RNA干扰抑制高氧暴露下A549细胞中硫氧还蛋白-2表达的研究

Effects of RNA interference on mRNA expression of thioredoxin-2 in A549 cells exposed to hyperoxia

目的研究小分子干扰RNA（small interference RNA，SiRNA）抑制高氧暴露下人肺腺癌A549细胞硫氧还蛋白-2（thioredoxin-2，Trx-2）的表达及其与细胞凋亡的关系，探讨Trx-2对高氧肺损伤线粒体的保护作用。方法体外传代培养A549细胞系，将Trx-2序列特异性的SiRNA通过Lipo—fectamine 2000转染A549细胞，随机分为干扰后空气组、无干扰高氧组和干扰后高氧组。三组分别于高氧或空气暴露12、24、48h后采用RT-PCR技术测定Trx-2和细胞色素c（cytochromec，Cytc）mRNA的表达；采用流式细胞术检测三组A549细胞凋亡情况。结果（1）RT-PCR检测显示序列特异性SiRNA可显著降低Trx-2 mRNA的表达。（2）干扰后高氧组24hTrx-2 mRNA水平为0．2774±0．0174，48h为0．25874-0．0069，均低于无干扰高氧组（分别为0．7799±0．1249，0．4050±0．0842）（P均〈0．05）。无干扰高氧组24hTrx-2 mRNA水平高于干扰后空气组（O．4046±0．0161）（P〈0．05）。（3）无干扰高氧组24hCytc mRNA水平为0．3957±0．0903，48h为0．3438±0．0092，均低于干扰后高氧组（分别为0．6620±0．0395，0．4806±0．0977）和干扰后空气组（分别为0．5197±0．0377，0．4902±0．1079）（P均d0．05）。（4）干扰后高氧组A549细胞凋亡率明显高于干扰后空气组。结论SiRNA能显著下调A549细胞Trx-2 mRNA的表达；高氧暴露诱导SiRNA干扰后A549细胞Trx-2和Cytc mRNA表达的变化表明Trx-2对高氧肺损伤线粒体可能起重要的保护作用。

Objective To study the mRNA expression of thioredoxin-2 suppressed by small interference RNA （SiRNA） in A549 cells exposed to hyperoxia and the relationship with cell apoptosis. To investigate the protection effect of thioredoxin-2 Trx-2 on mitochondria under hyperoxia induced lung injury. Methods A549 cell line was cultured and transfected with synthetic Trx-2-sequence-specific siRNA by Lipofectamine 2000. These cells were randomly divided into three groups as air group with interference, hyperoxia group without interference and hyperoxia group with interference. After exposure to oxygen or room air for 12, 24 and 48 h, Trx 2 and Cytc mRNA were detected by reverse transcription polymerase chain reaction. Flow cytometry was used to detect the apoptosis of A549 ceils in the three groups. Results （1） Trx-2-sequence-specific SiRNA significantly down-regulated the Trx-2 mRNA expression in A549 cells. （2） Trx-2 mRNA levels in hyperoxia group with interference at 24 h and 48 h （0. 2774±0. 0174 and 0. 2587± 0. 0069） were lower than those in hyperoxia group without interference （0. 7799 ± 0. 1249 and 0. 4050 ±0. 0842）（P〈0. 05）. Trx-2 mRNA in hyperoxia group without interference at 24 h was higher than that in air group with interference （0. 4046 ± 0. 0161 ） （P 〈 0. 05）. （3） Cytc mRNA in hyperoxia group without interference at 24 h and 48 h （0. 3957±0. 0903 and 0. 3438±0. 0092） were lower than those in hyperoxia group with interference （0. 6620 ± 0. 0395 and 0. 4806±0. 0977） and air group with interference （0. 5197±0. 0377 and 0. 4902±0. 1079） （P〈0.05）. （4） The apoptosis rate of A549 cells in hyperoxia group with interference was significantly higher than that in air group with interference （P〈 0. 05）. Conclusions Sequence-specific SiRNA targeting Trx-2 significantly down-regulated its expression in A549 cells; Exposure to high concentration of oxygen can significantly change the mRNA expression of Trx-2 and Cytc, which indicates that Trx-2 may protect mitochondria of A549 cells from hyperoxia.