Effects of down-regulation of Netrin-1 expression by RNA interference on a cutaneous squamous cell carcinoma cell line SCL-1 / 中华皮肤科杂志

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-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 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.