Abstract:

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 line SCL-1. Methods Unspecific （control） small interference RNA （siRNA） and Netrin-1-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-1-targeting siRNA （experiment group） by using Lipofectamine 2000 respectively. After 48 hours of additional culture, flow cytometry was performed to detect the apoptosis of cells, 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 cells 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-1-targeting siRNA significantly downregulated the proliferation of SCL-1 cells, but induced the apoptosis in SCL-1 cells 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. Conclusions 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.

Key words: cell migration