DNA methylation at hepatitis B virus integrants and flanking host mitochondrially encoded cytochrome C oxidase III

Oikawa,Ritsuko; Watanabe,Yoshiyuki; Yotsuyanagi,Hiroshi; Yamamoto,Hiroyuki; Itoh,Fumio

doi:10.3892/ol.2022.13544

DNA methylation at hepatitis B virus integrants and flanking host mitochondrially encoded cytochrome C oxidase III

Authors:
- Ritsuko Oikawa
- Yoshiyuki Watanabe
- Hiroshi Yotsuyanagi
- Hiroyuki Yamamoto
- Fumio Itoh
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Affiliations: Division of Gastroenterology, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216‑8511, Japan, Department of Infectious Diseases, Graduate School of Medicine, University of Tokyo, Tokyo 108‑8639, Japan
Published online on: October 11, 2022 https://doi.org/10.3892/ol.2022.13544
Article Number: 424
Copyright: © Oikawa et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

It is widely accepted that hepatitis B virus (HBV) integrants in the human genome are one of the key factors in liver carcinogenesis. Although it is difficult to observe pre/post‑HBV infection genomic‑level changes in the same clinical sample pairs, they can be observed using artificially infected HBV cell lines such as HepG2.2.15. A detailed HBV integration analysis comparing HepG2.2.15 with HepG2 cells, especially their mitochondrial (mt) DNA, was conducted using next‑generation sequencing (NGS)‑based integration analysis. Following target DNA enrichment for elements of the HBV genome, NGS was used to identify HBV integration sites in the mtDNA and DNA methylation was analyzed using semi‑quantitative pyrosequencing at the boundaries of the integrated region. The results revealed the HBV integration site in the mtDNA of HepG2.215, most notably the insertion of the HBV preCore, X gene fragment in exon 1 of mitochondrially encoded cytochrome C oxidase III (MT‑CO3; ChrM 9652), along with a ‘CACCA’ microhomology sequence. Both boundaries of the integrated region were concordant and highly methylated (HBV side, 92.3%; MT‑CO3 side, 95.5%) relative to those observed in nonintegrated HepG2 (4.3%), HepG2.2.15 (3.0%) and PLC/PRF/5 (4.0%) cells. In conclusion, HBV integration sites were successfully identified in the MT‑CO3 gene along with a ‘CACCA’ microhomology sequence using NGS‑based analysis and mitochondrial heteroplasmy was identified. The present study also revealed that the HBV/MT‑CO3‑integrated boundary DNA was hypermethylated at both the HBV and MT‑CO3 sides.

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