敲低鞘氨醇激酶-1对非小细胞肺癌细胞增殖及线粒体凋亡途径的影响及机制

The effect and mechanism of sphingosine kinase-1 knockdown on non-small cell lung cancer cell proliferation and mitochondrial apoptotic pathway

本文旨在探讨敲低鞘氨醇激酶-1(sphingosine kinase-1,SPHK1)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖、周期和凋亡的影响,并探讨其可能机制。用定量逆转录聚合酶链反应(qRT-PCR)检测人胚肺成纤维细胞系(MRC-5)和4种NSCLC细胞中SPHK1 mRNA表达水平。利用SPHK1-shRNA和相应的阴性对照转染A549和H1299细胞。用CCK8法测定细胞增殖,用Annexin V-FITC/PI双重染色试剂盒评价细胞凋亡,用细胞周期检测试剂盒测定细胞周期分布,用JC-1线粒体膜电位测定试剂盒检测线粒体膜电位,用蛋白质印迹法检测细胞周期和线粒体凋亡途径相关蛋白、MEK/ERK信号通路的蛋白表达水平。结果显示,NSCLC细胞SPHK1 mRNA表达水平高于MRC-5细胞。SPHK1-shRNA显著抑制A549和H1299细胞的增殖,并促进线粒体途径的细胞凋亡,将细胞周期阻滞在G0/G1期;与对照组相比,SPHK1-shRNA组MEK和ERK蛋白磷酸化水平显著下调。MEK/ERK抑制剂可抑制A549和H1299细胞增殖,并促进细胞凋亡。上述结果提示,SPHK1敲低可能通过抑制MEK/ERK信号通路来抑制NSCLC的增殖和促进线粒体途径的细胞凋亡。

The purpose of the present study was to investigate the effect and potential mechanism of knockdown of sphingosine kinase-1(SPHK1)on the proliferation,cell cycle and apoptosis of non-small cell lung cancer(NSCLC)cells.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect SPHK1 mRNA expression in human healthy lung fibroblasts(MRC-5 cells)and four NSCLC cell lines.Then,A549 and H1299 cells were transfected with SPHK1-shRNA and corresponding negative control.CCK-8,Annexin V-FITC/PI dual staining and cell cycle assay were performed to evaluate cell proliferation,apoptosis and cell cycle distribution,respectively.JC-1 mitochondrial membrane potential measurement kit was adopted to measure mitochondrial membrane potential.Western blot was used to detect the protein expression levels of cell cycle and mitochondrial apoptotic pathwayrelated proteins,as well as MEK/ERK signaling pathway.The results showed that the mRNA expression of SPHK1 in NSCLC cells was higher than that in MRC-5 cells.SPHK1-shRNA significantly inhibited the proliferation of A549 and H1299 cells,blocked the cell cycle in G0/G1 phase,and promoted cell apoptosis through the mitochondrial pathway.Compared with the control group,the expression of p-MEK and p-ERK proteins in the SPHK1-shRNA group was significantly down-regulated.Moreover,MEK/ERK inhibitor could dramatically suppress cell proliferation and promote cell apoptosis.These results suggest that SPHK1 knockdown can inhibit the proliferation of NSCLC cells and might promote mitochondrial apoptotic pathway by inhibiting MEK/ERK signaling pathway.