Abstract
Integration of hepatitis B virus (HBV) DNA into the human genome is one of the most important steps in HBV-related carcinogenesis. This study attempted to find the link between HBV DNA, the adjoining cellular sequence, and altered gene expression in hepatocellular carcinoma (HCC) with integrated HBV DNA. We examined 15 cases of HCC infected with HBV by cassette ligation-mediated PCR. The human DNA adjacent to the integrated HBV DNA was sequenced. Protein coding sequences were searched for in the human sequence. In five cases with HBV DNA integration, from which good quality RNA was extracted, gene expression was examined by cDNA microarray analysis. The human DNA sequence successive to integrated HBV DNA was determined in the 15 HCCs. Eight protein-coding regions were involved: ras-responsive element binding protein 1, calmodulin 1, mixed lineage leukemia 2 (MLL2), FLJ333655, LOC220272, LOC255345, LOC220220, and LOC168991. The MLL2 gene was expressed in three cases with HBV DNA integrated into exon 3 of MLL2 and in one case with HBV DNA integrated into intron 3 of MLL2. Gene expression analysis suggested that two HCCs with HBV integrated into MLL2 had similar patterns of gene expression compared with three HCCs with HBV integrated into other loci of human chromosomes. HBV DNA was integrated at random sites of human DNA, and the MLL2 gene was one of the targets for integration. Our results suggest that HBV DNA might modulate human genes near integration sites, followed by integration site-specific expression of such genes during hepatocarcinogenesis.
Highlights
Chromosomal Sequence no.size differentiation localization accession no
Cases 68 and 96 seem to be located close to each other and far from the other samples, indicating that the gene expression patterns of cases 68 and 96 differ from those of the other cases. This result suggests that the samples of hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) integrated into mixed lineage leukemia 2 (MLL2) had characteristic gene expression profiles compared with HCC with HBV integrated into other loci of human chromosomes
We identified new integration sites of HBV DNA in all 15 cases of HCC associated with HBV infection by cassette ligation – mediated PCR
Summary
PLC/PRF/5 cells were grown in DMEM supplemented with 10% fetal bovine serum This cell line was used as a positive control of HBV DNA integration into human genome [11]. Genomic DNA was extracted from cell lines and tumor tissues by proteinase K digestion followed by phenol/chloroform extraction, as previously described [12]. The cloning double-stranded DNA was sequenced by the dideoxy method with fluorescently labeled 2V,3Vdideoxynucleoside 5V-triphosphate with the cassette- or HBV-specific primer. 2 AL of the cDNA obtained were amplified in 40 AL of a reaction buffer containing 20 pmol of the two appropriate primers, four deoxynucleotides each at a concentration of 100 mmol/L, PCR buffer, and 2.5 units of human recombinant Taq polymerase (Takara Bio). Subtracting the background signals from the spot signals, we obtained (R, G) type of gene expression data,
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