Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, 1277 Jiefang Avenue, Wuhan, 430022, People's Republic of China.
J Exp Clin Cancer Res. 2023 Oct 16;42(1):267. doi: 10.1186/s13046-023-02847-2.
Long non-coding RNAs (LncRNAs) have been extensively studied to play essential roles in tumor progression. However, more in-depth studies are waiting to be solved on how lncRNAs regulate the progression of hepatocellular carcinoma (HCC).
Different expression levels of lncRNAs in HCC cells were compared by analysis of Gene Expression Omnibus and The Cancer Genome Atlas databases. The effects of lncRNA FTO Intronic Transcript 1 (FTO-IT1) on HCC cells were assessed by gain- and loss-of-function experiments. Colony formation assay, Edu assay, glucose uptake and lactic acid production assay were performed to evaluate the regulation of proliferation and glycolysis of HCC cells by FTO-IT1. The binding between protein interleukin enhancer binding factor 2/3 (ILF2/ILF3) and FTO-IT1 was determined by RNA pull-down, mass spectroscopy and RNA immunoprecipitation experiments. RNA stability assay, quantitative reverse transcription PCR and Western blot were employed to determine the regulatory mechanisms of FTO-IT1 on fat mass and obesity-associated (FTO). Methylated RNA immunoprecipitation assay was used to assessed the regulation of key enzymes of glycolysis by FTO. The role of FTO-IT1/FTO in vivo was confirmed via xenograft tumor model.
LncRNA FTO-IT1, an intronic region transcript of FTO gene, was highly expressed in HCC and associated with poor prognosis of patients with HCC. FTO-IT1 was related to proliferation and glycolysis of HCC cells, and contributed to the malignant progression of HCC by promoting glycolysis. Mechanistically, FTO-IT1 induced stabilization of FTO mRNA by recruiting ILF2/ILF3 protein complex to 3'UTR of FTO mRNA. As a demethylase for N-methyladenosine (mA), FTO decreased mA modification on mRNAs of glycolysis associated genes including GLUT1, PKM2, and c-Myc which alleviated the YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated mRNA degradation. Therefore, the upregulated expression of FTO-IT1 leaded to overexpression of GLUT1, PKM2, and c-Myc by which enhanced glycolysis of HCC. Meanwhile, it was found that c-Myc transcriptional regulated expression of FTO-IT1 by binding to its promoter area under hypo-glucose condition, forming a reciprocal loop between c-Myc and FTO-IT1.
This study identified an important role of the FTO-IT1/FTO axis mediated mA modification of glycolytic genes contributed to glycolysis and tumorigenesis of HCC, and FTO-IT1 might be served as a new therapeutic target for HCC.
长链非编码 RNA（lncRNA）在肿瘤进展中发挥重要作用，已有广泛研究。然而，lncRNA 如何调控肝细胞癌（HCC）进展等问题仍有待深入研究。
通过基因表达综合分析数据库和癌症基因组图谱数据库分析 HCC 细胞中不同 lncRNA 的表达水平。通过 gain- 和 loss-of-function 实验评估 lncRNA FTO 内含子转录物 1（FTO-IT1）对 HCC 细胞的影响。通过集落形成实验、Edu 实验、葡萄糖摄取和乳酸生成实验评估 FTO-IT1 对 HCC 细胞增殖和糖酵解的调节作用。通过 RNA 下拉、质谱和 RNA 免疫沉淀实验确定蛋白质白细胞介素增强子结合因子 2/3（ILF2/ILF3）与 FTO-IT1 之间的结合。通过 RNA 稳定性实验、定量逆转录 PCR 和 Western blot 实验确定 FTO-IT1 对脂肪量和肥胖相关（FTO）的调节机制。通过甲基化 RNA 免疫沉淀实验评估 FTO 对糖酵解关键酶的调节作用。通过异种移植肿瘤模型证实 FTO-IT1/FTO 在体内的作用。
lncRNA FTO-IT1 是 FTO 基因的内含子转录物，在 HCC 中高表达，与 HCC 患者的预后不良相关。FTO-IT1 与 HCC 细胞的增殖和糖酵解有关，并通过促进糖酵解促进 HCC 的恶性进展。机制上，FTO-IT1 通过募集 ILF2/ILF3 蛋白复合物到 FTO mRNA 的 3'UTR，诱导 FTO mRNA 的稳定。作为 N6-甲基腺苷（m6A）的去甲基酶，FTO 降低了包括 GLUT1、PKM2 和 c-Myc 在内的糖酵解相关基因的 m6A 修饰，减轻了 YTH N6-甲基腺苷 RNA 结合蛋白 2（YTHDF2）介导的 mRNA 降解。因此，FTO-IT1 的上调表达导致 GLUT1、PKM2 和 c-Myc 的过表达，从而增强 HCC 的糖酵解。同时，研究发现 c-Myc 在低糖条件下通过结合其启动子区域转录调控 FTO-IT1 的表达，在 c-Myc 和 FTO-IT1 之间形成一个正反馈循环。
本研究确定了 FTO-IT1/FTO 轴介导的糖酵解基因 m6A 修饰在 HCC 糖酵解和肿瘤发生中的重要作用，FTO-IT1 可能成为 HCC 的新治疗靶点。
