Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells

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, 72 h 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 Notch1-shRNA group was significantly 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-1 cells 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. Key words: Receptor, Notch1; Neoplastic stem cells; Carcinoma, non-small-cell lung; Cell proliferation; Apoptosis