The Study Of Hepatitis B Virus DNA Integration From HBV-related Hepatocellular Carcinomas By HBV-Alu-PCR

Objective To study the relationship of hepatitis B virus(HBV)infection and hepatocellular carcinoma.To investigate the integration role of HBV DNA on the host chromosome and its work on the integration in the formation of hepatocellular carcinoma.To study the mechanism of hepatocellular carcinoma.To apply the method of HBV-Alu-PCR detecting the HBV DNA integration among the HCC tissues.Methods DNA was isolated from the liver tissue samples.Genomic DNA was amplified using three independent primer sets targeting the HBV core,S,and X regions. To investigate the state of HBV DNA in such HCC,HBV DNA was examined by polymerase chain reaction(PCR) between HBV DNA and human Alu sequence (HBV-Alu PCR),which could detect integrated form of HBV DNA only,and by conventional HBV PCR,which could detect both integrated and episomal forms of HBV DNA.Through specially designed primers:U-base primer was introduced. According to the known genes and human Alu repeat sequences(Alu repeat),primers were designed respectively.Using touchdown PCR technology,a highly sensitive PCR derived assay(Alu-PCR) was employed to examine the cloning integration of HBVDNA and the adjacent cell gene sequences.PCR products were sequenced off biotechnology companies.NCBI(national center for biotechnology information) BLAST and MapViewer search were used for accurate identification of HBV location on human genomes.Results In 24 HBsAg positive Hepatocellular carcinoma samples,HBV DNA was detected almost in both the X and pre-C/core regions,even the S region in 17 of 24 (70.8%) by conventional HBV PCR.A highly sensitive PCR derived assay(Alu-PCR) was employed to detect integrated form of HBVDNA.15 samples were showed to have the integrations of HBV fragment.And another 9 samples didn't acquiredthe integration. Among 14 samples integrated,11 samples were found that HBV positively inserted into the host chromosomal DNA.While there were 8 samples inserted inversely.Among these integrations.5 samples were found interrupted both positively and inversely. Analysis from viral-cellular junctions,X gene was found to be interrupted at any length. All were found it inserted at the truncated viral sequences.Conclusion The incidence of hepatocellular carcinoma and the integration of HBV DNA is closely related.HBV X gene is the most common in the integration of HBV regions.HBV integration is not distributed evenly throughout the host genome.