Effect of emodin combined with survivin shRNA on proliferation of human breast cancer MCF-7 cells

Abstract

Objective To investigate the effect of emodin combined with survivin shRNA on the proliferation of human breast cancer MCF-7 cells. Methods The human breast cancer MCF-7 cells were cultured and treated with different concentrations of emodin (0, 10, 20, 40, 80 μmol/L). The effect of emodin on the proliferation of human breast cancer MCF-7 cells was determined by MTT assay to select suitable concentration (40 μmol/L) for subsequent experiments. Then the human breast cancer MCF-7 cells were divided into five groups: blank control group, negative control group (NC shRNA group, treated by empty plasmid), emodin treatment group (treated by 40 μmol/L emodin), survivin shRNA treatment group (transfected with survivin shRNA plasmid) and combined treatment group (treated by 40 μmol/L emodin plus survivin shRNA). The proliferation (optical density) and apoptosis of human breast cancer MCF-7 cells in each group were analyzed by MTT assay and flow cytometry, respectively. Because the result of MTT assay showed no significant difference in cell proliferation activity between blank control group and negative control group, no negative control group was established for the flow cytometry. Survivin mRNA and protein expressions were also assessed using real-time PCR and Western blot analysis, respectively. The apoptosis rate and survivin mRNA and protein expression were compared among multiple groups by one-way analysis of variance. The optical density was compared by repeated measurement analysis of variance. Pairwise comparison was performed using LSD method. Results The result of MTT assay showed that after 24, 48, and 72 h treatment of emodin at different concentrations (0, 10, 20, 40, 80 μmol/L), the optical density of each group was significantly different (F=1 873.565, P 50.0%), so this concentration of emodin was used for the following experiments. After the cells were treated with differeht methods for 24, 48, and 72 h, the optical density presented a significant difference among blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group (F=1 686.953, P<0.001), the optical density was significantly different at different time points (F=288.790, P<0.001), and there was an interaction between grouping and time points (F=67.916, P<0.001). The results of flow cytometry showed that the apoptosis rate was (0.57±0.10)%, (5.96±0.56)%, (9.00±0.73)% and (18.33±0.98)% in blank control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=114.848, P<0.001). The apoptosis rate of emodin treatment group was significantly higher than that of blank control group (P<0.001); the apoptosis rate of combined treatment group was significantly higher than that of any other group (P<0.001). The results of real-time PCR showed that survivin mRNA expression was 1.000±0.000, 0.968±0.033, 0.774±0.049, 0.483±0.026 and 0.364±0.012 in blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=284.199, P<0.001). Pairwise comparison showed that survivin mRNA expression in combined treatment group was significantly lower than that in emodin treatment group (P<0.001) or survivin shRNA treatment group (P=0.001). Western blot analysis showed that survivin protein expression was 1.000±0.000, 0.987±0.031, 0.696±0.050, 0.534±0.065 and 0.312±0.039 in blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=142.311, P<0.001). Pairwise comparison showed that survivin protein expression in combined treatment group was significantly lower than that in emodin treatment group or survivin shRNA treatment group (both P<0.001). Conclusion The combination of emodin and survivin shRNA can inhibit the proliferation of human breast cancer MCF-7 cells. Key words: Breast neoplasms; Emodin; Survivin; shRNA; Cell proliferation