Emerging evidence indicates that circular RNAs (circRNAs) and m6A RNA methylation participate in the pathogenesis and metastasis of multiple malignancies including hepatocellular carcinoma (HCC). However, it remains undocumented how circRNAs form a feedback loop with the m6A modification contributing to HCC. A novel hsa_circ_0017114 (circGPR137B) was identified from three pairs of primary HCC and adjacent normal tissues by circRNA expression profiling. The association of circGPR137B and miR-4739 with clinicopathological parameters and prognosis in patients with HCC was analyzed by RT-qPCR, fluorescence in situ hybridization and TCGA cohorts. The role of circGPR137B in HCC was estimated in vitro and in vivo. RT-qPCR, western blot, m6A dot blot, RIP, MeRIP and dual-luciferase reporter assays were used to validate the reciprocal regulation of the feedback loop among circGPR137B, miR-4739 and m6A demethylase FTO. Meanwhile, the expression, function and prognosis of FTO in HCC were investigated by RT-qPCR, western blot, TCGA and rescue experiments. We identified a new dramatically downregulated circGPR137B in HCC tissues, and found that downregulation of circGPR137B or upregulation of miR-4739 was associated with poor prognosis in patients with HCC. Ectopic expression of circGPR137B strikingly repressed the proliferation, colony formation and invasion, whereas knockdown of circGPR137B harbored the opposite effects. Moreover, restored expression of circGPR137B inhibited tumor growth and lung metastasis in vivo. Further investigations showed that circGPR137B, co-localized with miR-4739 in the cytoplasm, acted as a sponge for miR-4739 to upregulate its target FTO, which mediated m6A demethylation of circGPR137B and promoted its expression. Thus, a feedback loop comprising circGPR137B/miR-4739/FTO axis was formed. FTO suppressed cell growth and indicated favorable survival in patients with HCC. Our results demonstrate that circGPR137B inhibits HCC tumorigenesis and metastasis through the circGPR137B/miR-4739/FTO feedback loop. This positive feedback mechanism executed by functional coupling between a circRNA sponge and an m6A modification event suggests a model for epigenetics.

中文翻译：

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CircGPR137B/miR-4739/FTO反馈回路抑制肝细胞癌的发生和转移

新出现的证据表明环状 RNA (circRNA) 和 m6A RNA 甲基化参与了包括肝细胞癌 (HCC) 在内的多种恶性肿瘤的发病机制和转移。然而，关于 circRNAs 如何与 m6A 修饰形成反馈回路导致 HCC 尚无文献记载。通过 circRNA 表达谱从三对原发性 HCC 和邻近的正常组织中鉴定出一种新的 hsa_circ_0017114 (circGPR137B)。通过 RT-qPCR、荧光原位杂交和 TCGA 队列分析 circGPR137B 和 miR-4739 与 HCC 患者临床病理参数和预后的关系。在体外和体内估计了 circGPR137B 在 HCC 中的作用。RT-qPCR、蛋白质印迹、m6A 斑点印迹、RIP、MeRIP 和双荧光素酶报告基因检测用于验证 circGPR137B、miR-4739 和 m6A 去甲基化酶 FTO 之间反馈回路的相互调节。同时通过RT-qPCR、western blot、TCGA和抢救实验研究FTO在HCC中的表达、功能及预后。我们在 HCC 组织中发现了一种新的显着下调的 circGPR137B，并发现 circGPR137B 的下调或 miR-4739 的上调与 HCC 患者的不良预后相关。circGPR137B 的异位表达显着抑制了增殖、集落形成和侵袭，而 circGPR137B 的敲低则具有相反的效果。此外，circGPR137B 的恢复表达抑制了体内肿瘤生长和肺转移。进一步的研究表明，circGPR137B，与细胞质中的 miR-4739 共定位，充当 miR-4739 的海绵上调其靶 FTO，从而介导 circGPR137B 的 m6A 去甲基化并促进其表达。因此，形成了一个包含circGPR137B/miR-4739/FTO轴的反馈回路。FTO 抑制了细胞生长，并表明 HCC 患者的存活率良好。我们的研究结果表明，circGPR137B 通过 circGPR137B/miR-4739/FTO 反馈回路抑制 HCC 肿瘤发生和转移。这种通过 circRNA 海绵和 m6A 修饰事件之间的功能耦合执行的正反馈机制为表观遗传学提供了一个模型。FTO 抑制了细胞生长，并表明 HCC 患者的存活率良好。我们的研究结果表明，circGPR137B 通过 circGPR137B/miR-4739/FTO 反馈回路抑制 HCC 肿瘤发生和转移。这种通过 circRNA 海绵和 m6A 修饰事件之间的功能耦合执行的正反馈机制为表观遗传学提供了一个模型。FTO 抑制了细胞生长，并表明 HCC 患者的存活率良好。我们的研究结果表明，circGPR137B 通过 circGPR137B/miR-4739/FTO 反馈回路抑制 HCC 肿瘤发生和转移。这种通过 circRNA 海绵和 m6A 修饰事件之间的功能耦合执行的正反馈机制为表观遗传学提供了一个模型。