The unique expression pattern endows oncofetal genes with great value in cancer diagnosis and treatment. However, only a few oncofetal genes are available for clinical use and the underlying mechanisms that drives the fetal-like reprogramming of cancer cells remain largely unknown. Microarray assays and bioinformatic analyses were employed to screen for potential oncofetal long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC). The expression levels of MIR4435-2HG, NOP58 ribonucleoprotein (NOP58), insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) and stem markers were detected by quantitative polymerase chain reaction. The 2′-O-methylation (2′-O-Me) status of rRNA were detected through reverse transcription at low dNTP concentrations followed by PCR. The regulation of MIR4435-2HG by IGF2BP1 was explored by RNA immunoprecipitation (RIP), methylated RIP (MeRIP) and dual-luciferase assays. The interaction between MIR4435-2HG and NOP58 was investigated by RNA Pulldown, RIP and protein stability assays. In vitro and in vivo function assays were performed to detect the roles of MIR4435-2HG/NOP58 in HCC. MIR4435-2HG was an oncofetal lncRNA associated with poor prognosis in HCC. Functional experiments showed that overexpression of MIR4435-2HG remarkably enhanced the stem-cell properties of HCC cells, promoting tumorigenesis in vitro and in vivo. Mechanically, MIR4435-2HG directly bound NOP58 and IGF2BP1. IGF2BP1 upregulated MIR4435-2HG expression in HCC through N6-methyladenosine (m6A) modification. Moreover, MIR4435-2HG protected NOP58 from degradation, which raised rRNA 2’-O-Me levels and promoted internal ribosome entry site (IRES)-dependent translation of oncogenes. This study identified an oncofetal lncRNA MIR4435-2HG, characterized the role of MIR4435-2HG/NOP58 in stemness maintenance and proliferation of HCC cells, and confirmed m6A as a ‘driver’ that reactivated MR4435-2HG expression in HCC.

中文翻译：

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N6-甲基腺苷修饰的癌胎lncRNA MIR4435-2HG通过调节rRNA 2'-O甲基化促进肝细胞癌细胞的干性特征

独特的表达模式赋予癌胎基因在癌症诊断和治疗中具有重要价值。然而，只有少数癌胎儿基因可用于临床，并且驱动癌细胞胎儿样重编程的潜在机制仍然很大程度上未知。采用微阵列分析和生物信息学分析来筛选肝细胞癌 (HCC) 中潜在的癌胚长非编码 RNA (lncRNA)。采用定量聚合酶链式反应检测MIR4435-2HG、NOP58核糖核蛋白（NOP58）、胰岛素样生长因子2 mRNA结合蛋白1（IGF2BP1）和茎标志物的表达水平。rRNA 的 2'-O-甲基化 (2'-O-Me) 状态通过低 dNTP 浓度下的逆转录和 PCR 检测。通过 RNA 免疫沉淀 (RIP)、甲基化 RIP (MeRIP) 和双荧光素酶测定探索 IGF2BP1 对 MIR4435-2HG 的调节。通过 RNA Pulldown、RIP 和蛋白质稳定性测定研究了 MIR4435-2HG 和 NOP58 之间的相互作用。进行体外和体内功能测定来检测MIR4435-2HG/NOP58在HCC中的作用。MIR4435-2HG 是一种与 HCC 不良预后相关的癌胎儿 lncRNA。功能实验表明，MIR4435-2HG 的过表达显着增强了 HCC 细胞的干细胞特性，促进体外和体内肿瘤发生。从机械角度来看，MIR4435-2HG 直接结合 NOP58 和 IGF2BP1。IGF2BP1 通过 N6-甲基腺苷 (m6A) 修饰上调 HCC 中 MIR4435-2HG 的表达。此外，MIR4435-2HG 保护 NOP58 免于降解，从而提高 rRNA 2'-O-Me 水平并促进癌基因的内部核糖体进入位点 (IRES) 依赖性翻译。这项研究鉴定了一种癌胎儿 lncRNA MIR4435-2HG，表征了 MIR4435-2HG/NOP58 在 HCC 细胞干性维持和增殖中的作用，并证实 m6A 是重新激活 HCC 中 MR4435-2HG 表达的“驱动程序”。