METTL14 介导的 circORC5 的 mA 修饰通过调节 miR-30c-2-3p/AKT1S1 轴抑制胃癌进展。
METTL14-mediated mA modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.
机构信息
Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Yishan Road 600, Shanghai, China.
出版信息
Mol Cancer. 2022 Feb 14;21(1):51. doi: 10.1186/s12943-022-01521-z.
BACKGROUND
N6-methyladenosine (mA) RNA methylation and circular RNAs (circRNAs) have been shown to act vital roles in multiple malignancies including gastric cancer (GC). However, there is little knowledge about how mA modification of circRNAs contributes to GC progression.
METHODS
The association of METTL14 expression with the clinicopathological characteristics and prognosis in patients with GC was assessed by Western blot, Immunohistochemistry and public datasets. In vitro and vivo function experiments were conducted to investigate the role of METTL14 in GC. Furthermore, mA-circRNA epitranscriptomic microarray was utilized to identify METTL14-mediated mA modification of circRNAs, which were validated by methylated RNA immunoprecipitation (Me-RIP), RT-qPCR and rescue experiments in GC cells. The sponge of circORC5 with miR-30c-2-3p was confirmed by luciferase gene report and RNA immunoprecipitation assays. The expression, localization and prognosis of circORC5 in GC were evaluated by fluorescence in situ hybridization. The effects of METTL14 and (or) circORC5 on miR-30c-2-3p-mediated AKT1S1 and EIF4B were estimated by RT-qPCR and Western blot analyses.
RESULTS
We found that METTL14 was downregulated in GC tissue samples and its low expression acted as a prognostic factor of poor survival in patients with GC. Ectopic expression of METTL14 markedly repressed growth and invasion of GC cells in vitro and in vivo, whereas knockdown of METTL14 harbored the opposite effects. Mechanically, mA-circRNA epitranscriptomic microarray and Me-RIP identified circORC5 as the downstream target of METTL14. Silencing of METTL14 reduced the mA level of circORC5, but increased circORC5 expression. Moreover, circORC5 could sponge miR-30c-2-3p, and reverse METTL14-caused upregulation of miR-30c-2-3p and downregulation of AKT1S1 and EIF4B. In addition, circORC5 possessed a negative correlation with miR-30c-2-3p and indicated a poor survival in GC.
CONCLUSION
Our findings demonstrate that METTL14-mediated mA modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.
背景
N6-甲基腺苷（m6A）RNA 甲基化和环状 RNA（circRNA）已被证明在包括胃癌（GC）在内的多种恶性肿瘤中发挥重要作用。然而，关于 circRNA 的 m6A 修饰如何促进 GC 进展的知识还很少。
方法
通过 Western blot、免疫组织化学和公共数据集评估 METTL14 表达与 GC 患者临床病理特征和预后的关系。进行体外和体内功能实验以研究 METTL14 在 GC 中的作用。此外，利用 m6A-circRNA 表转录组微阵列鉴定 METTL14 介导的 circRNA 的 m6A 修饰，通过甲基化 RNA 免疫沉淀（Me-RIP）、RT-qPCR 和 GC 细胞中的挽救实验进行验证。通过荧光原位杂交评估 circORC5 在 GC 中的表达、定位和预后。通过 RT-qPCR 和 Western blot 分析评估 METTL14 和（或）circORC5 对 miR-30c-2-3p 介导的 AKT1S1 和 EIF4B 的影响。
结果
我们发现 METTL14 在 GC 组织样本中下调，其低表达是 GC 患者预后不良的预后因素。METTL14 的异位表达显著抑制了 GC 细胞的体外和体内生长和侵袭，而 METTL14 的敲低则产生了相反的效果。机制上，m6A-circRNA 表转录组微阵列和 Me-RIP 鉴定 circORC5 是 METTL14 的下游靶标。沉默 METTL14 降低了 circORC5 的 m6A 水平，但增加了 circORC5 的表达。此外，circORC5 可以海绵 miR-30c-2-3p，并逆转 METTL14 引起的 miR-30c-2-3p 上调和 AKT1S1 和 EIF4B 下调。此外，circORC5 与 miR-30c-2-3p 呈负相关，并且在 GC 中预示着不良的预后。
结论
我们的研究结果表明，METTL14 介导的 circORC5 的 m6A 修饰通过调节 miR-30c-2-3p/AKT1S1 轴抑制胃癌的进展。
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