Genome-Wide Extrachromosomal Circular DNA Profiling of Paired Hepatocellular Carcinoma and Adjacent Liver Tissues

SIMPLE SUMMARY: Hepatocellular carcinoma is a severe cancer with various underlying causes. Extrachromosomal circular DNA, first identified in the 1960s, has gained significant attention in recent years with the development of sequencing techniques, revealing its presence in various cancer types. However, the distribution and significance of extrachromosomal circular DNA in hepatocellular carcinoma remain poorly understood. In this study, we collected eight pairs of hepatocellular carcinoma and adjacent non-tumor tissue samples, and conducted a comprehensive analysis of extrachromosomal circular DNA profiles. The results provide evidence of the extrachromosomal circular DNA expression patterns and their correlation with transcriptome dysregulation in hepatocellular carcinoma. ABSTRACT: Hepatocellular carcinoma (HCC) develops through multiple mechanisms. While recent studies have shown the presence of extrachromosomal circular DNA (eccDNA) in most cancer types, the eccDNA expression pattern and its association with HCC remain obscure. We aimed to investigate this problem. The genome-wide eccDNA profiles of eight paired HCC and adjacent non-tumor tissue samples were comprehensively elucidated based on Circle-seq, and they were further cross-analyzed with the RNA sequencing data to determine the association between eccDNA expression and transcriptome dysregulation. A total of 60,423 unique eccDNA types were identified. Most of the detected eccDNAs were smaller than 1 kb, with a length up to 182,363 bp and a mean sizes of 674 bp (non-tumor) and 813 bp (tumor), showing a greater association with gene-rich rather than with gene-poor regions. Although there was no statistical difference in length and chromosome distribution, the eccDNA patterns between HCC and adjacent non-tumor tissues showed significant differences at both the chromosomal and single gene levels. Five of the eight HCC tissues showed significantly higher amounts of chromosome 22-derived eccDNA expression compared to the non-tumor tissue. Furthermore, two genes, SLC16A3 and BAIAP2L2, with a higher transcription level in tumor tissues, were related to eccDNAs exclusively detected in three HCC samples and were negatively associated with survival rates in HCC cohorts from public databases. These results indicate the existence and massive heterogeneity of eccDNAs in HCC and adjacent liver tissues, and suggest their potential association with dysregulated gene expression.