Effects And Mechanism Of Hepatitis B Virus X Gene On Mitochondrial Function Of Health Adult Liver Cell Line HL-7702

AIM: To investigate the effects of hepatitis B virus (HBV) X gene and X protein (HBx) on mitochondrial function in health adult liver cell line HL-7702 by RNA interference and discuss the possible mechanisms in the pathway.METHODS: Molecular cloning technique was used to construct and identify recombinant expression plasmid pX1 and pX2, which of small interfering RNA(siRNA)targeting HBx, and pScr specific to unrelated sequence served as a control. After transfection into HL-7702/HBx cells line by liposome, reverse transcription-polymerase chain reaction (RT-PCR) and western blot were applied to identify the down regulation of HBx expression by siRNAs. And the level of reactive oxygen species (ROS) and mitochondrial membrane potential(?Ψm) were detected by flow- cytometry. Western blot was used assess signal conduction pathway associated protein of oxidative stress: Bax,Bcl-2,cytochrome C (cyt C),caspase-9 and caspase -3 in group HL-7702/HBx, group pX1, group pScr and group N-Acetylcysteine (NAC).RESULTS: Enzyme cutting and sequencing results confirm the construction of pX1 and pX2 successfully. Compared to blank group, after transfection into HL-7702/HBx cells for 48h, the expression level of HBx mRNA and protein in group pX1 and pX2 were decreased obviously (P<0.05). But the inhibition of group pX1 was stronger than that of pX2 (P<0.05). The analysis of flow-cytometry show RNA interference could obviously decrease ROS and increase ?Ψm in HL-7702/HBx cells. There was a significant distinction (P<0.05) when compared with blank group. By western blot, mitochondrial Bax, cytosolic cyt C, cleavage of caspase-9 and caspase-3 were lower in group pX1 and NAC than in group HL-7702/HBx. The expression of mitochondrial Bcl-2 and cyt C were upregulated. The distinction was significant (P<0.05).Conclusions: HBx can induce mitochondrial oxidative stress of host cell through decreasing the level of ROS and increasing the level ofΔΨm,which in association with mitochondrial- injured endogenous apoptosis signal conduction pathway.