Department of General Surgery, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, School of Medicine, South China University of Technology, Guangzhou, 510080, China.
Shantou University of Medical College, Shantou, 515000, Guangdong Province, China.
Oncogene. 2020 Jun;39(23):4507-4518. doi: 10.1038/s41388-020-1303-7. Epub 2020 May 4.
N-methyladenosine (m6A) RNA methylation contributes to the cancer stem cell (CSC) phenotype through regulating gene expression. YTHDF2, an m6A reader, was shown to be associated with hepatocellular carcinoma (HCC) patient prognosis. However, the effect of YTHDF2 on liver CSC and cancer metastasis and the molecular mechanism of this effect have not been documented. Here, we show that YTHDF2 expression is negatively correlated with HCC patient survival in both data from the Cancer Genome Atlas (TCGA) database and clinical data from our center. By detecting CD133 cells and carrying out sphere culture assays, we found that knockdown of YTHDF2 led to impaired stemness in Hep3B and Huh7 cells. In contrast, overexpression of YTHDF2 increased the CSC phenotype. Mechanistically, the knockdown and overexpression of YTHDF2 in liver cancer cells resulted in decreased and increased m6A levels in the 5'-untranslated region (UTR) of OCT4 mRNA, respectively, leading to decreased and increased OCT4 protein expression, respectively. A luciferase activity assay showed that mutation of the corresponding m6A methylation sequence in the 5'-UTR of OCT4 mRNA caused significantly decreased gene expression, suggesting a role for YTHDF2-dependent m6A methylation in protein translation. Polysome profiling results also indicated the knockdown and overexpression of YTHDF2 could decrease and increase OCT4 translation, respectively. In particular, overexpression of OCT4 rescued the impaired stemness caused by YTHDF2 depletion, which confirmed the effect of YTHDF2 on CSC phenotype is dependent on OCT4. In vivo, the loss of YTHDF2 reduced tumor burden and inhibited lung metastasis following orthotopic transplantation in nude mice. Last, we demonstrated that YTHDF2 expression is positively correlated with OCT4 expression and m6A levels in the 5'-UTR of OCT4 mRNA in clinical HCC specimens. In conclusion, YTHDF2 promotes the CSC liver phenotype and cancer metastasis by modulating the m6A methylation of OCT4 mRNA.
N6-甲基腺苷（m6A）RNA 甲基化通过调节基因表达，有助于癌症干细胞（CSC）表型。YTHDF2 是一种 m6A 读码器，与肝细胞癌（HCC）患者的预后相关。然而，YTHDF2 对肝 CSC 和癌症转移的影响及其分子机制尚未有文献记录。在这里，我们显示 YTHDF2 的表达与 TCGA 数据库中的 HCC 患者生存时间和我们中心的临床数据呈负相关。通过检测 CD133 细胞并进行球体培养实验，我们发现 YTHDF2 的敲低导致 Hep3B 和 Huh7 细胞的干性受损。相反，YTHDF2 的过表达增加了 CSC 表型。在机制上，肝癌细胞中 YTHDF2 的敲低和过表达分别导致 OCT4 mRNA 5'-非翻译区（UTR）的 m6A 水平降低和增加，导致 OCT4 蛋白表达降低和增加。荧光素酶活性测定表明，OCT4 mRNA 5'-UTR 中相应 m6A 甲基化序列的突变导致基因表达显著降低，表明 YTHDF2 依赖性 m6A 甲基化在蛋白翻译中起作用。多核糖体谱分析结果也表明，YTHDF2 的敲低和过表达分别可以降低和增加 OCT4 的翻译。特别是，OCT4 的过表达挽救了 YTHDF2 耗竭引起的干性受损，这证实了 YTHDF2 对 CSC 表型的影响依赖于 OCT4。在体内，裸鼠原位移植后，YTHDF2 的缺失减少了肿瘤负担并抑制了肺转移。最后，我们证明 YTHDF2 的表达与临床 HCC 标本中 OCT4 mRNA 5'-UTR 的 OCT4 表达和 m6A 水平呈正相关。总之，YTHDF2 通过调节 OCT4 mRNA 的 m6A 甲基化促进 CSC 肝表型和癌症转移。
