E6-AP基因在乳腺癌MDA-MB-231细胞中调节膜联蛋白A2的表达

E6-AP gene regulates Annexin A2 expression in breast cancer MDA-MB-231 cells

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 study the expressions of E6-AP gene and Annexin A2in breast cancer MDA-MB-231cells,and explore their effects on the proliferation,apoptosis and invasion and metastasis of cancer cells.Methods Negative-siRNA was transfected into MDA-MB-231cells as negative control,3designed E6-AP-siRNA sequences were transfected into MDA-MB-231cells 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 A2in MDA-MB-231cells were detected by RT-PCR after E6-AP interference.The E6-AP-siRNA1group 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 A2in MDA-MB-231cells after E6-AP interference were detected by Western blot.