Résumé
A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse tran-scriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of inte-grated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC 157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 re-spectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.
Résumé
To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHα and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma,the gene-transfected cells HepG2/HBx which stably expressed HBx was established,and the effect of HBx on the cell cycle and proliferation of HepG2 was examined.By using the β-actin as the interior control,real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx,the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1).The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1.Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx (0.021±0.007) was significantly lower than that of HepG2 (0.099±0.041) (P<0.05) and HepG2/pDNA3.1 (0.121±0.005) (P<0.05).However,the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains (P>0.05).The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05).It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage,to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function,thus participate in the occurrence and development of hepatocellular carcinoma.