Cao Yu-Hua, Yin Wu, Lyu Yan-Ru
Clinical Tumor Center, People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China.
Department of Pathology, People's Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, China.
Sheng Li Xue Bao. 2021 Dec 25;73(6):893-900.
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 pathway-related 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.
本研究的目的是探讨敲低鞘氨醇激酶-1（SPHK1）对非小细胞肺癌（NSCLC）细胞增殖、细胞周期和凋亡的影响及潜在机制。采用定量逆转录聚合酶链反应（qRT-PCR）检测人健康肺成纤维细胞（MRC-5细胞）和四种NSCLC细胞系中SPHK1 mRNA的表达。然后，将SPHK1-shRNA和相应的阴性对照转染至A549和H1299细胞。分别进行CCK-8、Annexin V-FITC/PI双染和细胞周期检测，以评估细胞增殖、凋亡和细胞周期分布。采用JC-1线粒体膜电位检测试剂盒检测线粒体膜电位。通过蛋白质印迹法检测细胞周期和线粒体凋亡途径相关蛋白以及MEK/ERK信号通路的蛋白表达水平。结果显示，NSCLC细胞中SPHK1的mRNA表达高于MRC-5细胞。SPHK1-shRNA显著抑制A549和H1299细胞的增殖，使细胞周期阻滞于G0/G1期，并通过线粒体途径促进细胞凋亡。与对照组相比，SPHK1-shRNA组中p-MEK和p-ERK蛋白的表达显著下调。此外，MEK/ERK抑制剂可显著抑制细胞增殖并促进细胞凋亡。这些结果表明，敲低SPHK1可抑制NSCLC细胞的增殖，并可能通过抑制MEK/ERK信号通路促进线粒体凋亡途径。
