成纤维细胞生长因子13通过ROS/Caspase-3通路调控非小细胞肺癌A549细胞的凋亡

Fibroblast growth factor 13 regulates apoptosis of A549 cells through the ROS/Caspase-3 pathway

目的:探讨成纤维细胞生长因子13(fibroblast growth factor 13,FGF13)对非小细胞肺癌A549细胞活性氧(reactive oxygen species,ROS)的生成和凋亡的影响及其调控机制。方法:WB法检测FGF13在人正常肺上皮细胞BEAS-2B和肺癌A549、H460细胞中的本底表达量。采用FGF13过表达载体转染BEAS-2B和A549细胞;设计两组靶向FGF13的shRNA序列,构建慢病毒干扰载体,包装病毒后侵染A549细胞,采用qPCR和WB法检测干扰效果,DCFH-DA探针结合荧光酶标仪分析敲减FGF13对A549细胞内ROS水平的影响,MitoSOX与WB法检测对线粒体ROS水平及烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphate oxidase 4,NOX4)蛋白表达量的影响,Annexin V-FITC-PI双染法检测对细胞凋亡和Caspase-3及Cleaved Caspase-3蛋白表达的影响。结果:与BEAS-2B细胞相比,FGF13蛋白在两种肺癌细胞中均高表达(均P<0.05)。成功构建FGF13过表达、低表达的A549细胞系。过表达FGF13后,BEAS-2B和A549细胞内ROS水平显著降低(P<0.05);敲减FGF13表达后,A549细胞内ROS水平显著升高(P<0.05);然而过表达及干扰FGF13对A549细胞内线粒体ROS水平无显著影响,但NOX4蛋白表达量显著下调(P<0.05)及显著上调(P<0.05)。FGF13干扰后A549细胞凋亡率显著升高(P<0.01),Caspase-3及Cleaved Caspase-3蛋白表达量显著上调(P<0.05)。结论:FGF13可能通过NOX家族途径调控ROS的生成,并通过ROS/Caspase-3通路调控A549细胞凋亡。

Objective:To explore the effect of fibroblast growth factor13(FGF13)on the generation of reactive oxygen species(ROS and apoptosis of non-small cell lung cancer A549 cells and its regulatory mechanism.Methods:WB was used to detect the background expression of FGF13 in human normal lung epithelial BEAS-2 B cells and lung cancer A549 and H460 cells.BEAS-2 B and A549 cells were transfected with FGF13 over-expression vector.Two groups of shRNA sequences targeting FGF13 were designed to construct lentivirus interference vector.The packaged lentivirus were used to infect A549 cells.qPCR and WB were used to detect the interference efficiency.DCFH-DA probe combined with fluorescence microplate reader was used to analyze the effect of FGF13 knockdown on the level of ROS in A549 cells.MitoSOX and WB were used to detect mitochondrial ROS levels and nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)protein expression levels.Annexin V-FITC-PI double staining method was used to detect the cell apoptosis and expressions of Caspase-3 and Cleaved Caspase-3 protein.Results:FGF13 protein was highly expressed in lung cancer cells compared with BEAS-2 B cell(both P<0.05).A549 cell line with over-expression and low-expression of FGF13 were successfully constructed.Over-expression of FGF13 significantly reduced the level of ROS in A549 and BEAS-2 B cells(P<0.05),while knockdown of FGF13 significantly increased the level of ROS in A549 cells(P<0.05).Though over-expression and interference of FGF13 had no significant effect on mitochondrial ROS levels in A549 cells,NOX4 protein expression was significantly down-regulated(P<0.05)after FGF13 over-expression and significantly upregulated after FGF13 knockdown(P<0.05),respectively.The interference of FGF13 significantly increased the apoptosis rate(P<0.01)of A549 cells and significantly upregulated the protein expression levels of Caspase-3 and Cleaved Caspase-3(P<0.05).Conclusion:FGF13 regulates ROS production possibly through the NOX family pathway and regulates apoptosis of A549 cells by the ROS/Caspase-3 pathway.