Chromatin Structure Analysis Of Hepatitis B-related Liver Cancer Cells Based On Three-dimensional Genomics Technology

Hepatitis B Virus(HBV)infection is an important factor in the development of hepatocellular carcinoma.Despite the widespread availability of preventive vaccines,6%of the population in China still carries HBV.Therefore,studying the pathogenic mechanisms of long-term chronic HBV infection remains important.The main oncogenic mechanisms of hepatitis B include:(1)the expression of viral antigens affects cellular functions and activates oncogenic signaling pathways,leading to increased sensitivity of hepatocytes to cancer-inducing factors;(2)the integration of viral DNA in the host genome,allowing HBV viruses to actively integrate into oncogenes in host cells,such as TERT,CCNE1,and MLL4;(3)the interference of HBV DNA in the host genome.The active integration of HBV DNA into the host genome and the expression of viral proteins lead to an increase in genomic instability;however,the exact mechanism remains unclear.Over the past decade,high-throughput chromatin conformation-capture technologies based on next-generation sequencing(NGS)have been increasingly used to study genomic structural variation.The aim of this study was to investigate the specific effects of HBV infection on genomic structure using threedimensional(3D)genomic techniques to more closely explore the genomic structural alterations caused by genomic instability.In this study,we used the HBV stable transitional cell line HEPG2.cw and the parental cell Hep G2 as the target,and analyzed the 3D spatial organization of the genomes of the two groups of cells using the mammalian bridging Hi-C technique,and the results showed small-scale changes in the3 D spatial organization throughout the day;however,there were single chromosomes with significant shifts in spatial structure(chr1,chr4,chr21,and chr22).Subsequently,we analysed the transcriptomes of the two samples using transcriptome sequencing,and the results showed that the presence of HBV caused the cells to show a large number of differentially expressed genes(DEGs)compared to the background cells.In addition to the information on the regions of structural alteration found in the three-3D spatial structure analysis of the genome,we obtained DEGs specific to chromatin regions,and their analysis revealed a high correlation with extracellular matrix(ECM)-related functions.The analysis of DEGs also revealed the presence of a series of survival prognosis-related genes with expressions that are closely related to hepatocellular carcinoma in the organism.1.Identification and differential analysis of the 3D spatial structure of the genomeWe used the HBV stable transitional cell line HEPG2.cw and the parental cell Hep G2 as the target,and analyzed the 3D spatial organization of the genomes of the two groups of cells using the mammalian bridging Hi-C technique to investigate alterations in the 3D spatial structure of the nuclear genome in the presence of HBV,we performed library analysis of two cell samples using the mammalian bridging Hi-C technique.The results showed that the two cell lines did not exhibit significant differences in the overall structural characteristics of the chromosomes;however,chromosomes(chr1,chr4,chr21,and chr22)with significantly altered chromatin compartmentalization were detected.Compared to Hep G2 cells,HBV-stable cells exhibited a B2 A transition signature,with results showing higher cellular transcriptional activity in HBV-stable cells and the presence of more chromatin-open,actively expressed chromosomal compartment A.A more refined topologically related structural domain analysis was completed following comparisons of compartment identification,with no significant differences in the number and average length of structural features,similar to the topologically associating domain(TAD).These differences were reflected in the MOTIF enrichment analysis(MEA)of the TAD boundaries.In the MEA,we analyzed the top five MEA-related transcription factors according to the relative levels of different motifs at the TAD boundary.The results showed that the HBV stable transcriptome had differences in the TEAD2,SOX12,CREB3L1,and ZSCAN29 transcription factors combined with MEA.2.Comparative analysis of transcriptome differencesTo investigate the effect of stable HBV expression on host transcript levels,we analyzed the transcriptomes of two cell lines using transcriptome sequencing technology.The results showed that 1737 DEGs appeared between the two cell lines,and Gene Ontology(GO)functional enrichment analysis revealed that the DEGs were enriched in functions such as regulation of ECM,external cellular spatial construction,phylogenic classification,and regulation of biological processes.The presence of HBV results in altered overall transcript levels in the host,mainly related to biological processes,such as ECM functions and biogenesis.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that the ECM receptor response,cancer pathway,WNT signaling pathway,and PPAR signaling pathway were enriched,suggesting that the overall altered transcript levels were close to three.related to cancer-related signaling pathways.3.Combined analysis to assess region-specific DEGsTo comprehensively evaluate the relationship between the transcriptome and 3D spatial structural alterations,we performed a joint analysis of the transcriptome and chromatin conformation capture data.For distinctly different single chromosomes(chr1,chr4,chr21,and chr22),266 region-specific DEGs were screened,and GO functional enrichment of the gene set revealed that the three ECM-related biological functions shared the same 23 differential genes.We then shifted the focus of differential gene studies to this functionally related gene and screened for six DEGs(NID1,SPP1,FGA,FGB,FGG,and TMPRSS6)within hepatocellular carcinoma with the help of tumour expression profile data in The Cancer Genome Atlas Program(TCGA).Further analysis revealed that overexpression of NID1,SPP1,and TMPRSS6 resulted in a higher survival risk for patients,whereas overexpression of FGA,FGB,and FGG resulted in better overall survival.In summary,the present study revealed that stable HBV expression resulted in significant alterations in single chromosomes in host cells,as well as significant alterations in transcriptional expression of genes in the transformed regions,with significant enrichment of extracellular matrix(ECM)and cancer-related signaling pathways in differentially expressed genes.Combining spatial structure and transcriptome analysis revealed a link between differentially expressed genes associated with extracellular matrix(ECM)and genomic alterations,suggesting that HBV may reshape ECM to promote cancer development by altering chromatin spatial structure,leading to alterations in gene expression patterns such as NID1,SPP1,FGA,FGB,FGG,and TMPRSS6.In this study,we investigated the effects of HBV genomic integration on host chromatin spatial structure,providing new insights into the pathogenesis of HBVassociated HCC.