Circ-ZNF609 regulates G1-S progression in rhabdomyosarcoma

Francesca Rossi,Carlo Dominici,Alessio Colantoni,Tiziana Santini,Francesca Megiorni,Mariangela Morlando,Gaia Di Timoteo,Irene Bozzoni,Dario Dattilo,Ivano Legnini

doi:10.1038/s41388-019-0699-4

Francesca Rossi, Carlo Dominici + Show 8 more

Open Access

https://doi.org/10.1038/s41388-019-0699-4

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Abstract

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS.

Highlights

CircRNAs are eukaryotic transcripts produced through a peculiar splicing reaction, which causes the circularization of one or more exons
We analyzed E2F1, CDK1, cyclin A2 (CycA2), cyclin B1 (CycB1), and cyclin B2 (CycB2), which are related to the cell cycle
The p53 gene is mutated in both RD and RH4 cells [46,47,48]; p53 expression levels are significantly lower in RH4 than in RD cells (Table S1). These features might explain the significantly high expression of the p53 target genes involved in cell cycle regulation in RH4 cells as well as the lack of an effect of circ-ZNF609 depletion on the alveolar rhabdomyosarcoma (ARMS) cell line. These results suggest that circ-ZNF609 can control a specific part of the transcriptome in RD cells, to what is observed in human primary myoblasts, by inducing, when downregulated, a specific gene expression response related to the cell cycle and fully consistent with the observed G1-S arrested phenotype

Summary

Introduction

CircRNAs are eukaryotic transcripts produced through a peculiar splicing reaction (back-splicing), which causes the circularization of one or more exons. The resulting molecules are characterized by a back-splicing junction and lack 5ʹ and 3ʹ free termini [1, 2]. Having such a shape, circRNAs are typically very stable molecules [3], a trait which has captivated a broad interest concerning their possible molecular activity.

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