TGF-β1诱导上皮间质转化过程中线粒体合成与功能的改变

Alteration of mitochondrial synthesis and function in epithelial-mesenchymal transition by TGF-β1

目的:研究在转化生长因子-β1(TGF-β1)诱导上皮间质转化过程中线粒体合成与功能的改变。方法:体外培养人肺癌A549细胞,经不同浓度(0、2.5、5、10、20、40 ng/m L)TGF-β1处理24和48 h后观察其形态变化;采用CCK-8法检测经TGF-β1处理48 h后A549细胞的活力;流式细胞术检测细胞凋亡、活性氧(ROS)、线粒体活性氧及线粒体膜电位的变化;酶标仪检测ATP含量;Western blot分别检测细胞中上皮间质转化(EMT)相关标记物(E-cadherin和α-SMA)及线粒体合成相关蛋白(NRF-1及mt-TFA)的表达水平。结果:与对照组相比,随着TGF-β1浓度增加,A549细胞活力逐步上升,呈剂量-效应关系(r=0.941,P<0.05);5~20 ng/m L TGF-β1处理组凋亡率明显升高(P均<0.05);不同浓度TGF-β1处理A549细胞48 h后大部分细胞呈现明显的间质细胞形态,出现上皮间质转化;Western b lot结果显示,与对照组比较,经TGF-β1处理48 h后上皮标记物E-cadherin蛋白表达水平逐渐降低(P<0.05),间质标记物α-SMA表达水平逐渐升高(P<0.05);流式细胞术检测结果显示,5~20 n g/m L T GF-β1处理后细胞内ROS含量上升(P<0.05),线粒体活性氧上升(P<0.05),线粒体膜电位荧光强度下降(P<0.05),ATP含量降低(P<0.05),线粒体合成相关蛋白NRF-1和mt-TFA表达水平均下调(P均<0.05)。结论:TGF-β1诱导细胞发生上皮间质转化过程中线粒体的合成与功能受到影响,并由此促进了EMT的发生。

OBJECTIVE： To investigate changes of ROS and mitochondria on epithelial-mesenchymal transition. METHODS：Human lung cancer A549 cells were divided into control and treated groups. The latter were treated with different concentrations of TGF-β1 （0,2.5,5,10,20,40 ng/mL） and morphological changes were observed after 48 hours. In addition,cell viability was detected by using the CCK-8 kit and cell apoptosis by flow cytometry. DCFH fluorescent probe,MitoSOX fluorescent probe and Rhodamine-123 staining were used to detect ROS and changes in mitochondrial membrane potential, respectively. The content of ATP was detect by using the enhanced ATP kit. The epithelial-mesenchymal transition-related markers and mitochondrial-related proteins were detected by Western blot. RESULTS：The treated groups showed major differences from the control group：cell viability increased gradually and showed a dose-effect relationship with increased concentrations of TGF-β1 （r=0.941,P〈0.05）;there was a significant difference on cell apoptosis after treatment with 5-20 ng/mL TGF-β1;mesenchymal cells were observed after treatment with TGF-β1 for 48 hours, indicating the appearance of epithelial mesenchymal transition;Western blot showed that the expression of epithelial marker E-cadherin decreased gradually and the expression of mesenchymal marker α-SMAincreased gradually （P〈0.05）;the detection of intracellular reactive oxygen showed that treatment with 5-20 ng/mL TGF-β1 induced generation of ROS and mitochondrial ROS （P〈0.05）;mitochondrial membrane potential declined as indicated by fluorescence intensity （P〈0.05）;the content of ATP also decreased （P〈0.05）;and expression of mitochondrial-relatedproteins were downregulated （P〈0.05）. CONCLUSION：TGF-β1 caused increase of reactive oxygen species which led to the occurrence of mitochondrial dysfunction and promotion of EMT.