induced by chemical. CRISPR/Cas9 technology was used to construct Ythdf3 and Mettl14 knockout mice. Hepatic cell population characteristics was determined by means of 10X single-cell RNA-seq and flow cytometry. Cell proliferation and DNA damage were evaluated by immunofluorescence, immunohistochemistry, and western blot. Liver organoids were cultured to examine liver stem cells function. MeRIP-seq was used to reveal alterations in m6A methylation patterns impacted by chemical-induced liver injury. RIP-seq and Ribo-seq were applied to identify YTHDF3 targets and determine translation efficiency. Small interfering RNAs and dCas13b-FTO-sgRNA plasmids were used to evaluate the function of YTHDF3 and CEBPA in vitro. RESULTS: YTHDF3 depletion exacerbated chemical-induced liver injury with a reduction in functional hepatocytes and stem cells. Furthermore, METTL14 and YTHDF3-dependent RNA m6A dysregulation induced DNA damage and promoted development of HCC. Mechanistically, knockout of Ythdf3 impeded the translation of CCAAT/enhancer-binding protein-alpha (CEBPA), subsequently inhibited expression of PARP1 and PRDX2 to promote DNA damage and induce genomic instability, finally leading to liver injury and HCC. CONCLUSIONS: m6A/YTHDF3/CEBPA regulatory axis plays an essential role in governing cell fates and genomic stability, thereby preventing liver injury and HCC, and offers potential therapeutic avenue for targeting YTHDF3 and CEBPA in the treatment of HCC. Overall design: RIP-seq were applied to identify YTHDF3 targets in mouse with or without CCL4 administration

研究背景与研究目的：由慢性肝损伤诱发的肝细胞癌（hepatocellular carcinoma, HCC）是癌症相关死亡的主要诱因之一，但其精确的表观遗传机制尚未得到充分研究。此外，N6-甲基腺嘌呤（N6-methyladenine, m6A）阅读蛋白YTHDF3在人类疾病中的功能仍有待阐明。
研究方法：通过化学物质诱导小鼠发生肝损伤及肝细胞癌变。利用CRISPR/Cas9技术构建Ythdf3与Mettl14基因敲除小鼠。采用10X单细胞RNA测序（10X single-cell RNA-seq）及流式细胞术分析肝细胞群体的特征。通过免疫荧光、免疫组织化学及蛋白质印迹法评估细胞增殖与DNA损伤水平。构建肝类器官以检测肝干细胞的功能。使用MeRIP-seq揭示化学物质诱导肝损伤后m6A甲基化修饰模式的变化。通过RIP-seq与Ribo-seq鉴定YTHDF3的靶标基因，并测定其翻译效率。体外实验中，采用小干扰RNA及dCas13b-FTO-sgRNA质粒验证YTHDF3与CEBPA的生物学功能。
研究结果：敲除YTHDF3会加重化学物质诱导的肝损伤，同时伴随功能性肝细胞与干细胞数量的减少。此外，依赖METTL14与YTHDF3的RNA m6A修饰失调可诱导DNA损伤，并促进肝细胞癌的发生发展。机制层面，敲除Ythdf3会抑制CCAAT/增强子结合蛋白α（CCAAT/enhancer-binding protein-alpha, CEBPA）的翻译过程，进而下调PARP1与PRDX2的表达，最终促进DNA损伤并引发基因组不稳定，最终导致肝损伤及肝细胞癌。
研究结论：m6A/YTHDF3/CEBPA调控轴在调控细胞命运与基因组稳定性中发挥关键作用，可预防肝损伤及肝细胞癌的发生，为以YTHDF3与CEBPA为靶点的肝细胞癌治疗提供了潜在的治疗策略。
实验整体设计：本研究通过RIP-seq鉴定经或未经四氯化碳（CCL4）处理的小鼠体内YTHDF3的靶标基因。