Identification of Extrachromosomal Circular DNA Isolated from Cell Culture Supernatant and Its Potential Applications for Hepatocellular Carcinoma Diagnosis

Abstract

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy worldwide and is associated with a high mortality rate. The unique genetic features and structural properties of extrachromosomal circular DNA (eccDNA) offer a promising approach for early cancer diagnosis. OBJECTIVE: This study aims to investigate the potential of eccDNA as a diagnostic marker by identifying its distinguishing characteristics using a two-dimensional cell culture model. METHODS: EccDNA was isolated from the supernatant of HepG2 and THLE-2 cell cultures. Long-read sequencing and bioinformatic analysis were employed to detect and characterize eccDNA. The size distribution, chromosomal origin, and genomic annotation of eccDNA from both cell types were compared. PCR was performed to validate DNA fragments at the junctions of the closed-circular forms. RESULTS: The total number of cell-free eccDNA derived from HepG2 cells averaged 2,669,673 bases, encompassing 2,542 sequences, while THLE-2 cells averaged 857,718 bases with 975 sequences. Size distribution analysis revealed that most cell-free eccDNA from both cell types ranged from 351 to 600 bp. Bioinformatic analysis revealed that approximately 80% of eccDNAs corresponded to gene coding regions. Ten eccDNAs were the most frequently detected in HepG2 cells, with four eccDNAs harboring CDC27P11, RAC1P3, LOC112268123, and LOC124902279 genes successfully validated and uniquely detected in HepG2 cells, suggesting their potential as biomarkers for HCC diagnosis. CONCLUSION: Distinct eccDNA types were identified in HepG2 cells, which may serve as promising biomarkers for HCC diagnosis.