乙型肝炎病毒感染相关性肝癌中HBV cccDNA水平与突变关系的研究

Association between intrahepatic levels and mutation of hepatitis B virus covalently closed circular DNA in hepatitis B virus-related hepatocellular carcinoma

目的:研究乙型肝炎病毒(hepatitis B virus,HBV)感染相关性肝癌(hepatocellular carcinoma,HCC)中HBV共价闭合环状DNA(covalently closed circular DNA,ccc DNA)水平与突变关系。方法:运用特异性荧光定量PCR定量分析了12例肝癌组织和癌旁组织中乙肝病毒复制水平的表征因子HBV ccc DNA,HBV total DNA及前基因组RNA(pregenome RNA,pg RNA)的表达水平,再用滚环扩增(rolling circle amplification,RCA)特异性扩增癌组织中HBV ccc DNA,测序分析了病毒基因组4个编码调控区域pre C/C、RT、X、pre S中ccc DNA序列的突变。最后检测了肿瘤组织ccc DNA的AP位点。结果:HBV ccc DNA在癌组织的表达水平明显低于癌旁组织(3.32 copies/cell vs.9.64 copies/cell,P=0.029),但是HBV总DNA和pg RNA的表达没有明显差异(16.94 copies/cell vs.12.18 copies/cell,P=0.325,75.54 copies/cell vs.22.46 copies/cell,P=0.128);在肝癌组织中,HBV ccc DNA pre C/C和X区间,G-A突变是主要的突变类型;其次,pre C/C和X区域发生G-A突变的位置上,均更倾向于GA二联核苷酸组合方式,在肝癌中所占比例为37.1%和40%。最后,肿瘤组织中APE1酶处理后,HBV ccc DNA水平明显下降(1 011 copies/μl vs.501.8 copies/μl,P=0.058)。结论:HBV ccc DNA水平在癌组织中明显低于癌旁组织,可能与癌组织中HBV ccc DNA发生大量GA突变及形成的AP位点有关。

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.