RESUMO
The integration of Hepatitis B Virus (HBV) is now known to be closely associated with the occurrence of liver cancer and can impact the functionality of liver cells through multiple dimensions. However, despite the detailed understanding of the characteristics of HBV integration and the mechanisms involved, the subsequent effects on cellular function are still poorly understood in current research. This study first systematically discusses the relationship between HBV integration and the occurrence of liver cancer, and then analyzes the status of the viral genome produced by HBV replication, highlighting the close relationship and structure between double-stranded linear (DSL)-HBV DNA and the occurrence of viral integration. The integration of DSL-HBV DNA leads to a certain preference for HBV integration itself. Additionally, exploration of HBV integration hotspots reveals obvious hotspot areas of HBV integration on the human genome. Virus integration in these hotspot areas is often associated with the occurrence and development of liver cancer, and it has been determined that HBV integration can promote the occurrence of cancer by inducing genome instability and other aspects. Furthermore, a comprehensive study of viral integration explored the mechanisms of viral integration and the internal integration mode, discovering that HBV integration may form extrachromosomal DNA (ecDNA), which exists outside the chromosome and can integrate into the chromosome under certain conditions. The prospect of HBV integration as a biomarker was also probed, with the expectation that combining HBV integration research with CRISPR technology will vigorously promote the progress of HBV integration research in the future. In summary, exploring the characteristics and mechanisms in HBV integration holds significant importance for an in-depth comprehension of viral integration.
RESUMO
Hepatitis B virus (HBV) integration into human genome causes hepatocellular carcinoma (HCC). The present study used inverse nested PCR; the full sequence of HBV DNA fragments of the chrX: 111009033 integration site was detected (987 bp), containing two fragments of doublestranded linear DNA with the same orientation (1,7441,094 and 1,5651,228 nt). By reverse transcriptionquantitative PCR, HBVcell fusion transcript was observed in HepG2.2.15 cells. The mean copy number of this site in cells with H2O2 treatment (8.73x102±1.65x102 copies/cell) was significantly higher than that in the cells without H2O2 treatment (3.02x102±2.33x102 copies/cell; P<0.0001). The mean levels of P21activated kinase 3 (PAK3) were 15.67±5.65 copies/cell in HepG2.2.15 cells with H2O2 treatment, significantly higher than in the cells without H2O2 treatment (11.34±4.58 copies/cell, P=0.0076) and in HepG2 cells (5.92±1.54 copies/cell, P<0.0001). Significant difference of PAK3 levels was also found between HepG2.2.15 cells without H2O2 treatment and HepG2 cells (11.34±4.58 vs. 5.92±1.54 copies/cell, P<0.0001). The average copy numbers of the integration site chrX: 111009033 were positively correlated with the average levels of PAK3 (P=0.0013). The overall trend of PAK3 expression was significantly increased in HepG2.2.15 cells with H2O2 treatment compared with that in HepG2.2.15 cells without H2O2 treatment (37.63±8.16 and 31.38±7.94, P=0.008) and HepG2 cells (21.67±7.88, P<0.0001). In summary, the chrX: 11009033 integration site may originate from primary human hepatocytes, occurrence and clonal expansion of which may upregulate PAK3 expression, which may contribute to hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular , Hepatite B Crônica , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , DNA Viral/genética , Peróxido de Hidrogênio/farmacologia , Neoplasias Hepáticas/patologia , Vírus da Hepatite B/genética , Quinases Ativadas por p21RESUMO
BACKGROUND: Hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) is heterogeneous and frequently contains multifocal tumors, but how the multifocal tumors relate to each other in terms of HBV integration and other genomic patterns is not clear. METHODS: To interrogate heterogeneity of HBV-HCC, we developed a HBV genome enriched single cell sequencing (HGE-scSeq) procedure and a computational method to identify HBV integration sites and infer DNA copy number variations (CNVs). RESULTS: We performed HGE-scSeq on 269 cells from four tumor sites and two tumor thrombi of a HBV-HCC patient. HBV integrations were identified in 142 out of 269 (53%) cells sequenced, and were enriched in two HBV integration hotspots chr1:34,397,059 (CSMD2) and chr8:118,557,327 (MED30/EXT1). There were also 162 rare integration sites. HBV integration sites were enriched in DNA fragile sites and sequences around HBV integration sites were enriched for microhomologous sequences between human and HBV genomes. CNVs were inferred for each individual cell and cells were grouped into four clonal groups based on their CNVs. Cells in different clonal groups had different degrees of HBV integration heterogeneity. All of 269 cells carried chromosome 1q amplification, a recurrent feature of HCC tumors, suggesting that 1q amplification occurred before HBV integration events in this case study. Further, we performed simulation studies to demonstrate that the sequential events (HBV infecting transformed cells) could result in the observed phenotype with biologically reasonable parameters. CONCLUSION: Our HGE-scSeq data reveals high heterogeneity of HCC tumor cells in terms of both HBV integrations and CNVs. There were two HBV integration hotspots across cells, and cells from multiple tumor sites shared some HBV integration and CNV patterns.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Variações do Número de Cópias de DNA , DNA Viral/genética , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Integração ViralRESUMO
Hepatocellular carcinoma (HCC) is one of the most lethal malignancies that is closely associated with the Hepatitis B virus (HBV). HBV integration into host genomes can induce instability and the aberrant expression of human genomic DNA. To directly assess HBV integration breakpoints at whole genome level, four small sequencing libraries were constructed and the HBV integration profiles of four patients with HCC were characterized. In total, the current study identified 11,800,974, 11,216,998, 11,026,546 and 11,607,842 clean reads for patients 1-3 and 4, respectively, of which 92.82, 95.95, 97.21 and 97.29% were properly aligned to the hybrid reference genome. In addition, 220 HBV integration events were detected from the tumor tissues of four patients with HCC and an average of 55 breakpoints per sample was calculated. The results indicated that HBV integration events may be implicated in HCC physiologies and diseases. The results acquired may also provide insight into the pathogenesis of HCC, which may be valuable for future HCC therapy.
RESUMO
The present study surveyed the characteristics of hepatitis B virus (HBV) integration in the liver genomes of patients with acute hepatitis B (AHB), carriers of inactive hepatitis B surface antigen (HBsAg), and patients with chronic hepatitis B (CHB) receiving antiviral treatment. 'Shortread' whole genome sequencing (WGS) with an average of 4,879x coverage for HBV integration was performed in three patients with AHB, two carriers of inactive HBsAg, and 13 patients with CHB receiving antiviral treatment. Conventional polymerase chain reaction and Sanger sequencing were used to verify integration breakpoints supported by at least two pairedend reads, and viralhost chimeric transcripts were surveyed simultaneously. HBV integration breakpoints were 100% identified with an average of 138.2±379.9 breakpoints per sample. The numbers of HBV integration breakpoints were positively associated with the sequencing depth coverage numbers and levels of intrahepatic covalently closed circular DNA, respectively (P<0.0001 and P<0.0001). Four types of viralhost junction in 14 HBV integration breakpoints were detected (two viral junctions mapped in the HBs gene, one in the Precore gene, and others within the HBx gene): Forward simple junction, reverse simple junction, forward and reverse complicated junction, and microhomology were found in many of the junctions. Expression of viralhuman chimeric transcripts was observed in several breakpoints, including the HBs gene. As a result, HBV can integrate into the host gene in the same manner as nonhomologous end joining and microhomologymediated end joining with numerous sites, and a close association may exist between HBV integration and patient prognosis. HBx integration may be indispensable for viralhost chimeric transcription and HBsAg may be produced from integrated DNA.