While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

中文翻译：

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METTL16通过控制核糖体生物发生和mRNA翻译促进肝癌干细胞自我更新

虽然肝癌干细胞 (CSC) 在肝细胞癌 (HCC) 的发生、进展、复发和治疗耐药中发挥着至关重要的作用，但肝脏 CSC 自我更新的机制仍然难以捉摸。我们的目标是表征甲基转移酶 16 (METTL16)（一种最近鉴定的 RNA N6-甲基腺苷 (m6A) 甲基转移酶）在 HCC 发展/维持、CSC 干性以及正常肝发生中的作用。生成肝脏特异性 Mettl16 条件 KO (cKO) 小鼠以评估其在 HCC 发病机制和正常肝发生中的作用。利用水动力尾静脉注射 (HDTVi) 诱导的从头肝癌发生和异种移植模型来确定 METTL16 在 HCC 发生和进展中的作用。利用有限稀释测定来评估 CSC 频率。通过对多组学数据的综合分析，包括 RNA-seq、RNA 免疫沉淀 (RIP)-seq 和核糖体分析，揭示了功能上重要的靶点。METTL16 在肝脏 CSC 中高表达，其消耗显着降低体外和体内 CSC 频率。Mettl16 KO 显着减弱 HCC 的发生和进展，但仅轻微影响正常肝发生。包括高通量测序在内的机制研究揭示了 METTL16 作为核糖体 RNA (rRNA) 成熟和 mRNA 翻译的关键调节因子，并确定真核翻译起始因子 3 亚基 a (eIF3a) 转录物是 METTL16 在 HCC 中的真正靶标。此外，通过CRISPR基因平铺扫描揭示了METTL16的功能必需区域，这将为潜在抑制剂的开发铺平道路。我们的研究结果强调了 METTL16 在促进 HCC 发病机制和通过提高 mRNA 翻译效率来增强肝脏 CSC 自我更新方面的关键致癌作用。