Association Of Hepatitis B Virus Covalently Closed Circular DNA And Human Apobec3 In Hepatitis B Virus-Related Hepatocellular Carcinoma

Obejective:HBV cccDNA is the key of persistent infection. To identify the potential role of host factor in cccDNA degration in HCC,we measured intrahepatic cccDNA levels, and investigated the association between cccDNA levels and expression of APOBEC3 in HCC patients.Methods:We first measured the levels of intrahepatic cccDNA and HBV total DNA in cancerous and contiguous noncancerous of 49 HCC patients by Tag Man probe qPCR, and analysis difference with Wilcoxon Matched-Pairs Signed-Ranks Test.Intrahepatic cccDNA was amplified by rolling circle amplification (RCA),and the preC/C, RT, X, preS region of cccDNA were sequenced.3D-PCR was used to analyse hypermutations rates of G-to-A or C-to-T in matched cancerous and contiguous noncancerous liver tissues. The transcription levels of APOBEC3 were measured with RT-qPCR, its expression levels were confirmed by Western blot. Finally, APOBEC3B proteins was over expressed in HepAD38 cell,and cccDNA in the APOBEC3B-transfection HepAD38 cell were detected by Tag Man probe qPCRResults:.Among 49 HCC patients,35 paired cancerous and contiguous noncancerous liver tissues had detectable cccDNA, and the median intrahepatic cccDNA in the cancerous tissues was significantly lower than that in the contiguous noncancerous tissues (p= 0.0033). Furthermore, G-to-A hypermutations accumulated in cancerous tissues compared with that in contiguous noncancerous tissues (3.9/1000bp vs 0.12/1000bp), and the dinucleotide context showed preferred editing in the 5’GpA,which indicated APOBEC3 would be responsible for cccDNA editing and degradation in cancerous tissues. Among 7 APOBEC3s genes, the APOBEC3A, APOBEC3B and APOBEC3D were up-regulated in the cancerous tissues, while the APOBEC3C, APOBEC3F, APOBEC3G and APOBEC3H were down-regulated.APOBEC3B was up-regulated in cancerous tissues both in transcriptional levels with p value< 0.05 and in expression levels.The overexpression of APOBEC3B in HepAD38 lead to degradation of cccDNA,the inhibition ratio is 42.1%.Conclusion:APOBEC3 play an important role on the innate immune system.APOBEC3B inhibit HBV cccDNA levels depending on cytidine-deamination. This implies APOBEC3 would be responsible for cccDNA editing and degradation in HCC patiens.