Abstract:

Objective

To study the expressions of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells, and explore their effects on the proliferation, apoptosis and invasion and metastasis of cancer cells.

Methods

Negative-siRNA was transfected into MDA-MB-231 cells as negative control,3 designed E6-AP-siRNA sequences were transfected into MDA-MB-231 cells as experimental groups, the untreated cells served as blank control and the cells with liposome treatment served as liposome group. The mRNA expressions of E6-AP and Annexin A2 in MDA-MB-231 cells were detected by RT-PCR after E6-AP interference. The E6-AP-siRNA1 group with the highest transfection efficiency, along with negative control group and blank control group, was selected for the following experiments. The protein expressions of E6-AP and Annexin A2 in MDA-MB-231 cells after E6-AP interference were detected by Western blot. The proliferation, apoptosis and invasion of MDA-MB-231 cells were detected by CCK-8 kit, flow cytometry and Transwell assay respectively after E6-AP interference. Comparison between groups was performed using analysis of variance, pairwise comparison using LSD method. The optical density was compared by repeated measurement analysis of variance.

Results

At 72 h after transfection, E6-AP mRNA expressions in E6-APsiRNA1 group, E6-AP-siRNA2 group, E6-AP-siRNA3 group, blank control group, negative control group and liposome group were 0.159±0.003, 0.325±0.006, 0.229±0.007, 0.593±0.031, 0.594±0.012, 0.612±0.016 respectively, Annexin A2 mRNA expressions were 0.929±0.017, 1.013±0.082, 0.992±0.024,1.341±0.037,1.323±0.010, 1.326±0.012 respectively, indicating statistically significant differences (F=850.792,417.447, both P＜0.050). E6-AP and Annexin A2 protein expressions in E6-AP-siRNA1 group,blank control group and negative control group were 0.271±0.017,0.492±0.018,0.477±0.016 and 0.447±0.034,0.887±0.022, 0.849 ± 0.033, respectively, indicating statistically significant differences (F =256.850,350.149, both P ＜0.050). There was a significant difference in optical density among E6-APsiRNA1 group, negative control group and blank control group at 24,48,72,96 h after the transfection (F=524.828, P＜0.001), and there was also a significant difference in optical density among the different time points (F= 904.079, P＜0.001). There was an interaction between the grouping and the time points (F=28.116, P＜0.001). The apoptosis rates in blank control group, negative control group and E6-AP-siRNA1 group were 2.959±0.117, 3.097±0.070 and 10.812±0.199 respectively, and the difference was significant among groups (F=3110.005, P＜0.050). The number of cells which went through Matrigel gel into lower Transwell chamber in E6-AP-siRNA1 group, blank control group and negative control group was 99±5,96±6,62±7,respectively,suggesting a significant difference among groups (F= 55.404,P＜0.001).

Conclusion

Interference with E6-AP gene may down-regulate the expression of Annexin A2, induce the apoptosis of MDAMB-231 cells, inhibit the proliferation and invasion ability of MDA-MB-231 cells.

Key words: Breast neoplasms, Cell proliferation, Apoptosis, Neoplasm invasiveness