미토콘드리아 DNA는 HBV 통합의 표적입니다

커뮤니케이션 생물학 6권, 기사 번호: 684(2023) 이 기사 인용

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B형 간염 바이러스(HBV)는 감염된 세포의 게놈에 통합되어 간암 발생에 기여할 수 있습니다. 그러나 간세포암종(HCC) 발생에서 HBV 통합의 역할은 아직 불분명합니다. 이 연구에서는 HBV 통합 사이트를 민감하게 식별하고 통합 클론을 열거할 수 있는 높은 처리량의 HBV 통합 시퀀싱 접근 방식을 적용합니다. 우리는 HCC 환자 7명의 쌍을 이루는 종양 및 비종양 조직 샘플에서 3339개의 HBV 통합 부위를 식별합니다. 우리는 2107개의 클론 확장된 통합(종양에서 1817개, 비종양 조직에서 290개)과 산화적 인산화 유전자(OXPHOS) 및 D-루프 영역에서 우선적으로 발생하는 미토콘드리아 DNA(mtDNA)에서 클론성 HBV 통합의 상당한 농축을 감지합니다. 우리는 또한 HBV RNA 서열이 폴리뉴클레오티드 포스포릴라제(PNPASE)와 관련하여 간암 세포의 미토콘드리아로 유입되고 HBV RNA가 HBV가 mtDNA로 통합되는 과정에서 역할을 할 수 있음을 발견했습니다. 우리의 결과는 HBV 통합이 HCC 발달에 기여할 수 있는 잠재적 메커니즘을 제시합니다.

만성 B형 간염 바이러스(HBV) 감염은 간세포암종(HCC) 발병의 주요 위험 요소입니다. 건강한 개인에 비해 만성 B형 간염(CHB) 환자는 간세포암종 발병 위험이 최대 100배 더 높습니다. 간세포암종은 전 세계적으로 암 관련 사망의 4번째 주요 원인이며 연간 약 780,000명이 사망합니다1,2. 통합된 바이러스 DNA는 HBV 관련 간세포암종의 85~90%에서 발견되었으며, 어린이나 젊은 성인의 비간경변성 간에서 발생하는 종양에서 이 DNA의 존재는 간암 발생에서 바이러스 DNA 통합의 역할을 더욱 뒷받침합니다3,4,5, 6,7. HBV DNA가 숙주 게놈에 통합되면 염색체 불안정성, 삽입 돌연변이 유발, 숙주 유전자 발현 조절 완화, 잘린 표면 및 발암 특성이 알려진 HBx 단백질과 같은 돌연변이 바이러스 단백질의 생성이 발생할 수 있습니다8,9. HBV DNA 삽입 부위는 숙주 게놈 전체에 무작위로 분포되어 있는 것처럼 보이지만 최근 NGS(차세대 염기서열분석) 접근법을 사용하면 TERT, MLL4, CCNE1 및 CCNA2, 종양 조직7,10,11,12,13,14,15,16. 또한, 최근 연구에 따르면 암 유발 유전자의 반복적인 복제 수 변경이 원격 바이러스 통합과 관련될 수 있는 것으로 나타났습니다. 상당수의 사례가 연구되었지만 알려진 암 관련 유전자는 HBV 관련 간세포 암종 중 극히 일부에서만 HBV 통합에 의해 변경됩니다. 또한 수많은 연구를 통해 반복 서열, 비암호화 RNA의 DNA 서열, 레트로트랜스포존과 같은 특정 게놈 요소가 HBV 통합의 표적이 된다는 사실이 입증되었습니다. HBV 양성 HCC 세포주를 분석한 홍콩의 한 연구에서는 특정 키메라 HBx-LINE1 전사체의 발현이 종양 촉진 기능을 가지며 평가된 HCC의 상당 부분이 이 전사체를 발현하는 것으로 나타났습니다17. 그럼에도 불구하고 유럽 환자의 대규모 HBV 관련 간세포암종에서는 HBx-LINE1 발현이 확인되지 않았습니다.

HBV DNA 통합에 대한 연구와 관련된 진전에도 불구하고 많은 주요 측면이 불분명합니다. 전반적으로, HBV 통합을 위한 대체 검출 방법의 개발은 HBV 통합에 의해 유발된 발암 과정과 관련된 메커니즘에 대한 더 나은 통찰력을 얻는 데 도움이 될 수 있습니다.

이 연구에서 HBIS(High-throughput HBV 통합 시퀀싱) 방법을 적용하여 HCC 환자의 종양 및 비종양 간 조직 모두에서 미토콘드리아 DNA(mtDNA)의 바이러스 통합이 농축되는 것을 확인했습니다. 또한, 우리는 HBV 유발 HepAD38 세포에서 정제된 미토콘드리아에 HBIS와 RNASeq를 적용하고 HBV-미토콘드리아 융합 전사물뿐만 아니라 mtDNA로의 다중 HBV 통합을 감지했습니다. 종양 조직의 모든 미토콘드리아 통합은 클론적으로 확장되었으며 산화적 인산화(OXPHOS) 미토콘드리아 유전자와 D-루프 영역이 모두 포함되었습니다. 우리는 또한 HBV RNA 서열이 간암 세포의 미토콘드리아로 유입되고 폴리뉴클레오티드 포스포릴라제(PNPASE)가 바이러스 전사물 유입에 관여할 수 있음을 발견했습니다.

15 kb, with more than 20% having a length shorter than 100 bp31,46,55. Considering that mtDNA averages several thousand copies per hepatocyte compared to the two copies of numts in nuclear DNA (nDNA), by isolating mitochondria from cells, it is possible to completely dilute out numts, leading to numt-free mtDNA sequences. Therefore, to study mtDNA HBV integration, we isolated nuclei, cytoplasm, and mitochondria from HBV-producing HepAD38 cells and applied both HBIS and RNASeq. According to HBIS, several HBV integration sites were identified in DNA isolated from mitochondria, whereas no integration was detected in numts from nDNA. Furthermore, RNASeq revealed the presence of chimeric HBV-mitochondrial transcripts within mitochondria but not in cytoplasm or nuclei of HBV-producing HepAD38 cells, and mtDNA insertion sites may be transcriptionally active in these cells. Both HBIS and RNAseq analysis also revealed that MMEJ have a major role in HBV integrations occurring in mitochondrial genomes. Therefore, taken together, our data clearly demonstrate that HBV can integrate into mtDNA of tumour and non-tumour hepatocytes. Some previous studies have reported data concerning HBV integration in mtDNA16,56,57,58,59,60. All these studies have utilised high-throughput HBV genome-enrichment sequencing approaches to study HBV integration, and most of them have analysed hepatoma cell lines stably expressing HBV DNA56,57,58,59.To the best of our knowledge, only one16 of the papers has reported data on HBV integration in mtDNA from human liver tissues. In particular, in the supplementary dataset of this paper, 58 different HBV integration sites in mtDNA from tumour and/or non-tumour liver tissue specimens of 11 patients with HBV-related HCC have been listed16. The mitochondrial genomic regions most frequently targeted by HBV integration in the 11 patients were the D-loop region, ND4, ND5, RNR2, CYTB, ND6, ND1, ND2 and COX3 genes16. HBV integration events have also been described in mtDNA from humanised-liver tissue samples of chimeric mice60. In this study, Furuta et al.60 have identified 50 distinct HBV integration sites in mtDNA from chimeric mice. These integrations (a) have been associated with higher levels of HBV replication, (b) occurred at higher frequency in the D-loop region, and (c) appeared to rely on MMEJ60. No detailed information on virus-mtDNA junctions has been provided in studies performed on PLC/PRF/5 cell lines56,59. However, the fact that HBV integration in mtDNA may occur in these cells—which do not replicate HBV and only express multiple distinct viral RNAs from HBV integrants56,59—suggests that viral RNA might be involved in the process of HBV integration in mtDNA. Despite a number of studies documenting interaction between HBV proteins and mitochondria and consequent alteration of mitochondrial functions61,62,63, whether HBV nucleic acids may translocate into mitochondria has only minimally been addressed. Based on our results, HBV transcripts, but not viral full-length genome or cccDNA, can localise to mitochondria. In addition, PNPASE, a mitochondrial protein considered the first RNA import factor for mammalian mitochondria35,36,39, possibly mediates viral RNA delivery into the mitochondrial matrix. A PNPASE-dependent RNA import sequence that we identified for the preS1 transcript as well as known stem-loop structures specific to HBV transcripts appear to mediate mitochondrial targeting of viral RNAs. Localisation of HBV transcripts to mitochondria leads us to hypothesise that viral RNA may represent a possible substrate for HBV integration in mtDNA. The mitochondrial genome is more prone to damage and double-strand break (DSB) formation than the nuclear genome due to frequent exposure to the ROS generated by mitochondrial oxidative phosphorylation and the lack of protective histones. Considering that several reports have shown that RNA molecules can directly act as a template for the repair of mitochondrial DSBs in human cells64, it is tempting to speculate that viral exploitation of this pathway may lead to HBV sequences being inserted into the mitochondrial genome. In summary, we found that HBV may integrate into mtDNA, with tumours and non-tumour liver tissues showing distinct profiles of viral integration into the mitochondrial genome. Moreover, our results indicate that HBV RNA may be actively imported into mitochondria and that viral RNA sequences might be involved in the process of HBV integration into mtDNA. In spite of the relatively limited sample of patients, this study offers new insight into the HBV-hepatocyte interaction and provides a new basis for investigative analyses that may lead to further comprehension of the mechanisms by which HBV insertion can drive HCC development and progression./p> 5 exo− (5000 U/mL) (New England Biolabs, Ipswich, MA) for 1 h at 37 °C. All reactions were purified by a MinElute Reaction Clean-up kit (Qiagen). Each aliquot of blunted, A-tailed DNA fragments was then ligated to 200 pmol annealed linkers (LinkerTop + LinkerBottom) (Supplementary Table 2) with 4 μL pLinker, 5 μL NEB T4 DNA ligase buffer and 1 μL T4 DNA ligase (2 × 106 U/mL, high concentration) (New England Biolabs) for 1 h at 25 °C and then overnight at 16 °C. The ligase was inactivated by incubation at 70 °C for 20 min, and the reactions were purified using a MinElute Reaction Clean-up kit (Qiagen). Finally, all six reactions were pooled, and the pooled linker-ligated DNA was aliquoted into two equal parts to perform semi-nested ligation-mediated PCR with forward or reverse HBV primers (Fig. 1 and Supplementary Table 2). The forward and reverse enrichment sequences were kept separate throughout the remainder of the protocol. The DNA was divided into 1-µg aliquots; each aliquot was mixed with 20 µL Phusion HF buffer (5×), 3 μL dNTPs (10 mM), 1 μL biotinylated forward (20 μM) or reverse HBV primer (Supplementary Table 2) (2.5 μM), 1 μl Phusion Taq (2000 U/ml) (New England Biolabs) and H2O to 50 μL. Single-primer PCRs were performed as follows: 98 °C for 1 min; 12 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 45 s; 72 °C for 1 min); and a hold at 4 °C. Each tube was then spiked with 1 μL pLinker (Supplementary Table 2) (2.5 μM) and subjected to additional cycles of PCR, as follows: 98 °C for 1 min; 35 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 45 s; 72 °C for 5 min; and a hold at 4 °C. Forward and reverse PCRs were purified using the QIAquick PCR purification kit (Qiagen). The purified products were well separated on a 2% agarose gel, and fragments of 300–1000 bp were excised. The DNA was purified using a QIAquick gel extraction kit, and gel-based size selection and purification was repeated once. Then, 100 μL T1 magnetic streptavidin beads (Invitrogen) were added to each forward and reverse PCR product, and the mixture was incubated for 1 h with gentle rocking at room temperature. The beads were magnetically isolated, washed three times in 500 μL 1× B&W buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl) and once in H2O and resuspended in 50 μL H2O. Subsequently, 25 μL of the beads from each of the forward and reverse PCRs were separately mixed with 10 µL Phusion HF buffer (5×), 1.5 μL dNTPs (10 mM), 1 μL forward (20 µM) or reverse MiSeq HBV primer (20 μM), 1 μL forward or reverse MiSeq-pLinker (20 μM) (all MiSeq primers contain an adaptor for Illumina flow cell surface annealing) (Supplementary Table 2), 0.5 μL Phusion Taq (2000 U/mL), and 11 μL H2O and subjected to PCR (98 °C for 1 min, 35 cycles of 98 °C for 10 s, 65 °C for 40 s, and 72 °C–40 s, followed by 72 °C for 5 min and a hold at 4 °C). The PCR products were magnetically separated from the beads and purified using the QIAquick PCR purification kit (Qiagen). The adaptor-ligated fragments were enriched by 25 cycles of PCR with Illumina primers Index 1 and Index 2, as follows: 98 C° for 1 min, 25 cycles of 98 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), and 72 C° for 5 min. Forward and reverse libraries for the same sample were mixed in equimolar ratios and sequenced by 250-bp paired-end sequencing using an Illumina MiSeq. A total of 340 integration libraries were constructed from liver tissue samples of the nine individuals analysed (7 patients with HBV-related HCC and 2 HBsAg-negative subjects as a control) and from the PLC/PRF/5, HepAD38 and Vero cell lines./p>

3.0.CO;2-E" data-track-action="article reference" href="https://doi.org/10.1002%2F1097-0142%28195405%297%3A3%3C462%3A%3AAID-CNCR2820070308%3E3.0.CO%3B2-E" aria-label="Article reference 24" data-doi="10.1002/1097-0142(195405)7:33.0.CO;2-E"Article CAS PubMed Google Scholar /p>

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