1 Introduction

In most cases of HCC (HCC, hepatocellular carcinoma), the HBV DNA has been found integrated into the DNA of the tumorous hepatocytes (Di Bisceglie, 2009). And the integrated HBV DNA can exist as whole genomes or reorganized subgenomic fragments. Dejean et al. sequenced the host-viral DNA junctions of two integrated HBV DNA sequences cloned from two different human liver tumors (Dejean et al., 1984). In each clone, one junction mapped to within one of two identical 11- base-pair (bp) 5' T-T-C-A-C-C-T-C-T-G-C cohesive - end sequences (DR1 and DR2) that are directly repeated near the extremities of the viral (+) and (-) strands, which start at nucleotides 1590 and 1824, were termed DR (DR, direct repeat region)1 and DR2, respectively. It is known by us that this HBV DR region was a crucial region about the integration of HBV, but it is also a crucial region about the replication of HBV. Between DR1 and DR2 region, there exists a gap, when the virus replicates, the gap will be filled up ahead of time. As far as the integration site of the viral DNA is concerned, reconmbinational mechanism via the single - stranded gap region of the viral genome have been proposed the integrated HBV DNA appears to have one fixed end at the virus - cell junction within the DRs. However, the function of this region was known very little by us, and the formation mechanism of HBV cccDNA (covalently closed circular DNA) and the integration mechanism of HBV DR region has not yet been reported, so the research on this region is very important. So we constructed the pcDNA3.1(+) - HBV Creg eukarya expression vector, in this Creg fragment, it contained DR region, X gene, C gene, and the regulatory sequence, then we made it transfect into HepG2 cells to find out the integration mechanism of HBV DR region.

The HBV Creg DNA fragment from nt1087 to nt2488 including DR region, X gene and C gene that will be cloned into eukarya expression vector pcDNA3.1(+) is in the DNA model graph below that was illustrated as Figure 1.

2 Materials and Methods

Primer design: The primers were designed on the basis of HBV ayr subtype genome from gene bank (ID: 5536), using the software primer5, synthesized by Invitrogen company. EcoR I and Nde I restriction sites were added on 5' end sequences of forward and reverse primers respectively, P1F 5'-AGTGTATCATATGCTTTCACTTTCTCGCCAACTTAC-3'(1087-1111), P1R 5'-GCCTACAGCCTCCTAGTACAAAGACCTT-3'(1760-1788), P2F 5'-GTTAAAGGTCTTTGTACTAGGAGGCTGTAGG-3'(1757---1788), P2R 5'-TGGAATTCCAGTAAAGTTTCCCACCTTATGAGT-3'(2462-2488), the outline and italic letters were restriction sites.

DNA extraction and PCR amplification: Hepatitis B virus DNA was extracted from HBsAg, HBeAg and anti - HBc positive serum of HBV infected patient, and then submitted to recombinant PCR amplification. PCR reaction was carried out for amplification of HBV nt1087-2488 DNA fragment.

The PCR reaction can be described as:

The first PCR reaction: The PCR reaction contained 10 μl of DNA, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The nt1087-1788 PCR reaction use primer p1F and p1R, the nt1788-2488 PCR reaction use primer p2F and p2R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.

PCR reaction was carried out within 30 cycles: denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, and elongation at 72℃ for 50 sec.

Electrophoresis and DNA purification: PCR product was submitted to electrophoresis using 1% agarose gel, stained by ethidium bromide (EB) and visualized under ultraviolet light (UV Trans illuminator), and then recovered by gel DNA extraction kit (TIANGEN Cat. No I7804).

The second PCR reaction: The PCR reaction contained 0.05 mM dNTP, 5μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV nt1087-1788 PCR purified product and nt1788-2488 PCR purified product, 2.5 unit of Taq DNA polymerase in 50 μl final volume.

PCR reaction was carried out within 20 cycles: denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, and elongation at 72℃ for 50 sec.

The third PCR reaction: The PCR reaction contained 10 μl of the second PCR reaction product, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR reaction use primer p1F and p2R), 2.5 unit of Taq DNA polymerase in 50 μl final volumes.

PCR reaction was carried out within 30 cycles: denaturation at 94℃ for 30 sec, annealing at 56℃ for 30 sec, and elongation at 72℃ for 90 sec.

Electrophoresis: PCR product was submitted to electrophoresis using 1% agarose gel, stained by ethidium bromide (EB) and visualized under ultraviolet light (UV Trans illuminator).

Cloning: pcDNA3.1(+) and HBV Creg DNA fragment was digested by blunt end cutter EcoRⅠand NdeⅠrestriction enzyme, the double digested product of HBV Creg DNA fragment was recovered by DNA purified kit (Promaga 230262), and the double digested product of pcDNA3.1(+) was submitted to electrophoresis using 0.5% agarose gel and then the larger DNA fragment was recovered by gel DNA extraction kit (TIANGEN I7804), Then the two DNA fragment that was purified were ligated together by T4 DNA ligation enzyme and transformed in E. Coli DH5αstrain, and then selected on LB agar containing 100 μg/ml of ampicillin. The transformant colony was inoculated into 3 ml LB medium and allowed to grow at 37℃ in a shaker at 200 rpm overnight. The next day, recombinant plasmid was extracted by plasmid extraction kit (TIANGEN Cat. No I7723) and digested with EcoRⅠand NdeⅠenzymes and electrophoresed through 0.8% agarose gel. And also, the recombinant plasmid was examined by PCR. Gel contained DNA fragment (containing HBV Creg DNA fragment) was seizured by scalpel under long wave UV (Sambrook and Russell, 2001).

DNA sequencing: The colony was sent to TIANGEN corporation (Beijing), so as to submit for sequencing by dideoxy chain termination method.

Assay of gene integration: Briefly, the HepG2 cells was transfected with recombinant plasmid by Lipofectamine^TM2 000 (Invitrogen 455 000) and selected in RPMI1640 medium containing 100 μg/ml of G418 (MDBio Inc 813 089). The transfected cells was inoculated into 25 ml RPMI 1 640 medium to grow at 37℃ 5% CO2 in a incubater for about a month and the medium was changed every 2 days. The total DNA of HepG2 cells was extracted and detected in the day when the HepG2 cells were transfected 4 days and 30 days, Untransfected control culture was analyzed in parallel.

The total DNA was extracted from the HepG2 cells that were transfected 4 days, 30 days and Untransfected control culture, then the HBV Creg DNA fragment, HBV X gene fragment and HBV C gene fragment were detected by PCR respectively, The PCR reaction can be described as:

The HBV Creg DNA extending PCR reaction: The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR reaction use primer P1F and P2R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.

PCR reaction was carried out within 30 cycles: denaturation at 94℃ for 30 sec, annealing at 60℃ for 30 sec, and elongation at 72℃ for 40 sec.

The HBV X DNA extending PCR reaction: The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR reaction use primer P1F and P1R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.

PCR reaction was carried out within 30 cycles: denaturation at 94℃ for 30 sec, annealing at 60℃ for 30 sec, and elongation at 72℃ for 40 sec.

The HBV C DNA extending PCR reaction: The PCR reaction contained 0.5 μl of DNA, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The PCR reaction use primer P2F and P2R), 2.5 unit of Taq DNA polymerase in 50 μl final volume.

PCR reaction was carried out within 30 cycles: denaturation at 94℃ for 30 sec, annealing at 60℃ for 30 sec, and elongation at 72℃ for 40 sec.

Electrophoresis: PCR product was submitted to electrophoresis using 1% agarose gel, stained by ethidium bromide (EB) and visualized under ultraviolet light (UV Trans illuminator).

3 Results

The HBV Creg DNA fragment was isolated by recombinant PCR.

The HBV Creg DNA fragment was amplified from extracted DNA of hepatitis B infected patient serum by using recombinant PCR. The PCR product was sequenced and then tested by the software chromas based on GenBank accession number ID: 5536.

Figure 2 (A) and Figure 2 (B) illustrated the recombinant PCR product at 1 401 bp parallel with the DNA size marker (DNA marker D2000). The PCR product was cloned into the pcDNA3.1 (+) expression vector and named it pcDNA3.1 (+) HBV Creg, then the recombinant plasmid was transformed in E. coli DH5αstrain, and the transfected into HepG2 cells.

Recombinant plasmid pcDNA3.1 (+) HBV Creg was constructed.

The recombinant plasmid pcDNA3.1 (+) HBV Creg was extracted from E. coli DH5αstrain, and then detected by PCR and double digested respectively. Figure 3 (A) illustrated the detection result of the recombinant plasmid pcDNA3.1 (+) HBV Creg by PCR, and Figure 3 (B) illustrated the detection result of the recombinant plasmid pcDNA3.1 (+) HBV Creg by double digested using blunt end cutter EcoRⅠand NdeⅠrestriction enzyme, both Figure 3 (A) and Figure 3 (B) illustrated a DNA fragment at 1 401 bp, that indicated the recombinant plasmid pcDNA3.1 (+) HBV Creg was successfully constructed.

Detection of HBV integration into HepG2 cells.

The total DNA was extracted from HepG2 cells when the day after the HepG2 cells were transfected 4 days and 30 days, and then detected by PCR. Figure 4 (A) illustrated the whole HBV Creg DNA sequence can be amplified by PCR in the day when the HepG2 cells were transfected 4 days (before integrated into host genome), Figure 4 (B) illustrated in the day when the HepG2 cells were transfected 30 days (after integrated into host genome), the whole HBV Creg DNA sequence cannot be amplified by PCR using the primer P1F / P2R, but 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, C gene which is in the downstream of DR region can be detected too. So the HBV Creg DNA sequence integrates into the HepG2 genome not as a whole but upstream or downstream from DR region.

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 at least two groups with virus-cell junction at DR1 and DR2 downstream through the core gene or upstream through the X gene, this may related to the replication and integration function of HBV DR region. Since DRs are 5’ ends of the minus and plus DNA strands of the linearized HBV genome, it is reasonable to argue that the frequent use of the DRs as integration sites is likely due to the preferred use of the free ends of replication intermediates of HBV. Although fusions can occur close to both DR1 and DR2, the viral human fusion transcripts were strongly biased to regions near DR1. But it is still known little by us how the gap is filled up to form cccDNA, whether the gap filled up by recombinant PCR separated again when the DNA fragment integrated into host genome that we don’t know. So whether the four groups included in the HepG2 cells, it needs to further research. This DNA fragment that we isolated has a significance to find out the mechanism of how the gap is filled up and it is very important to make it clear that what is the relationship between the DR region and the integration of HBV. By researching this HBV DR region, maybe we can know more about the formation mechanism of HBV cccDNA, Furthermore, we can know more about the replication and integration mechanism of HBV DNA. In addition, researching this DNA fragment maybe has great significance for preventing the replication of HBV.

5 Conclusions

The HBV Creg fragment can integrate into HepG2 cells genome. And the integration of HBV is related to DR region, the way of HBV Creg DNA fragment integrated into HepG2 cells is from DR region to both sides.

Competing interest

The authors declare that they have no competing interests.

Compliance with ethics guidelines

This article does not contain any studies with human or animal subjects performed by any of the authors.

Author contributions

Haoxiang Luo conceived the study, performed the experiments, and wrote the paper. Long Gu and Lihong He participated in the study design, experiments and literatures. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the Science and Technology Department of Guizhou Province of China (No. J [2013]2268).

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