Effects of small interfering RNA targeting hepatitis B virus X protein on mitochondrial function in healthy liver cell line steadily expressed X gene
10.3760/cma.j.issn.1000-6680.2009.08.002
- VernacularTitle:靶向乙型肝炎病毒X蛋白小干扰RNA对稳定表达X基因的正常人肝细胞株线粒体功能的影响
- Author:
Rongfeng HUANG
;
Na LIN
;
Hongying CHEN
;
Zhixin CHEN
;
Xiaozhong WANG
- Publication Type:Journal Article
- Keywords:
Hepatitis B virus;
Viral proteins;
RNA,small interfering;
Oxygen;
Membranepotentials;
Mitochondria,liver
- From:
Chinese Journal of Infectious Diseases
2009;27(8):454-460
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and identify recombinant expression plasmid of small interfering RNA (siRNA)targeting hepatitis B virus X protein(HBx), and observe its effect on mitoehondrial function in healthy liver cell line steadily expressed HBx gene (HL-7702/HBx). Methods Two siRNA sequences containing short hairpin structure, which target on the total length HBx gene, were synthesized and cloned into the vector psiRNA-Hh1GFPzeo to eonstruct recombinant expression plasmids pX1 and pX2. Non-specific recombinant pScr plasmid served as control. After siRNA transfected into HL-7702/HBx cells line by liposome, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to identify the suppressive effect on HBx expression. Levels of intraeellular reactive oxygen species (ROS) and mitochondrial membrane potential (△ m) were determined by flow cytometry. The experimental results were compared by analysis of variance. Results Successful constructions of pX1 and pX2 were confirmed by restriction enzyme digestion and sequencing. The expressions of HBx mRNA and protein after 48 h of transfection into HL-7702/HBx cells in control group were 0.65± 0.12 and 0.62± 0.09, respectively, which were both higher than those (0.33±0.10 and 0.19±0.08, respectively) in group pX1 (t=4.73, P<0.05; t=7.53, P<0.05) and those (0.48±0.10 and 0.37±0.11, respectively) in group pX2 (t=2.39, P<0.05;t=4.43,P<0.05). But the inhibition of group pX1 was stronger than that of pX2 (t=2.28,P<0.05). Levels of ROS and △ m after RNA interference were 5.00±0.38 and 33.86±0.50, respectively, while those in control group were 72. 10±0. 55 and 3. 57±0.26, respectively (ROS: t=276.22, P<0.05; △ m: t=107.15, P<0.05). Conclusions siRNA targeting HBx can efficiently and specifically suppress the HBx expression in HL-7702/HBx cells, and decrease the level of ROS and increase the level of △ m, thus relieve cellular oxidative stress.
