RNA干扰神经突起导向因子Netrin-1表达下调对鳞状细胞癌SCL-1细胞的影响

Effects of down-regulation of Netrin-1 expression by RNA interference on a cutaneous squamous cell carcinoma cell line SCL-1

目的探讨神经突起导向因子Netrin-1表达下调对皮肤鳞状细胞癌（鳞癌）SCL-1细胞增殖、凋亡和迁移的影响。方法脂质体2000将Netrin-1siRNA和对照siRNA转染皮肤鳞癌SCL-1细胞。将细胞分为对照1组（未处理组）、对照2组（siRNA对照组）、实验组（Netrin-1siRNA组）。Western印迹检测转染Netrin-1siRNA后SCL-1细胞Netrin-1蛋白的表达。人胆囊收缩素／缩胆囊素8肽（CCK一8）ELISA试剂盒分析细胞增殖的变化，流式细胞仪检测细胞凋亡情况。Western印迹检测caspase-3、caspase-9及MMP-2的表达。结果Netrin-1siRNA明显下调SCL-1细胞中Netrin-1蛋白的表达。Netrin-1表达下调明显抑制SCL-1细胞的增殖和诱导细胞凋亡，增加caspase-3和caspase-9的表达，同时可显著抑制MMP-2的表达。结论Netrin-1表达下调可介导鳞癌细胞增殖抑制、增高细胞凋亡率和降低细胞迁移能力。

Objective To investigate the effects of down-regulation of Netrin-1 expression on cell proliferation, apoptosis and migration of a cutaneous squamous cell carcinoma （CSCC） cell fine SCL-1. Methods Unspecific （control） small interference RNA （siRNA） and Netrin-l-targeting siRNA were designed and constructed. SCL-1 cells were divided into 3 groups to remain untreated （control group 1）, be transfected with control siRNA （control group 2） and Netrin-l-targeting siRNA （experiment group） by using Lipofectamine 2000 respectively. After 48 hours of additional culture, flow cytometry was performed to detect the apoptosis of ceils, Western blot to determine the protein expression of caspase-3 and -9, Netrin-1 and matrix metalloproteinases （MMP） in these cells, and Boyden chamber was utilized to evaluate the migration of cells. Enzyme linked immunosorbent assay （ELISA） was performed with cholecystokinin-8 （CCK-8） kit to estimate the proliferation of cells at 24, 48, 72 and 96 hours after the transfection. Results The expression of Netrin-1 protein was significantly downregulated in SCL-1 ceils in the experiment group compared with the control group 1 and 2 （0.207 ,± 0.044 vs. 0.741 ± 0.111 and 0.716 .± 0.109, F = 31.43, P 〈 0.05）. The Netrin-l-targeting siRNA significantly downregulated the proliferation of SCL-1 cells, but induced the apoptosis in SCL-1 ceils with significant differences in the percentage of early apoptotic cells and late apoptotic cells between the control group 1, 2 and experiment group （11.74 ± 0.33 vs. 11.55 ± 1.22 vs. 24.74 ,± 2.43, F = 68.63, P 〈 0.01; 3.82 ± 0.32 vs. 4.69 ± 0.68 vs. 5.33 ,± 0.18, F = 8.58, P 〈 0.05）. The experiment group showed a significant increase in the expression of caspase-3 and -9 （1.458 ± 0.111 vs. 0.398 ,± 0.057 and 0.412 ± 0.049, F = 183.99, P 〈 0.01; 0.967 ± 0.099 vs. 0.234 ± 0.036 and 0.205 ± 0.033, F = 138.11, P 〈 0.01）, but a statistical decrease in the expression of MMP-2 （0.235 ± 0.038 vs. 0.862 ± 0.040 and 0.892 ,± 0.035, F = 291.07, P 〈 0.01） compared with the control group 1 and 2. Coneluslons The down-regulation of Netrin-1 expression can suppress the proliferation and migration of SCL-1 cells, but promote the apoptosis in SCL-1 cells.