Elsevier

Cellular Signalling

Volume 72, August 2020, 109622
Cellular Signalling

circ_ARF3 regulates the pathogenesis of osteosarcoma by sponging miR-1299 to maintain CDK6 expression

https://doi.org/10.1016/j.cellsig.2020.109622Get rights and content

Highlights

  • circ_ARF3 is upregulated in osteosarcoma cell lines and tumor tissues.

  • circ_ARF3 plays pivotal roles in osteosarcoma cell growth, colony formation and cell cycle progression.

  • circ_ARF3 acts as a sponge of miR-1299 to maintain CDK6 expression

Abstract

Increasing evidence suggests that circular RNAs are emerging biomarkers or targets for early cancer diagnosis and treatment. However, the studies of circular RNA in osteosarcoma (OS) are limited. In this study we found that circ_ARF3 were highly expressed in osteosarcoma cell lines and tumor tissues. Knocking down circ_ARF3 greatly ceased OS cell growth, impaired cell colony formation and halted cell cycle transition from G1 to S phase. Bioinformatic analysis suggested that miR-1299 is the target of circ_ARF3. Luciferase assay and biotin labeled circ_ARF3 pull down assay confirmed their interactions in OS cells. The regulatory roles of circ_ARF3 on miR-1299 was also investigated. Further bioinformatic analysis showed that CDK6 is the target of miR-1299. Overexpressing miR-1299 in OS cells decreased CDK6 expression and arrested OS cell growth and cell cycle progression. However, the roles of miR-1299 in regulating CDK6 expression, OS cell growth and cell cycle progression were greatly impaired in the presence of circ_ARF3. In general, our study demonstrated that in the OS, highly expressed circ_ARF3 acts as a sponge of miR-1299 to inhibit miR-1299 mediated CDK6 downregulation which further promoted OS pathogenesis. circ_ARF3 could be a potential target for OS treatment in the future.

Section snippets

Background

Osteosarcoma is the most common primary sarcoma of bone which occurred predominantly in children and adolescence [1,2]. Recent years advancement in non-metastatic osteosarcoma treatment, including surgical resection, neoadjuvant and adjuvant combinational chemotherapy have improved the 5-year survival rate approaches 70% [3]. However, for the metastatic or relapsed osteosarcoma the prognosis remains poor and the 5 year survival rates is less than 20%, with no improvement over the past 20 years [4

Cell lines and antibodies

Osteosarcoma cell lines Saos2, U2OS and MG-63 were purchased from ATCC. Normal osteoblast cell line NHOst was purchased from Chinese Academy of Sciences, Shanghai china. Saos2 and U2OS were cultured in McCoy's 5a Medium supplemented with 10% FBS (Gibco), MG63 was grown in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS (Gibco). NHOst was cultured in DMEM with 10% FBS added. All the cell lines were subjected to incubation in a humidified environment with 5% CO2 at 37 °C.

Characterize of circ_ARF3 expression in OS cell lines

Previous genome wide transcriptome sequencing in lung cancer showed that circ_ARF3 was highly expressed in lung cancer tissues than in normal adjacent tissues. However, the roles and expression of circ_ARF3 in OS are still unknown. To determine if circ_ARF3 also expresses in OS, we first checked the circular RNA database from circinteractome, results showed that circ_ARF3 is back spliced by exon 3,4 and 5 of ARF3 transcripts (Fig. 1A). To further confirm the circular form of ARF3 transcript is

Discussion

CircRNAs are recently discovered noncoding RNAs characterized by covalently closed loop structures with no polyadenylated tail [18]. Most circRNAs are generated through pre-mRNA back-splicing with upstream 3′ splice site connected to the downstream 5'splice site [19]. Recent studies have shown that circRNAs play important roles in multiple biological process, including carcinogenesis, and were associated with many cancer progressions, for example, in liver cancer, circ_100395 was found

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

This project was supported by The China Scholarship Council.

Credit author statement

Gao Ai-Mei: Conceptualization, writing first draft, methodology and data analysis. Yuan Chunyan: Data curation and validation. Ai-Xin Hu: Western blots and in vivo assay. Liu Xiang-Sheng supervision, writing-review and editing.

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