ABSTRACT
To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha 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 beta-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 hMYHalpha 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 hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P0.05). 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 hMYHalpha 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.
Subject(s)
DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Hep G2 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/geneticsABSTRACT
Objective To study the effects of the HBV x gene (HBx) on the biological characteristics and the expression of DNA repair enzyme hMTH1 mRNA of the L02/HBx transgene cell model. Methods Light microscopy was used to observe the morphologic characteristics of gene-transfected cell strain Lff2/HBx that stably expressed the HBx protein and the control groups of L02 and L02/PcDNA3.1. The changes of L02/HBx on the proliferation, cell cycle and apoptosis were observed by MTT assays and flow cytometry analysis respectively. Moreover, the malignant transformation of L02/HBxwas assayed by colony formation in soft agar and the expression of DNA repair enzyme hMTH1 mRNA was assayed in each group by real-time qPCR. Results Inversion phase contrast microscope showed that the morphologic characteristics of L02/HBx had changed obviously compared with control groups. The MTT showed that L02/HBx proliferated more quickly and flow cytometry analysis indicated that HBx could accelerate the progression of cell cycle and inhibit apoptosis. Colony formation in soft agar demonstrated that the rate of colony formation of L02/HBx was remarkably higher than the L02 and the L02/peDNA3. 1 cells (P<0. 05). The real-time qPCR detection showed that the expression of hMTH1 mRNA in L02/HBx was significantly higher than that in the control groups ( P < 0. 05 ). Conclusion HBx could play an important role in the malignant transformation of L02/HBx and the over expression of hMTH1 mRNA.