Use Reverse Polymerase Chain Reaction Detection Mhcc97-h Hepatoma Cells Integrated Hepatitis B Virus Sequences

Hepatocellular carcinoma(HCC) is a poorly prognostic malignant tumor and is one of the most frequently occurring cancers in Asia. A few of biological and chemical substances, for instance, hepatitis B virus (HBV), hepatitis C virus(HCV) and flavacin, are the risk agents of HCC. China is a prevalent region with HBV infection and HCC,about 10% of its population is infected with HBV and a portion of those individuals who are chronically infected will eventually develop into HCC. HBV infection is closely related to the occurrence and development of hepatocellular carcinoma,and HBV integrations into hepatocyte have great significance to the pathogenesis of HCC.The research of mechanisms and consequences of HBV DNA integration will further elucidate the molecular biologic mechanism of HCC development and lay the foundation of exploring new treatment techniques and methods.This work applies conventional polymerase chain reaction (PCR) to identify approximate location of HBV DNA breakpoint and accordingly designes two pairs of reverse primers in the viral gene adjacent to virus-host junction, and then digests the left-side and right-side of the viral genome with the endonuclease Apa I and Nco I Respectively. After circularization by self-ligation, inverse polymerase chain reaction (IPCR) is used to amplify the integration sequences of the human high metastatic potential hepatocellular carcinoma cell line MHCC97-H genome.The PCR products are then inserted into pGEM-T vector and sequencing analyzed.The sequencing data indicates that the breakpoint on the left-hand side of HBV integrant is at nucleotide 2220 of C gene, while on the right-hand side, it is at nucleotide 1590 of X gene, The integrated HBV DNA fragment is around 2570 bp, and contains the entire S gene and incomplete C gene, X gene and P gene. BLAST analyses of the integrated human genome fragments reveale that HBV DNA integrates into human keratin gene at chromosome 12.IPCR is a rapid,accurate and economic approach to investigate HBV DNA integration sequences.