The aim of this study was to elucidate the mechanism of action of the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) on the phenotype of the liver cancer stem cells (LCSCs). To gain insight into the mechanism of action of the IGF2BP1 on LCSCs, the IGF2BP1 shRNA sequences were transfected into hepatocellular carcinoma (HCC) cells. The LCSC phenotypes were measured by stemness gene expressions, spheroid formations, percentages of the CD133⁺ cells, colony formations, and tumorigenesis in vivo. Next, we screened for possible molecular mechanisms from the Cancer Genome Atlas (TCGA) database, and a methylated RNA immunoprecipitation-quantitative polymerase chain reaction (MeRIP-qPCR) was used to adjust the binding of IGF2BP1 to the target gene, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase (MGAT5). The MeRIP-qPCR was used to detect the binding of IGF2BP1 and MGAT5 through N⁶ methyladenosine (m6A) modification. Furthermore, we adjusted the attenuation of the mRNA of the MGAT5 using quantitative real-time PCR (qRT-PCR). The IGF2BP1 was upregulated in the LCSCs. Furthermore, the IGF2BP1 promoted self-renewal and chemoresistance in human LCSCs and tumorigenesis in mice and it enhanced the expression of stemness genes in the LCSCs compared with the HCC cells. Further exploration indicated that the IGF2BP1 binds directly to the MGAT5 and inhibits its mRNA attenuation, suggesting that the IGF2BP1 impacts MGAT5 mRNA stability through m6A modification. Thus, it can be concluded that the IGF2BP1 facilitated the LCSC phenotypes by promoting the MGAT5 mRNA stability through the upregulation of m6A modification of the MGAT5 mRNA.

中文翻译：

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IGF2BP1 通过 m6A RNA 甲基化调节 MGAT5 mRNA 稳定性促进肝癌干细胞表型

本研究旨在阐明胰岛素样生长因子 2 mRNA 结合蛋白 1 ( IGF2BP1 ) 对肝癌干细胞 (LCSCs) 表型的作用机制。为了深入了解IGF2BP1对 LCSCs 的作用机制，将IGF2BP1 shRNA 序列转染到肝细胞癌 (HCC) 细胞中。通过干性基因表达、球体形成、CD133 ⁺细胞的百分比、集落形成和体内肿瘤发生来测量 LCSC 表型。接下来，我们从癌症基因组图谱 (TCGA) 数据库中筛选可能的分子机制，并使用甲基化 RNA 免疫沉淀-定量聚合酶链反应 (MeRIP-qPCR) 来调整与IGF2BP1到靶基因，α-1,6-甘露糖基糖蛋白 6-β-N-乙酰氨基葡萄糖转移酶 ( MGAT5 )。MeRIP-qPCR 用于检测IGF2BP1和MGAT5通过 N ⁶甲基腺苷 (m6A) 修饰的结合。此外，我们使用定量实时 PCR (qRT-PCR)调整了MGAT5 mRNA 的衰减。IGF2BP1在 LCSCs 中上调。此外，与 HCC 细胞相比， IGF2BP1促进了人类 LCSCs 的自我更新和化学抗性以及小鼠的肿瘤发生，并且它增强了 LCSCs 中干性基因的表达。进一步的探索表明IGF2BP1直接与MGAT5结合并抑制其 mRNA 衰减，表明IGF2BP1通过 m6A 修饰影响MGAT5 mRNA 稳定性。因此，可以得出结论，IGF2BP1通过上调MGAT5 mRNA 的 m6A 修饰来促进MGAT5 mRNA 稳定性，从而促进 LCSC 表型。