Lentiviral Vector-mediated HBx Stably Expression In HL7702 Cell And The Role Of HBx On Mitochondria

Objective: To establish a HL7702 cell line stably expressing the HBx which transduced with a lentiviral vector. And to study the effect of HBx on a new HBx-interacted protein COXIII and mitochondria in HL7702 cells.Methods: Using the In-Fusion technology,the recombinant lentiviral vector was constructed by insert the lentiviral vectors with the HBV X gene fragment, which was isolated from pc DNA3.1-X. It was analysised with PCR and sequencing. The recombination lentiviral particles were produced by three plasmid transiently co-transfected into packaging 293 T cell.The viral titer was analysised by quantitative real-time PCR. HL7702 cells were then transfected with the packaged recombinant lentiviral particles, and then resistant cell were selected with puromycin. The expression of HBx in HL7702 cells was examined using RT-PCR and Western-blot. we examined the expression of COXIII,the active of mitochondrial cytochrome C oxidase(COX), the level of mitochondrial membrane potential, the level of intracellular ROS. The distributions of HBx and COXIII proteins were measured by confocal microscopy.The morphological changes of cell and mitochondria were observed by TEM.Results: We successfully established a HL7702 cell line stably expressing HBx in vitro using lentiviral vectors. Stable expression of HBx was verified by RT-PCR and Western-blot. HBx co-localized with the inner mitochondrial protein, COXIII, in HL7702 cells. the co-localization of HBx and COXIII leads to the changes of mitochondrial biogenesis and morphology. Our data shows the exprssion of COXIII protein is up-regulated in HL7702/HBx cells,however m RNA expression doesn’t change in HL7702/HBx cells.COX activity and ROS lever were higher in HL-7702/HBx cells comparesd with other two groups. But mitochondrial membrane potential level shows no difference between threet groups.Conclusion: We assume that HBx located to the inner mitochondrial membrane protein, COXIII, leading to the up-regulation of COXIII protein expression as well as COX activity and the changes of mitochondrial morphology to be slightly swelling.HBx elevated the generation of mitochondrial ROS without effect mitochondrial membrane potential. This provides a new insight into the molecular mechanism of HBV-associated HCC.