Extrachromosomal circular DNAs are common and functional in esophageal squamous cell carcinoma

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is the leading cause of cancer-related mortality.While recent studies have documented the presence of extrachromosomal circular DNAs (eccDNAs) in various tumors, to date, there have been no studies examining the distribution and function of eccDNAs in ESCC.Methods: The eccDNAs from three surgically matched ESCC tissue samples were extracted and amplified by rolling circle amplification after removal of linear DNA and mitochondrial circular DNA. Highthroughput eccDNA sequencing and bioinformatics analysis was performed to study the distribution pattern and the level of eccDNA expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the genes associated with the differentially expressed eccDNAs. Five up-regulated and five down-regulated candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. The nucleotides flanking the eccDNA junctions were analyzed to explore the mechanisms of eccDNA formation.Results: A total of 184,557 eccDNAs was identified. The overall length distribution ranged from 33 to 96,8842 base pairs (bp), with the peak at approximately 360 bp. These eccDNAs mainly originated from 5'-and 3'-untranslated regions (UTRs), and rarely from exons, introns, LINE, or Alu repeat regions. The chromosome distribution, length distribution, and genomic annotation of the eccDNAs were comparable between ESCC samples and matched normal epithelium. Nevertheless, 16,031 eccDNAs were found to be differentially expressed between ESCC and matched normal epithelium, including 10,126 up-regulated eccDNAs and 5,905 down-regulated eccDNAs. GO analysis and KEGG pathway analysis showed enriched in cancer pathways, mitogen-activated protein kinase (MAPK) pathway, GTPase-related activity, and cytoskeleton function. PCR, TOPO-TA cloning, and Sanger sequencing validated the junctional sites of five up-regulated candidate eccDNAs and four other unexpected eccDNAs. A repeat nucleotide pattern between the position flanking the start site and that flanking the end site was detected.Conclusions: This study demonstrated the genome-wide presence of eccDNAs, explored the differential expression of eccDNAs, and revealed the potential mechanisms of eccDNAs in ESCC. This work provides further insights into our understanding of genome plasticity, the role of eccDNAs in ESCC, and may contribute to the development of potential clinical therapies.

The important role of the eccDNA in tumors

“…In the article, the authors did not mention the total amount of detected eccDNAs in their samples and showed only 298 up-regulated and 71 down-regulated eccDNAs in AML patients compared to healthy controls, which is far less than the number in our work ( 1 , 2 ). Maybe it is due to tumor specificity or different sequencing methods.…”

“…Additionally, the study observed altered biological processes related to up-regulated eccDNAs and no process or network related to down-regulated eccDNAs. Nevertheless, our work demonstrated various biological processes playing key roles in ESCC progression by analysis of either up-regulated or down-regulated eccDNAs ( 1 , 2 ). These differences may be caused by less amount of differently expressed eccDNAs.…”

“…We would like to thank Qian et al for their interest in our published work about the potential function of extrachromosomal circular DNA (eccDNA) in esophageal squamous cell carcinoma (ESCC) ( 1 ). We have read their article revealing the characterization of the eccDNA in acute myeloid leukemia (AML) with great interest ( 2 ).…”

Identification and Characterization of Extrachromosomal Circular DNA in Plasma of Lung Adenocarcinoma Patients

“…We observed that eccDNA can be dropped from practically any point on the chromosome after visualizing the loci of the eccDNA chromosome, which is consistent with previous studies. 20 , 21 Although most eccDNA existed independently in various samples according to the sequencing data, some eccDNA were concurrently present in plasma samples of lung adenocarcinoma. We searched the eccDNA molecules that detected in at least 3 lung adenocarcinoma patients but not identified in samples of healthy people, and 9 kinds of eccDNA molecules were eligible (split reads ≥1).…”

Characterization of extrachromosomal circular DNA in patients with acute myeloid leukemia: proof-of-concept report using cohorts from Beijing and Shanghai

“…We have read with great interest the manuscript of Sun et al ( 2 ), in which they proved the gone-wide presence of extrachromosomal circulating DNA and showed its potential in the pathogenesis of esophageal squamous cell carcinoma. EccDNA elements are responsible for carrying DNA sequences that are homologous with demonic DNA ( 3 ).…”