应用滚环扩增研究肝癌组织中的HBV cccDNA

Amplification of hepatitis B virus covalently closed circular DNA in patients with hepatocellular carcinoma using rolling circle amplification

摘要:

目的：运用滚环扩增（rolling circle amplification，RCA）分离肝癌患者组织中乙型肝炎病毒（hepatitis B virus，HBV）共价闭合环状DNA（covalently closed circular DNA，cccDNA），研究pre-S区的缺失。方法：对RCA技术的特异性和灵敏度进行了研究，并应用该技术扩增13对癌组织和癌旁组织中HBV cccDNA，对pre-S区的缺失进行了研究。结果：RCA能够高效，特异的扩增组织中10拷贝/μl HBV cccDNA；26例样本中，22例样本的HBV cccDNA能够被RCA撤增测序，其中有8例样本（8/22，36.4%）在pre-S区域存在着缺失，pre-S2缺失比例高于 pre-S1（8/8 vs. 2/8，P=0.007）。结论：首次发现肝癌（hepatocellular carcinoma，HCC）组织中HBV cccDNA存在着pre-S区的缺失，有助于深入理解cccDNA pre-S区缺失与HCC的关系。

Abstract:

Objective：To analyze the intrahepatic hepatitis B virus（HBV） covalently closed circular DNA（cccDNA） in hepatocellular carcinoma（HCC） patients. Methods：The specificity and sensitivity of rolling circle amplification（RCA） were investigated. The intra－hepatic HBV cccDNA in 13 pairs of tumor and adjacent non-tumor tissues were amplified by RCA and pre-S deletions were deter－mined by sequencing. Results：The HBV cccDNA（10 copy/μl） could be amplified specifically by RCA. Among 26 samples，the HBV cccDNA in 22 tissues were amplified successfully，and the pre-S deletions were identified in 8 samples（8/22，36.4%）. In addition，the rate of pre-S2 deletion was higher than that of pre-S1（8/8 vs. 2/8，P=0.007）. Conclusion：The pre-S2 deletion of HBV cccDNA is first identified in hepatocellular carcinoma patients，which would be benefit for understanding the relationship between cccDNA pre-S deletion and HCC.