Objective:To analyze the association between intrahepatic levels and mutation of hepatitis B virus covalently closed circular DNA(HBV ccc DNA)in hepatitis B virus-related hepatocellular carcinoma. Methods:The levels of intrahepatic HBV ccc DNA

HBV total DNA and pg RNA were measured by real-time quantitative PCR. Intrahepatic ccc DNA was amplified by rolling circle amplification(RCA)

and the pre C/C

RT

X

pre S region of ccc DNA were sequenced. Finally

the AP sites of intrahepatic ccc DNA in cancerous tissues were measured. Results:The mean intrahepatic HBV ccc DNA level was significantly lower in the cancerous tissues than in the contiguous noncancerous tissues(3.32 copies/cell vs. 9.64 copies/cell

P=0.029)

while there was no significant difference in the median levels of intrahepatic HBV total DNA and pg RNA between the cancerous tissues and the contiguous noncancerous tissues(HBV total DNA:16.94 copies/cell vs. 12.18 copies/cell

P=0.325;pg RNA:75.54 copies/cell vs. 22.46 copies/cell

P=0.128). GA mutation was the main mutation pattern for pre C/C and X gene regions. The dinucleotide context of the G-to-A mutation in pre C/C and X gene regions showed preferred editing in the 5’Gp A

accounting for 37.1% and 40% in cancerous. Finally

the levels of intrahepatic HBV ccc DNA in cancerous tissues decreased with APE1 treatment(1 011 copies/μl vs. 501.8 copies/μl

P=0.058)

indicating HBV ccc DNA in the cancerous tissues contains AP site. Conclusion:The intrahepatic HBV ccc DNA level is lower in the cancerous tissues than in contiguous noncancerous tissues

which maybe associated with high mutations of G-A and AP site.