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Circular RNA circDNA2 upregulates CCDC6 expression to promote the progression of gastric cancer via miR-149-5p suppression.

ABSTRACT: Circular (circ)RNAs are widely involved in gastric cancer (GC) pathogenesis, and coiled-coil domain containing 6 (CCDC6) is a fused partner of multiple oncogenes; however, the underlying mechanisms of how circRNAs regulate CCDC6 expression in the progression and prognosis of GC remain unclear. Here, we discovered the circRNA derived from the DNA2 gene locus (circDNA2) through RNA sequencing. By performing quantitative real-time PCR and fluorescence in situ hybridization (FISH) assays with a human tissue microarray, circDNA2 was found to be highly expressed in GC tissues and associated with lymphatic invasion of GC patients. Knockdown of circDNA2 expression suppressed the proliferation of GC cells by reducing CCDC6 expression. Mechanistically, circDNA2 acted as a microRNA (miR)-149-5p sponge, which was confirmed to target CCDC6 by RNA pulldown and dual-luciferase reporter assays and rescue experiments. Both low miR-149-5p expression and high CCDC6 expression were related to unfavorable prognosis in GC patients. Moreover, GC patients with low miR-149-5p expression had shorter overall survival and a higher risk of chemotherapy resistance than those with high miR-149-5p expression. In summary, circDNA2 contributes to the growth and lymphatic metastasis of GC by upregulating CCDC6 expression by sponging miR-149-5p. The circDNA2/miR-149-5p/CCDC6 axis might be developed as a therapeutic target and prognostic indicator for GC.

SUBMITTER: Jin D

PROVIDER: S-EPMC8426470 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Circular RNA circDNA2 upregulates CCDC6 expression to promote the progression of gastric cancer via miR-149-5p suppression.

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