ミトコンドリア DNA は HBV 組み込みの標的です

Communications Biology volume 6、記事番号: 684 (2023) この記事を引用

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B 型肝炎ウイルス (HBV) は感染細胞のゲノムに組み込まれ、肝発がんに寄与する可能性があります。しかし、肝細胞癌 (HCC) の発生における HBV 組み込みの役割は依然として不明です。この研究では、HBV 組み込み部位の高感度な同定と組み込みクローンの計数を可能にするハイスループット HBV 組み込みシーケンスアプローチを適用します。我々は、7 人の HCC 患者から採取した腫瘍組織サンプルと非腫瘍組織サンプルのペアにおいて 3,339 個の HBV 組み込み部位を特定しました。我々は、2,107 個のクローン的に拡大した組み込み (腫瘍組織で 1,817 個、非腫瘍組織で 290 個)、および酸化的リン酸化遺伝子 (OXPHOS) および D ループ領域で優先的に発生するミトコンドリア DNA (mtDNA) におけるクローン HBV 組み込みの大幅な濃縮を検出しました。また、HBV RNA 配列がポリヌクレオチドホスホリラーゼ (PNPASE) の関与によって肝癌細胞のミトコンドリアに取り込まれること、および HBV RNA が HBV の mtDNA への組み込みのプロセスに役割を果たしている可能性があることも見出しました。我々の結果は、HBV の組み込みが HCC の発症に寄与する可能性がある潜在的なメカニズムを示唆しています。

慢性 B 型肝炎ウイルス (HBV) 感染は、肝細胞癌 (HCC) 発症の主要な危険因子です。健康な人と比較して、慢性B型肝炎（CHB）患者はHCCを発症するリスクが最大100倍高く、世界中でがん関連死亡の第4位となっており、年間約78万人が死亡しています1,2。組み込まれたウイルス DNA は HBV 関連 HCC の 85 ～ 90% で検出されており、小児または若年成人の非硬変肝臓で発生する腫瘍にウイルス DNA が存在することは、肝発がんにおけるウイルス DNA の組み込みの役割をさらに裏付けています 3,4,5。 6、7。 HBV DNA の宿主ゲノムへの組み込みは、染色体の不安定性、挿入突然変異誘発、宿主遺伝子発現の調節解除、および既知の発癌特性を持つ表面切断型タンパク質や HBx タンパク質などの突然変異ウイルスタンパク質の産生を引き起こす可能性があります 8,9。 HBV DNA 挿入部位は宿主ゲノム全体にランダムに分布しているように見えますが、最近の次世代シーケンス (NGS) アプローチの使用により、TERT、MLL4、CCNE1、および腫瘍組織における CCNA2、7、10、11、12、13、14、15、16。さらに、最近の研究では、がんドライバー遺伝子における反復的なコピー数の変化が、遠隔ウイルスの組み込みと関連している可能性があることが示されています 16。かなりの数の症例が研究されているが、既知の癌関連遺伝子は、HBV 関連 HCC のごく一部でのみ HBV 組み込みによって変化します。多くの研究では、反復配列、非コード RNA の DNA 配列、レトロトランスポゾンなどの特定のゲノム要素が HBV 組み込みの標的となることも実証されています 7、11、12、14、17、18、19。 HBV 陽性 HCC 細胞株を分析した香港での 1 つの研究では、特定のキメラ HBx-LINE1 転写物の発現には腫瘍促進機能があり、評価された HCC の大部分がこの転写物を発現していることが示されました 17。それにもかかわらず、HBx-LINE1 の発現は、ヨーロッパの患者由来の大規模な一連の HBV 関連 HCC では確認されませんでした 20。

HBV DNA 組込みに関する研究に関する進歩にもかかわらず、多くの重要な側面は依然として不明のままです。全体として、HBV 組み込みの代替検出法の開発は、HBV 組み込みによって誘発される発がんプロセスに関与するメカニズムについてのより良い洞察を得るのに役立つ可能性があります。

この研究では、ハイスループット HBV インテグレーションシークエンシング (HBIS) 法を適用することにより、HCC 患者の腫瘍および非腫瘍肝組織の両方からミトコンドリア DNA (mtDNA) におけるウイルスの組み込みの濃縮が検出されました。さらに、HBIS および RNASeq を HBV 誘導 HepAD38 細胞から精製したミトコンドリアに適用し、mtDNA への複数の HBV 組み込みおよび HBV ミトコンドリア融合転写物を検出しました。腫瘍組織におけるすべてのミトコンドリアの組み込みはクローン的に拡大され、酸化的リン酸化 (OXPHOS) ミトコンドリア遺伝子と D ループ領域の両方が関与していました。また、HBV RNA 配列が肝癌細胞のミトコンドリアに移入されること、およびポリヌクレオチドホスホリラーゼ (PNPASE) がウイルス転写物の移入に関与している可能性があることも発見しました。

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>

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