Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells / 国际肿瘤学杂志

ABSTRACT

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.