Abstract
BackgroundCircRNA 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.MethodsWe 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.ResultsWe 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.ConclusionsWe proved that circNRIP1 sponges miR-149-5p to affect the expression level of AKT1 and eventually acts as a tumour promotor in GC.
Highlights
CircRNA has emerged as a new non-coding RNA that plays crucial roles in tumour initiation and development
To further verify whether the expression level of circNRIP1 was high in Gastric cancer (GC) tumours according to the RNA-seq data, we detected higher circNRIP1 expression in 80 GC samples relative to adjacent normal samples via Quantitative real-time PCR (qRT-PCR), which was consistent with the RNA-seq data (Fig. 1d)
We found that circNRIP knockdown in vivo significantly blocked tumour growth in terms of tumour weight and volume relative to the negative control group, whereas overexpression of circNRIP1 promoted the growth of xenografted tumours. e. qRT-PCR was performed in Patient-derived xenograft (PDX) tumours, and we found increased and persistent levels of circNRIP1 in the circNRIP1 overexpression plasmid-treated groups relative to the NC group and lower levels of circNRIP1 in the long-term circNRIP1 Short interfering RNA (siRNA)-treated groups. f
Summary
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. The AKT/mTOR axis exerts a positive effect on EMT, which promotes tumour metastasis. Gastric cancer (GC), called stomach adenocarcinoma, has raised societal concerns worldwide, especially in East Asian countries in recent years. Many advancements have been achieved in terms of diagnostic methods and surgical procedures, the overall survival of GC patients has remained largely unsatisfactory in recent years. The 5-year overall survival is less than 30% in most countries [4]. There is an urgent need to gain an in-depth understanding of the molecular mechanisms explaining GC initiation and development
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