| BackgroundHepatocellular carcinoma?hepatocellular carcinoma,HCC?caused by chronic hepatitis B virus?HBV?infection?CHB?is one of the leading causes of death in China.Somatic mutation is the basis of HCC evolution,and HBV integration is a unique type of mutation in HBV-HCC.The accumulation of HBV integrations in HCC genome has been ovserved by many researchs,suggesting that these mutations can give cells the advantages of survival,and thus promote hepatocarcinogenesis.Current researchs in this area have several limitations:First,studies of HBV integration are limited to the genome level and the study on transcriptome level is rare.The mechanism and degree of HBV integration in genome affects the gene transcription are still elusive.The generation and reverse transcription of prgenomic RNA are essential to the HBV replication.It is unclear if the HBV prgenomic RNA can directly integrate into the RNA of host during the process of HBV replication.Second,the significant heterogeneity of HBV mutation spectrums among individuals obstructs the comprehensive detection and analysis of HBV integrations.Third,TERT and MLL4 were proved to be frequently attacked by HBV integrations.However,less then 2%of the total HBV integrations located in these two genes,and the carcinogenic functions of the others are still unclear.Last,the mechanism of the generation of HBV integration remains uncertain.The family of apolipoprotein B mRNA editing enzyme catalytic polypeptide 3?APOBECE3?is one of the major causes of somatic and viral mutations which can induce the break of nucleic acid strands.To the best of our knowledge,no studies have been published to investigate the effects of APOBEC3s on HBV integration.The development of HBV-HCC is an evolutionary process affected by the genetic,immune,and viral factors.The study on the effects of single nucleotide polymorphisms?SNPs?of APOBEC3s is important for clarifying the HBV-induced hepatocarcinogenesis.PurposeThe purpose of this study was to elucidate the distribution of HBV integration at the genome and transcriptome level,to explore the mechanism of HBV integration occurrence and its role in the HCC evolution process,to clarify the relationship between APOBEC3-induced mutation and HBV integration,and to analysis the correlations between APOBEC3s genetic polymorphisms and virus elimination,viral mutations,and HCC risk.This study can provide novel biomarkers for cancer prevention of HBV-infected population.MethodsHBV-capture sequencing and RNA sequencing were conducted for HCC tissues and paired adjacent tissues from 50 HCC patients.The personalized HBV reference sequences were generated from the standard sequences of different HBV genotypes which were revised with HBV mutations of each individual.All reads were aligned to the reference sequences of human nuclear genome,mitochondrial genome,and HBV genome to search chimeric reads of HBV integration mutaions.Eighty DNA and RNA integration sites were randomly selected and were verified with nest PCR and Sanger sequencing.The number of support PE-assembled reads?NNSS?and chimeric reads were used to described the signal intensity of HBV integration mutaions in DNA and RNA levels,respectively.HBV integrations in DNA strands with NNSS?1 and HBV integrations in RNA strands with reads?2 were selected for analysis.?2 test was used to investigate the distribution of HBV integration sites on human genome,on viral genome,and among populations with different clinical characteristics.To avoid the influence of low-frequency random HBV integration mutations,the upper quartile values of signal intensity were used as cut-off points to select high intensity HBV integration mutaions,which were used to verify the important distribution patterns.T test and ANOVA were used to evaluate the difference of gene expression.Wilcoxon test was used to analysed the difference of HBV integration levels.Pearson correlation test was used to investigated the relationship between the expression of APOBEC3 and the number of HBV integration.Log-Rank test was conducted to evaluate the effects of HBV integration on HCC prognosis.P<0.05 was considered as statistically significant.KEGG and GSRA datebase were used to identified the pathways and bio-functions that are closely related with HBV integration.Three sequencing or expression public databases of HCC patients were downloaded,including HBV capture sequencing database?SRP068532,426 samples?,HCC cohort database of The Cancer Genome Atlas?TCGA,LIHC cohort,366samples?,and HCC expression database of Gene Expression Omnibus?GEO,GSE14520,242 samples?.These data were used to verify the patterns and effects of HBV integration discovered in this study.Four SNPs in the APOBEC3A,APOBEC3B,and UNG were selected.SNP genotyping was performed in 1449 healthy controls and 3772 HBV infected patients?including 1271HCC patients?by fluorescence quantitative PCR.PreS and BCP segments of HBV were amplified by nested PCR,and MEGA software was applied to align and analysis HBV mutations.Logistic regression was used to calculate the correlation between SNP genotypes and virus clearance,virus mutation,and HCC risk.Results1.The patterns of HBV integration on DNA and RNA levels are significantly different.Due to the customization of the HBV reference sequence based on viral mutations,the detect rate of HBV integration mutaions was increased.A total of 2777 and 2918 HBV integration mutations were detected in the genome and the transcriptome,respectively.At the DNA level,the number of HBV integration in cancer tissue was significantly higher than that in adjacent tissues(P=1.0×10-4).The number of HBV integration in tumors consecutively increased among HCC patients without recurrence,patients with late-recurrence?>2 years?,and patients with early-recurrence??2 years?.At the RNA level,the number of HBV integration in cancer tissue was significantly lower than that in adjacent tissues(P=1.2×10-3)and it was similar among populations on different stages of HCC evolution.Only 0.5%of the HBV integration mutations at the DNA level were distributed in the mitochondrial genome,while19.43%of the RNA HBV integration mutations located in the mitochondrial genome.This difference was repeated in the validation cohort.2.HBV X is the main fragment integrated into human DNA/RNA.Integraion events of HBV X region?nt1374-nt1835?accounted for 31.6%,35.1%and 58%of all HBV integration events in the DNA of whole genome,RNA of mitochondrial genome,and RNA of nuclear genome,respectively.These rates are significantly higher than the random integration rate?14%?.3.HBV frequently integrated genes.At the DNA level,292 repeatedly integrated genes were determined?in the region of these genes,HBV integration mutations were detected in more than 2samples?,while only 11 of them were classified as repeatedly integrated genes on the RNA level as well.Only 2 genes?TERT and MLL4?were found to bear highly consistent HBV integration mutations in the DNA and RNA data from the same sample.On the DNA level,the detection rate of HBV integration in the TERT gene was the highest?16%?,which all located in the promoter region.On the RNA level,the HBV integration detection rate of ALB gene was the highest?51%?and all the 13 coding genes in the mitochondrial genome were integrated with HBV DNA with repeated detection rates ranging from13.2%to 43%.4.Correlation between HBV integration and gene expression.At the DNA level,HBV integration in the TERT promoter region was correlated with increased TERT gene expression in cancer tissues(P=3.89×10-8).The integration of the reverse fragment of HBV X at the 3kb upstream to TERT may also have the similar effect.The HBV integration in the other repeatedly integrated genes,such as MLL4 and MT-ND4,were not related with gene expression.On the RNA level,HBV integration in the MLL4,ALB,HP,MT-CO2 and MT-CYB gene was associated with elevated gene expression.5.Signaling pathways related to HBV integration.The HBV integration mutation on the DNA level was significantly enriched in the PI3K-mTOR pathway and chromatin remodeling pathway,with the overall integration rate to ranging from 30%to 20%,respectively.The integration mutation at the RNA level was significantly enriched in inflammatory pathways including IL2-STAT5 and IL6-STAT3.6.The prognostic effects of HBV integration on HCC.At the DNA level,the high total number of HBV integration mutaions in adjacent tissue indicated early relapse?P=0.012?,which was verified in the validation cohort?P=0.014?.On the RNA level,the high total number of HBV integration events in the nuclear genome and mitochondrial genome of adjacent tissue indicated poor overall survival of HCC patients?P=0.027and P=0.033,respectively?.7.The correlation between APOBEC3 expression and HBV integration.The expression of APOBEC3A in cancer tissue was positively related to the total number of HBV integration events both on the DNA and RNA level[DNA:Pearson correlation coefficient?r?=0.374,P=0.008;RNA:r=0.302,P=0.035];the expression of UNG in cancer tissue was negatively related to the total number of HBV integration events on the RNA level?RNA:r=-0.388.P=0.008?;the expression of APOBEC3B in adjacent tissue was positively related to the total number of HBV integration events on the RNA level?r=0.322,P=0.024?.In tumor tissues,the high expression of APOBEC3A,APOBEC3B,and UNG were related with the worse prognosis.The high level of APOBEC-signature mutaions can only indicate poor prognosis in HCC patients without HBV infection but not in HBV-HCC patients.8.The relationship between APOBEC3B and UNG SNPs and the risk of HCCIn HBV infected population,after adjustment for gender and age,rs2267401 TG+GG genotype of APOBEC3B can significantly increase the risk of HCC[AOR?95%CI?=1.52?1.226-1.889?];rs3890995 TC+CC genotype of UNG significantly increased the risk of HCC[AOR?95%CI?=1.28?1.109-1.477?].9.The relationship between APOBEC3B and UNG SNPs and HBV mutationIn subjects infected by genotype C HBV,after adjustment for age and sex,rs2267401 TG+GG genotype significantly increased the level of APOBEC related virus mutation as well as occurrence of A1762T/G1764A;rs3890995 TC+CC genotype significantly increased the frequency of 11 cancer promoting mutations in the virus.10.Interaction of APOBEC3B and UNG SNPs with HBV mutations in HBV-HCCIn people infected by genotype C HBV,after adjustment for age and sex,interaction of rs2267401TG+GG and A1762T/G1764A significantly increased the risk of HCC[AOR?95%CI?=6.05?3.407-10.726?,the interaction of rs3890995 TG+GG genotype and G146C significantly increased the risk of HCC[AOR?95%CI?=4.14?2.679-6.399?].Conclusions1.There is a significant difference between the distribution of HBV integration at DNA and RNA levels.The major source of HBV integration in RNA is the direct insertion of virus fragments to the host RNA,but the transcription of the chimeric genes generated by the HBV integration in DNA.2.On the RNA level,HBV integration significantly affects the genes related to mitochondrial aerobic oxygenation respiratory chain.At the DNA level,HBV integrated genes are significantly enriched in the carcinogenic pathway.The high numbers of HBV integration mutations in DNA and RNA were both significantly related to the poor prognosis.The carcinogenic effects of HBV integration were preliminarily defined.3.It is found that the occurrence of HBV integration at the RNA level is positively correlated with the expression of APOBEC3A and APOBEC3B,and it is also closely related to the inflammatory and immune pathways.These evidences preliminarily proved that the HBV integration at the RNA level is caused by increased expression of mutagenic molecules stimulated by inflammatory pathway.4.While the expression of APOBEC3A,APOBEC3B,and UNG can affect HCC prognosis,the APOBEC-signature mutation was only related with the prognosis of HCC patients without HBV infection,the SNPs of APOBEC3B and UNG?rs2267401 and rs3890995?can increase the HCC risk by promoting the generation of high risk HBV mutations,suggesting these genes promoting carcinogenesis by interacting with HBV rather than directly injure the human genome during the evolution of HBV-HCC. |
PDF Full Text Request