环状 RNA circNRIP1 通过 AKT1/mTOR 通路作为 microRNA-149-5p 的海绵促进胃癌进展。
Circular RNA circNRIP1 acts as a microRNA-149-5p sponge to promote gastric cancer progression via the AKT1/mTOR pathway.
机构信息
Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, No.300, Guangzhou Road, Nanjing, Jiangsu Province, China.
Department of General Surgery, The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University, Huaian, 223300, Jiangsu, China.
出版信息
Mol Cancer. 2019 Feb 4;18(1):20. doi: 10.1186/s12943-018-0935-5.
BACKGROUND
CircRNA has emerged as a new non-coding RNA that plays crucial roles in tumour initiation and development. 'MiRNA sponge' is the most reported role played by circRNAs in many tumours. The AKT/mTOR axis is a classic signalling pathway in cancers that sustains energy homeostasis through energy production activities, such as the Warburg effect, and blocks catabolic activities, such as autophagy. Additionally, the AKT/mTOR axis exerts a positive effect on EMT, which promotes tumour metastasis.
METHODS
We detected higher circNRIP1 expression in gastric cancer by performing RNA-seq analysis. We verified the tumour promotor role of circNRIP1 in gastric cancer cells through a series of biological function assays. We then used a pull-down assay and dual-luciferase reporter assay to identify the downstream miR-149-5p of circNRIP1. Western blot analysis and immunofluorescence assays were performed to demonstrate that the circNRIP1-miR-149-5p-AKT1/mTOR axis is responsible for the altered metabolism in GC cells and promotes GC development. We then adopted a co-culture system to trace circNRIP1 transmission via exosomal communication and RIP experiments to determine that quaking regulates circNRIP1 expression. Finally, we confirmed the tumour suppressor role of microRNA-133a-3p in vivo in PDX mouse models.
RESULTS
We discovered that knockdown of circNRIP1 successfully blocked proliferation, migration, invasion and the expression level of AKT1 in GC cells. MiR-149-5p inhibition phenocopied the overexpression of circNRIP1 in GC cells, and overexpression of miR-149-5p blocked the malignant behaviours of circNRIP1. Moreover, it was proven that circNRIP1 can be transmitted by exosomal communication between GC cells, and exosomal circNRIP1 promoted tumour metastasis in vivo. We also demonstrated that quaking can promote circNRIP1 transcription. In the final step, the tumour promotor role of circNRIP1 was verified in PDX models.
CONCLUSIONS
We proved that circNRIP1 sponges miR-149-5p to affect the expression level of AKT1 and eventually acts as a tumour promotor in GC.
背景
CircRNA 作为一种新的非编码 RNA，在肿瘤的发生和发展中起着至关重要的作用。“miRNA 海绵”是 circRNA 在许多肿瘤中最常报道的作用。AKT/mTOR 轴是癌症中的经典信号通路，通过能量产生活动（如瓦博格效应）维持能量稳态，并阻止分解代谢活动（如自噬）。此外，AKT/mTOR 轴对 EMT 有正向作用，促进肿瘤转移。
方法
我们通过 RNA-seq 分析发现胃癌中 circNRIP1 的表达升高。我们通过一系列生物学功能测定验证了 circNRIP1 在胃癌细胞中的肿瘤促进作用。然后，我们使用下拉测定和双荧光素酶报告测定来鉴定 circNRIP1 的下游 miR-149-5p。Western blot 分析和免疫荧光测定表明，circNRIP1-miR-149-5p-AKT1/mTOR 轴负责改变 GC 细胞中的代谢，并促进 GC 发展。然后，我们采用共培养系统通过外泌体通讯追踪 circNRIP1 的传递，并通过 RIP 实验确定 quaking 调节 circNRIP1 的表达。最后，我们在 PDX 小鼠模型中证实了 microRNA-133a-3p 的肿瘤抑制作用。
结果
我们发现，circNRIP1 的敲低成功地阻断了 GC 细胞的增殖、迁移、侵袭和 AKT1 的表达水平。miR-149-5p 的抑制可模拟 circNRIP1 在 GC 细胞中的过表达，而过表达 miR-149-5p 则阻断了 circNRIP1 的恶性行为。此外，证明 circNRIP1 可以通过 GC 细胞之间的外泌体通讯传递，外泌体 circNRIP1 促进体内肿瘤转移。我们还证明 quaking 可以促进 circNRIP1 的转录。在最后一步中，在 PDX 模型中验证了 circNRIP1 的肿瘤促进作用。
结论
我们证明 circNRIP1 作为 miRNA-149-5p 的海绵，影响 AKT1 的表达水平，最终在 GC 中发挥肿瘤促进作用。
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