E6-AP基因在乳腺癌MDA-MB-231细胞中调节膜联蛋白A2的表达

发布时间：2018-07-27 14:17

【摘要】：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,P0.001;F=904.079,P0.001);分组与时间点存在交互作用(F=28.116,P0.001)。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,P0.050)。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,P0.001)。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。
[Abstract]:Objective to detect the expression of E6-AP gene and Annexin A2 in breast cancer MDA-MB-231 cells, and to investigate the effects of Annexin A2 on the proliferation, apoptosis, invasion and metastasis of breast cancer cells. Methods A sequence of unrelated Negative-siRNA was designed as negative control group and three specific E6-AP-siRNAs fragments for E6-AP gene were transfected into MDA-MB-231 cells as experimental group, untreated cells as blank control group and liposome treated cells as liposome group. The relative expression of E6-AP and Annexin A2 mRNA in MDA-MB-231 cells after interfering with E6-AP was detected by RT-PCR. The highest transfection efficiency of E6-APsiRNA1 group and negative control group were selected, and the control group continued to do the following experiment. Western blot was used to detect the relative expression level of E6-AP and Annexin A2 in MDA-MB-231 cells after interfering with E6-AP. The proliferation, apoptosis and invasiveness of MDA-MB-231 cells after interfering with E6-AP were detected by CCK-8 kit and flow cytometry. The expression level of mRNA and protein, cell apoptosis rate and cell number were compared by ANOVA, LSD method and absorbance analysis were used to compare the expression level of mRNA and protein, the rate of apoptosis and the number of cells. Results after 72 h of transfection, each experimental group (E6-AP-siRNA2 group) and blank control group (E6-AP-siRNA2 group) were transfected with E6-AP gene interference. The relative expression level of E6-AP mRNA in the negative control group and the liposome group was 0.159 卤0.003n 0.325 卤0.006U 0.229 卤0.007 卤0.593 卤0.031U 0.594 卤0.0121.0.612 卤0.016Annexin A2 mRNA, respectively 0.929 卤0.0171.013 卤0.0820.92 卤0.0241.341 卤0.0371.323 卤0.0101.323 卤0.0101.323 卤0.0101.323 卤0.0101.3261.326 卤0.0122.There were significant differences between the two groups (FG 850.792417.447P = 0.050). After 72 hours of transfection, the relative expression levels of E6-AP and Annexin A2 protein in the E6-AP-siRNA1 group, the blank control group and the negative control group were 0.271 卤0.017, 0.492 卤0.018, 0.477 卤0.016 and 0.447 卤0.034, 0.887 卤0.022, 0.849 卤0.033, respectively. There were significant differences between the two groups (F256.850350.149, P = 0.050). E6-AP-siRNA1 group, negative control group and blank control group had significant difference in cell absorbance at different time points (FF524.828P0.001, F904.079, P0.001), and there was interaction between the group and the time point (F28.116, P0.001) after transfection for 24 ~ 448 ~ 72H ~ (72) h after transfection, the difference of cell absorbance was statistically significant between the negative control group and the blank control group (F _ (524.828) P _ (0.001) F _ (904.079) P _ (0.001). 72 h after transfection, the apoptotic rate of control group and negative control group were 2.959 卤0.117 卤3.097 卤0.070 ~ 10.812 卤0.199, respectively. The difference was statistically significant (F _ (3110.005) P _ (0.050) .Transwell detection of E6-AP-siRNA1 group, blank control group, and control group, the apoptosis rate of E6-AP-siRNA1 group was 2.959 卤0.117 卤3.097 卤0.070 卤10.812 卤0.199, respectively. In the negative control group, the number of cells penetrating the Matrigel gel to the lower chamber of Transwell was 99 卤50.96 卤60.62 卤7, respectively, and the difference was statistically significant (FF55.404, P0.001). Conclusion interfering with E6-AP gene can down-regulate the expression of Annexin A2, induce apoptosis of MDA-MB-231 cells, and inhibit the proliferation and invasion of MDA-MB-231 cells.
【作者单位】： 川北医学院附属医院甲状腺乳腺外科;川北医学院附属医院肝胆胰肠研究所;遂宁市中心医院乳腺甲状腺外科;第三军医大学附属西南医院乳腺外科;川北医学院附属医院解剖学教研室;
【基金】：国家自然科学基金资助项目(81172496) 四川省教育厅重点项目(13ZA0228);四川省科技厅科技创新苗子工程项目(2016060)
【分类号】：R737.9
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