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目的检测E6-AP基因及膜联蛋白A2(Annexin A2)在乳腺癌MDA-MB-231细胞中的表达,探讨其对癌细胞增殖、凋亡及浸润转移的影响。方法设计1条无关序列Negative-siRNA作为阴性对照组和针对E6-AP基因的3条特异性E6-AP-siRNAs片段转染至MDA-MB-231细胞内作为实验组,未经处理细胞作为空白对照组,加入脂质体处理的细胞为脂质体组,利用RT-PCR检测干扰E6-AP后在MDA-MB-231细胞中E6-AP和Annexin A2 mRNA相对表达水平。选择出转染效率最高的E6-APsiRNA1组及阴性对照组、空白对照组继续行后续实验。Western blot检测干扰E6-AP后E6-AP和Annexin A2在MDA-MB-231细胞中的蛋白的相对表达水平。利用CCK-8试剂盒法、流式细胞术、Transwell小室侵袭实验分别检测干扰E6-AP后MDA-MB-231细胞的增殖、凋亡、侵袭能力。基因的mRNA及蛋白表达水平、细胞凋亡率及细胞数的组间比较采用方差分析,两两比较采用LSD法,吸光度比较采用重复测量的方差分析。结果转染72 h后,E6-AP基因干扰后各实验组(E6-AP-siRNA1组、E6-AP-siRNA2组、E6-AP-siRNA3组)及空白对照组、阴性对照组及脂质体组中的E6-AP mRNA相对表达水平分别为0.159±0.003、0.325±0.006、0.229±0.007、0.593±0.031、0.594±0.012、0.612±0.016,Annexin A2 mRNA相对表达水平分别为0.929±0.017、1.013±0.082、0.992±0.024、1.341±0.037、1.323±0.010、1.326±0.012,差异均有统计学意义(F=850.792、417.447,P均<0.050)。转染72 h后,E6-AP-siRNA1组、空白对照组和阴性对照组中E6-AP及Annexin A2蛋白相对表达水平为分别为0.271±0.017、0.492±0.018、0.477±0.016及0.447±0.034、0.887±0.022、0.849±0.033,组间差异均有统计学意义(F=256.850、350.149,P均<0.050)。转染24、48、72、96 h后,E6-AP-siRNA1组、阴性对照组和空白对照组间比较,不同时间点之间比较,细胞吸光度差异均有统计学意义(F=524.828,P<0.001;F=904.079,P<0.001);分组与时间点存在交互作用(F=28.116,P<0.001)。转染72 h后,空白对照组、阴性对照组、E6-AP-siRNA1组的凋亡率分别为2.959±0.117、3.097±0.070、10.812±0.199,组间差异有统计学意义(F=3110.005,P<0.050)。Transwell检测E6-AP-siRNA1组、空白对照组、阴性对照组中细胞穿透Matrigel胶到达Transwell下室的细胞数分别为99±5、96±6、62±7,组间差异有统计学意义(F=55.404,P<0.001)。结论干扰E6-AP基因可使Annexin A2表达下调,同时可诱导MDA-MB-231细胞的凋亡,其增殖、侵袭能力也受到抑制。
Objective To detect the expression of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells and to explore its effect on proliferation, apoptosis and invasion and metastasis of breast cancer cells. Methods Negative siRNA was designed as negative control group and 3 specific E6-AP-siRNAs fragments targeting E6-AP gene were transfected into MDA-MB-231 cells as experimental group, untreated cells as blank In the control group, the liposome-treated cells were liposome group. The relative expression levels of E6-AP and Annexin A2 mRNA in MDA-MB-231 cells were detected by RT-PCR after interference with E6-AP. The E6-APsiRNA1 group and the negative control group with the highest transfection efficiency were selected, and the blank control group continued to follow-up experiments. Western blot was used to detect the relative expression of E6-AP and Annexin A2 in MDA-MB-231 cells after interference with E6-AP. The proliferation, apoptosis and invasion of MDA-MB-231 cells were detected by CCK-8 kit, flow cytometry and Transwell chamber invasion assay respectively. The mRNA and protein expression level, apoptosis rate and cell number of the two groups were compared by analysis of variance (ANOVA), pairwise comparison by LSD, and absorbance by repeated measures ANOVA. Results E6-AP-siRNA group, E6-AP-siRNA2 group, E6-AP-siRNA3 group and blank control group, negative control group and liposomes The relative expression levels of E6-AP mRNA 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. The relative expression levels of Annexin A2 mRNA were 0.929 ± 0.017 and 1.013 ± 0.082,0.992 ± 0.024,1.341 ± 0.037,1.323 ± 0.010,1.326 ± 0.012, the difference was statistically significant (F = 850.792,417.447, P <0.050). After 72 h of transfection, the relative expression levels of E6-AP and Annexin A2 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 respectively, 0.887 ± 0.022,0.849 ± 0.033, there was significant difference between the two groups (F = 256.850, 350.149, P <0.050). After transfection for 24, 48, 72 and 96 h, there were significant differences in cell absorbance between E6-AP-siRNA1 group, negative control group and blank control group at different time points (F = 524.828, P <0.001; F = 904.079, P <0.001). There was interaction between groups and time points (F = 28.116, P <0.001). The apoptosis rates of blank control group, negative control group and E6-AP-siRNA1 group after 72 h transfection were 2.959 ± 0.117, 3.097 ± 0.070 and 10.812 ± 0.199, respectively. The difference between the two groups was statistically significant (F = 3110.005, P <0.050). Transwell assay E6-AP-siRNA1 group, blank control group, negative control group cells through the Matrigel to reach the lower chamber of Transwell cells were 99 ± 5,96 ± 6,62 ± 7, the difference between the groups was statistically significant (F = 55.404, P <0.001). Conclusion The interference of E6-AP gene can down-regulate the expression of Annexin A2 and induce the apoptosis of MDA-MB-231 cells. The proliferation and invasion ability of E6-AP is also inhibited.