DNA Double-strand Breaks, Potential Targets For Hepatitis B Virus Integration

HBV is considerded as the most important etiological factor of HCC. Viral DNA integrations play an important role in the development of hepatocellular carcinoma. Studies have shown that virus integration can take place at multiple sites on various chromosomes. The integration would first lead to genomic instability ; then different locations in the host genome and different integrated viral DNA fragment can result in different effects. The studies on the virus potential integration targets will help to inhibit virus integration. Objective We chose the DNA double-strand breaks(DSBs), one of the most detrimental DNA damage, to see if the sites are specific targets for HBV DNA integration. If the damage can not be repaired timely or perfectly because of interventions of the virus fragment, it would promote the progress of the carcinogenesis, the results will provide new ways to investigate HBV related HCC.Methods We inserted a single 18-bp I-SceI recognition sequence into the EGFP gene of plasmid pEGFP-C1 to obtain the pEGFP/I-SceI vector. Then pEGFP/I-SceI was transfected into HepG2 cells to get the individual clone HepG2/I-SceI, containing the I-SceI endonuclease. The HepG2/I-SceI cells were treated with 2% DMSO and incubated with the patients'serum containing high copies of HBV DNA. Furthermore, pCMV (3×NLS)I-SceI, an I-SceI expression plasmid was tranfected to the HepG2/I-SceI cells after HBV infection.γ-H2AX, the molecular symbol of DSBs, was analyzed by immunofluorescence and western blot. HBV cccDNA and the HBcAg was deteced as the marker of HBV replication . Genomic DNA was then isolated from cell pellets and was digested with I-SceI restriction enzyme to enrich for altered I-SceI recognition sites, the nucleotide sequences of sites-specific integration was amplified by nest PCR and the DNA was excised from agarose gels. Finally by using DNA sequencing, we compared the inserted bases with HBV genome.Results The pEGFP/I-SceI vector was successfully constructed.γ-H2AX was mainly located in the nuclei. Compared with the control groups,γ-H2AX was remarkably increased in pCMV(3NSL)I-SceI group indicating that I-SceI recognition sequence was introduced into the HepG2/I-SceI cells. Significant amounts of HBV cccDNA and HBcAg were detected in the cells DMSO treated implying the possibility of the virus integration. The blasted results showed the homology between the nucleotides inserted into the site-specific DSBs and part of HBV genome is 92%. The nucleotides contained mainly 5'sequence of HBV x region and also some unknown base sequences.Conclusions The DSBs may serve as potential targets for hepadnaviral DNA insertion. The further research can focus on the precise molecular mechanism of virus integration into the DSBs and the relationship between integration and the repair proteins. Try to prevent the virus integration to avoid the genome instability and the series of liver cancer related effects.