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

摘 要 ] 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。

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

Abstract] 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-2B cells and lung cancer A549 and H460 cells. BEAS-2B 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 was 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 knock-down 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-2B cells (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-2B 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.