Extrachromosomal circular DNA containing DTX1 promotes cell growth in hydroquinone-induced malignantly transformed cells by regulating the transcription of DTX1.

Abstract

Background: Extrachromosomal circular DNA (eccDNA), a novel class of DNA with a circular topological structure, is present in a variety of cancer cells and tissues and may play broad roles in processes ranging from aging to cancer cell heterogeneity through multiple mechanisms. EccDNA has been characterized by profile, structure and function in several prominent studies but its effect on hydroquinone (HQ)-induced malignantly transformed cells (TK6-HQ) is still elusive.

Methods: Circle-seq was applied to determine the eccDNA counts and characteristics of TK6-HQ cells. DNA-fluorescence in situ hybridization was used to measure the abundance of eccDNA_DTX1. Differential gene expression analysis was carried out by RNA-seq. Gene expression was quantified by wertern blot and qPCR. Decircularization of eccDNA_DTX1 was achieved by CRISPR/Cas9. Tumorigenicity was evaluated by xenograft assay in BALB/c nude mice.

Results: In this study, we characterized the structure of eccDNAs and the function of DTX1-containing eccDNA (eccDNA_DTX1) in TK6-HQ cells. A total of 669,179 eccDNAs were identified, including 901 eccDNAs with different counts. Most of the eccDNAs were < 1000 bp in length and were enriched in four periodic peaks starting at 186 bp with an interval of ~ 180 bp. The genomic distribution of eccDNAs confirmed that eccDNAs could be observed across all chromosomes and had greater enrichment on chromosomes 17, 19, 20, and 22, with abundant Alu repeat elements, introns and CpG islands. By combining the results of the integrated circle-seq analysis of eccDNAs with those from the RNA-seq analysis (differentially expressed genes, 1064 upregulated and 427 downregulated), the authors showed that the transcription of 20 potential coding genes might be driven by eccDNAs. Finally, the knockdown of eccDNA_DTX1 by CRISPR/Cas9 inhibited the growth of TK6-HQ cells in vitro and in vivo by inhibiting the transcription of DTX1 and promoting ferroptosis, and ferroptosis inhibior, Ferrostatin-1, abrogated the proliferation inhibition of eccDNA_DTX1 knockdown.

Conclusions: EccDNA_DTX1 promotes cell growth in hydroquinone-induced malignantly transformed cells by regulating the transcription of DTX1 and ferroptosis. This study profiles eccDNA characteristics and defines the role and mechanism of eccDNA_DTX1 for the first time, shedding new light on the relationship between eccDNAs and carcinogenesis.