Mechanism Of APOBEC3 In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is the main cause of immature death(death before the average life span of a specific population)in China.Chronic hepatitis B virus(HBV)infection is the main risk factor for HCC in China.The coevolution of somatic cells and HBV is the basis for the occurrence and development of HBV related HCC(HBV-HCC).Somatic mutations,HBV genome mutations and HBV gene integration occur frequently and accumulate during the evolution of HCC,which endow cells with survival advantages and induce tumor occure.At present,studies on somatic mutations and HBV integration of HCC mainly focus on nuclear genome,while studies on mitochondrial genome mutations and HBV integration are lacking.Apolipoprotein B m RNA-editing enzyme catalytic polypeptide3(APOBEC3)is the main mutagenic molecule.APOBEC3 are activated in chronic inflammatory environment and induces APOBEC signature mutations in somatic cells and viruses.APOBEC signature mutations are significantly enriched in the nuclear genomes of cancers such as breast cancer,multiple myeloma,prostate cancer and head and neck squamous cell carcinoma,but less enriched in HBV-HCC nuclear genome.However,the enrichment of APOBEC signature mutations in HBV-HCC mitochondrial genome and its relationship with APOBEC3 expression remain unclear.Previous studies found that APOBEC3 expression was significantly associated with genome-wide HBV integration,but the relationship between APOBEC3 and mitochondrial HBV integration remains unclear.It has been confirmed that APOBEC3 expression is related to mitochondrial genome mutations,which is associated with abnormal metabolism and tumorigenesis.However,the roles and mechanisms of APOBEC3-related mitochondrial mutations and metabolic abnormalities in the development and progression of HBV-HCC have not been fully understood.Purpose:The purpose of this study is to explore the roles and mechanisms of HBV-HCC mediated by APOBEC3 through the occurrence of mitochondrial SNV,HBV integration and the related mitochondrial metabolism,and screening potential targeted inhibitors and predictive and early warning markers.APOBEC3A and APOBEC3B are most closely associated with APOBEC signature mutations in cancer genomes.Therefore,this study is mainly focused on APOBEC3A and APOBEC3B.We are aimed at as follows:to study the distribution of single nucleotide variant(SNV)and HBV integration in the transcriptome and genome and its role in HCC development;to elucidate the relationship between APOBEC3A expression and mitochondrial SNV and HBV integration;to investigate the effects of APOBEC3A overexpression on cell phenotype and mitochondrial metabolism in HCC cells;to screen small molecule inhibitors of APOBEC3A;to analyze the risk of single nucleotide polymorphism(SNP)of mitochondrial genes and APOBEC3A in HCC.Methods:1.The distribution of mitochondrial SNV and HBV integration and its relationship with APOBEC3 expression level were analyzed by high-throughput sequencing of HCC tissue samples.Transcriptome sequencing was performed on 48 paired HCC cancer and adjacent tissues.The difference between mitochondrial SNV and HBV integration was analyzed by paired t test.DESeq2 packet of R was used to analyze differences in gene expression levels.The correlation between APOBEC3A expression level and mitochondrial SNV and HBV integration was analyzed by Pearson correlation test.Log-rank test was used to evaluate the effect of mitochondrial SNV and HBV integration on HCC recurrence.GO and KEGG analysis were performed to determine the signaling pathways and biological functions significantly affected by APOBEC3A expression in organoids derived xenograft.2.Exploring the effects of APOBEC3A on malignant phenotype,metabolism and mitochondrial mutations in HCC cells;screening compounds that inhibit APOBEC3A cytidine deaminase activity.APOBEC3A was transfected into Hep G2 and Hep AD38 cells using lipofectamine 3000kit.Cell Counting Kit-8(CCK8)was used to detect cell proliferation.Transwell assay was used to detect cell migration;Cell cycle and apoptosis were detected by flow cytometry.Western blot and real-time quantitative PCR(q RT-PCR)were used to detect gene expression level.Metabolic detection kits(glucose,lactate,NAD~+/NADH,NADP~+/NADPHand glutamine)were used to detect cell metabolism.Compounds that can inhibit APOBEC3A cytidine deaminase activity were screened in vitro by detecting the deamination ability of APOBEC3A on single stranded DNA cytosine.3.A case-control study was conducted to analyze the correlation between the genetic polymorphism of mitochondrial genes and APOBEC3A and the risk of HCC.Two SNPs in APOBEC3A(rs12157810 and rs1014971)and two SNPs in mitochondria(D-loop region,rs41458645;ND3,rs2853826)were determined by q RT-PCR in 1159healthy controls and 2007 HBV infected patients(including 934 HCC patients),and logistic regression was used to analyze the correlation between SNP genotypes and the risk of HCC.Results:1.Distribution of mitochondrial SNV and HBV integration in HCC and its relationship with prognosis.Mitochondrial SNV and HBV integration were different between HCC and paired adjacent tissues.APOBEC3 associated C/G mutations(C>T/G or G>A/C mutation)accounted for 43.3%of mitochondrial SNV,which was significantly higher in cancer tissues than in adjacent tissues(P=0.028).Strictly APOBEC signature mutation(C>T mutation in TCW context and its corresponding G>A mutation)was also significantly higher in cancer tissues than in adjacent tissues(P=0.005).A total of 519 mitochondrial HBV integration mutations were detected in HCC and paired adjacent tissues,accounting for 18.9%of the total HBV integration mutations.The number of mitochondrial HBV integration in cancer tissues was significantly lower than that in adjacent tissues(P=0.011).The relationship between mitochondrial SNV and HBV integration and the prognosis of HCC is different.A higher number of mitochondrial C/G mutations in cancer and adjacent tissues suggests early HCC recurrence,while a lower number of mitochondrial HBV integration in adjacent tissues predicts early HCC recurrence.2.The relationship between the expression of mitochondrial DNA encoding genes,mitochondrial SNV and HBV integration and the prognosis of HCC.The expression of mitochondrial DNA coding genes in HCC tissues was lower than that in adjacent tissues,and its low expression predicted early recurrence.Mitochondrial APOBEC signature mutations were mainly concentrated in CYTB,ND5,TRNQ,ATP6 and ND1,but there was no correlation between the number of APOBEC characteristic mutations and the expression level of these genes.The mitochondrial genes with the highest HBV integration frequency included CYTB,COX1,ATP6,COX3 and ND4,and the number of mitochondrial HBV integration was positively correlated with the expression of these genes.3.Consistency of mitochondrial SNV and HBV integration in RNA and DNA.Mitochondrial SNV and HBV integration was examined at the DNA and RNA level.APOBEC characteristic mutations in mitochondria were found to be highly overlapping at the DNA and RNA levels(The average overlap rate was 86.8%).While HBV integration in mitochondria was not detected at the DNA level.4.The relationship between mitochondrial SNV and HBV integration and APOBEC3 expression.Differential gene expression analysis in HCC and adjacent tissues showed that APOBEC3A and APOBEC3C were down-regulated and APOBEC3B was up-regulated in cancer tissues.The number of mitochondrial C/G mutations and APOBEC signature mutation was negatively correlated with APOBEC3A expression in cancer and adjacent tissues,while the number of mitochondrial HBV integration was not correlated with APOBEC3 expression.5.Effect of APOBEC3A overexpression on malignant phenotype and metabolism of HCC cells.It was found that APOBEC3A inhibited cell proliferation and migration,promoted cell apoptosis and cell cycle arrest at S phase in Hep G2 cells.While APOBEC3A had no significant effect on cell proliferation,migration and apoptosis in Hep AD38 cells,however,cell cycle was also arrest at S phase.The concentration of glucose,lactate,glutamine and glutamate in cell culture supernatant of Hep G2 were increased,and the intracellular NAD~+/NADH was lower while NADP~+/NADPH was not significantly different.In Hep AD38 cells,the concentration of glutamine in cell culture supernatant and NADP+/NADPH in intracellular were lower,while the concentration of glucose,lactic acid,glutamic acid and NAD+/NADH were not significantly different.6.Effect of APOBEC3A overexpression on mitochondrial DNA encoding gene expression in HCC cells.After overexpression of APOBEC3A,the expression of high frequency APOBEC signature mutation and HBV integration gene(CYTB,ATP6,COX1,and ND4)in Hep G2and Hep AD38 cells was analyzed.It was found that the m RNA and protein levels of CYTB were down-regulated in Hep G2 cells.Both CYTB and COX1 were up-regulated in m RNA and protein levels in Hep AD38.7.Relationship between mitochondrial genes and APOBEC3A SNP and HCC risk.After adjusting for age and gender,the mitochondrial gene ND3 SNP rs2853826 was significantly associated with the development of HBV-HCC,and the G genotype of this locus significantly increased the risk of HBV-HCC in healthy people,cirrhosis,and patients with HBV infection(AOR were 1.45,2.35,and 1.45,respectively).The C allele of SNP rs12157810 in APOBEC3A promoter significantly reduced the risk of HCC in healthy subjects and HBV infected patients(AOR were 0.65 and 0.68,respectively).The T allele of SNP rs1014971 located 20kb upstream of APOBEC3A significantly reduced the risk of HBV-HCC in healthy people(AOR=0.82).The effect of rs2853826 and rs1014971 on HCC risk were analyzed after stratification according to gender or HBV genotype.Male and HBV type C infection were the main risk group.Conclusions:1.Mitochondrial SNV in RNA level is mainly derived from the transcription of DNA-level SNV.The level of mitochondrial RNA was highly coincident with the level of DNA SNV.RNA level SNV is transcripted from DNA level SNV,and mutations gradually accumulated during the development of HCC occurrence.Therefore,the number of SNV in HCC tissues is higher than that in adjacent tissues,and is positively correlated with poor prognosis.2.Mitochondrial HBV integration occurs at the RNA level and is independent of HBV integration at the DNA level.HBV integration at mitochondrial RNA level is highly inconsistent with HBV integration at DNA level,so it can be considered that HBV integration at mitochondrial RNA-level is caused by HBV directly attacking the transcript.Therefore,HBV integration at mitochondrial RNA level is correlated with m RNA level of mitochondrial DNA encoding genes,and the relationship between HBV integration quantity and mitochondrial gene expression at mitochondrial RNA level and HCC recurrence is consistent.3.The role of APOBEC3A on malignant phenotype and metabolism of HCC cells was influenced by HBV.In the absence of HBV,APOBEC3A mainly inhibited malignant phenotype.While,in the presence of HBV,APOBEC3A predisposes to attack HBV thus APOBEC3A did not significantly affect the malignant phenotype,but promoted the uptake and biosynthesis of glutamine in HCC cells.4.SNP of mitochondrial gene ND3(rs2853826)and SNP upstream of APOBEC3A(rs12157810)were associated with the risk of HBV-HCC.Rs2853826 G genotype mainly increased the risk of HBV-HCC in male population.Rs12157810 C allele mainly reduces the risk of HBV-HCC in male population and HBV C infected persons.