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

Effects of small interfering RNA targeting hepatitis B virus X protein on mitochondrial function in healthy liver ceil line steadily expressed X gene

目的构建并鉴定靶向HBVX蛋白（HBx）小干扰RNA（siRNA）重组表达质粒，观察其对稳定表达HBx基因的正常人肝细胞株HL-7702／HBx线粒体功能的影响。方法设计合成两条针对全长HBx基因具有短发夹结构的siRNA序列，克隆至载体psiRNA—Hh1GFPzeo中，构建重组表达质粒pX1和pX2，并以不针对任何序列的重组质粒pScr作为对照。通过脂质体介导转染入HL-7702／HBx肝细胞株，RT—PCR及Western印迹检测其对HBx的阻抑效应。流式细胞仪检测细胞内活性氧（ROS）和线粒体膜电位（Δψm）水平的变化。采用方差分析对实验结果进行统计分析。结果酶切鉴定和测序结果证实pX1和pX2构建成功。pScr、pX1、pX2分别转染入HL-7702／HBx细胞48h后，空白对照组HBxmRNA和蛋白表达水平分别为0．65±0．12和0．62±0．09，pX1组分别为0．33±0．10和0．19±0．08，明显低于空白对照组（t=4．73，P〈0．05；t=7．53，P〈0．05），pX2组分别为0．48±0．10和0．37±0．11，亦明显低于空白对照组（t=2．39，P〈0．05；t=4．43，P〈0．05），但pXl组抑制效果强于pX2组（t=2．28，P〈0．05）。RNA干扰后细胞内ROS水平为5．00±0．38，Aqrm水平为33．864-0．50；空白对照组ROS为72．10±0．55，AWm为3．57±0．26（t=276．22，P〈0．05；t=107．15，P〈0．05）。结论靶向HBx的siRNA能特异性抑制HBx在HL-7702／HBx细胞中的表达，并降低细胞内ROS水平，提高AWm水平，减轻细胞内氧化应激反应。

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 mitochondrial 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 HhlGFPzeo to construct 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 intracellular reactive oxygen species （ROS） and mitochondrial membrane potential （Δψm） 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 Δψm 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;Δψm 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 Δψm, thus relieve cellular oxidative stress.