Genomics and Applied Biology 2016, Vol.7, No.5, 1-8
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control using total DNA of untransfected cells as templet. (B) After the HBV CregDNA integrate into the HepG2 genome, in lane3,
the whole HBV CregDNA cannot be detected by PCR using primer P1F / P2R and using total DNA of transfected cells as templet,
but in lane1, X gene which is in the upstream of DR region can be detected using total DNA of transfected cells as templet and using
primer P1F / P1R, in lane2, C gene which is in the downstream of DR region can be detected too
4 Discussions
In the worldwide, more than three hundred million people are chronically infected with HBV (Hwang et al., 2003).
It is estimated that chronic hepatitis B will be at a higher risk of developing hepatocellular carcinoma. HBV DNA
has been shown to become integrated within the chromosomes of infected hepatocytes, the length and the
components in the HBV DNA integrant varies considerably and the viral DNA may be rearranged, deleted or
present in repeats, if the integration of HBV DNA has been observed within the retinoic acid receptor alpha gene
and within the human cyclin A gene, both playing crucial roles in cellular growth, but if the HBV DNA
integration site does not appear to be in a critical location, it is not the process of integration itself that leads to
HCC (John and Venetia, 2007). The association between HBV infection and the mechanism of HBV - related
chronic hepatitis and hepatocellular carcinoma remains unclear. Several viral and host factors have been suggested
to be involved in the chronicity and diversity of HBV - associated disease (Koshy et al., 1983). The sequence DR
region of HBV was considered to be the critical about HBV gene integrated into host genome, which is related to
hepatocellular carcinoma (Milich et al., 1990). But it is still unclear how this region integrates into the hepatocytes
genome. And also, the sequence DR region of HBV was considered to be the critical about replication of HBV.
As it is known, between DR1 and DR2 region, there exists a gap, when HBV replicated, the gap would be filled
up to form cccDNA. But it is also known little by us what the formation mechanism of cccDNA is. For these
reasons, we constructed the pcDNA3.1 (+) - HBV Creg eukarya expression vector including the DR region, the
aim of our study was to identify how the DR region integrated into host genome. Following these results showed
as Figure 4 (A), we found that the HBV Creg DNA fragment can be amplified entirely from X gene to C gene
including the DR region in the middle of it using the primer P1F and P2R when the HepG2 cells were transfected
4 days, and in this time, some of the HBV Creg DNA fragment have entered the cells, but without integration into
the hepG2 cells genome. However, as the results showed in Figure 4 (B), when the HepG2 cells were transfected
30 days, the HBV Creg DNA fragment had already integrated into hepG2 cells genome, and in this time, we
cannot amplify the entire HBV Creg DNA fragment from X gene to C gene, but we can amplify the X gene and
the C gene separately. In this study, we predicted that X gene that is upstream from DR region and C gene that is
downstream from DR region can be integrated into HepG2 genome separately, but Creg DNA sequence cannot be
integrated into HepG2 genome as a whole, it maybe for the reason of the DR region which is in the middle of the
HBV Creg DNA. Many data have indicated that the integration of HBV has the relation to homologous
recombination by DR region. Hino et al. suggest a mechanism for integration of the viral DNA molecule which
involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule
(at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by
the deletion of 11 base pairs of cellular DNA during the double recombination event (Hino et al., 1989). The
evidence suggests that the integrated HBV DNA appears to have one fixed end at the virus - cell junction within
the DRs, while the other end appears to be at variable positions (Jiang et al., 2012). In our study, the HBV Creg
DNA fragment which contained DR region can integrate into the hepG2 cells genome, DR1 and DR2 can mediate
viral integration, but X gene and C gene can be integrated into it respectively because of the DR region. To
explain the various structures of integrations, Shih et al. proposed that categorized viral integrations into four
groups according to the structure of the open circular molecule, their end specificity and strand polarity (Shih et
al., 1987). The four groups include integrations with one virus-cell junction at DR1 with the viral DNA extending
either downstream through the core gene (group I) or upstream through the X gene (group II) and integrations
with one virus-cell junction at DR2 with the viral sequences extending either through the core gene (group III) or
the X gene (group IV). In this study, we had detected the DNA integration fragment of HBV in the genome of
HepG2 cells, HBV X gene and HBV C gene could be detected separately, but the full length of HBV Creg DNA
fragment had not been detected all the time, so we predicted the integration of HBV Creg DNA fragment includes
