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. RIP-seq were applied to identify YTHDF3 targets in mouse with or without CCL4 administration

研究背景与研究目的：由慢性肝损伤诱导的肝细胞癌（Hepatocellular carcinoma, HCC）是癌症相关死亡的主要诱因，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）识别蛋白YTHDF3在人类疾病中的作用仍有待明确。
研究方法：通过化学诱导构建小鼠肝损伤与肝细胞癌模型。利用CRISPR/Cas9技术构建Ythdf3与Mettl14基因敲除小鼠。采用10X单细胞RNA测序与流式细胞术分析肝细胞群体特征。通过免疫荧光、免疫组化及蛋白质免疫印迹评估细胞增殖与DNA损伤水平。培养肝脏类器官以检测肝脏干细胞功能。采用MeRIP-seq揭示化学诱导肝损伤后m6A甲基化图谱的变化。通过RNA免疫沉淀测序（RIP-seq）与核糖体印记测序（Ribo-seq）鉴定YTHDF3的靶基因并确定其翻译效率。使用小干扰RNA与dCas13b-FTO-sgRNA质粒在体外评估YTHDF3与CEBPA的功能。
研究结果：YTHDF3缺失会加重化学诱导的肝损伤，伴随功能性肝细胞与干细胞数量减少。此外，依赖METTL14与YTHDF3的RNA m6A调控异常可诱发DNA损伤并促进肝细胞癌的发生发展。机制层面，Ythdf3基因敲除会阻碍CCAAT增强子结合蛋白α（CCAAT/enhancer-binding protein-alpha, CEBPA）的翻译，进而抑制多聚ADP核糖聚合酶1（PARP1）与过氧化物还原酶2（PRDX2）的表达，以促进DNA损伤并诱导基因组不稳定，最终引发肝损伤与肝细胞癌。
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，从而预防肝损伤与肝细胞癌，为靶向YTHDF3与CEBPA治疗肝细胞癌提供了潜在的治疗策略。本研究通过RIP-seq鉴定了经或未经四氯化碳（CCL4）处理的小鼠体内YTHDF3的靶基因。