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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2016, Vol. 10 ›› Issue (06) : 326 -332. doi: 10.3877/cma.j.issn.1674-0807.2016.06.002

论著

E6-AP 基因在乳腺癌MDA-MB-231 细胞中调节膜联蛋白A2 的表达
侯令密1,2, 谢少利1, 陈茂山3, 李敬东2, Emmanuel Ajedichiga Aduah2, 王明浩4, 邓世山5, 幸天勇1, 赵小波1,†()   
  1. 1.637000 四川南充,川北医学院附属医院甲状腺乳腺外科
    2.637000 四川南充,川北医学院附属医院肝胆胰肠研究所
    3.629000 遂宁市中心医院乳腺甲状腺外科
    4.400038 重庆,第三军医大学附属西南医院乳腺外科
    5.637000 四川南充,川北医学院附属医院解剖学教研室
  • 收稿日期:2015-11-10 出版日期:2016-12-01
  • 通信作者: 赵小波
  • 基金资助:
    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)

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

Lingmi Hou1,2, Shaoli Xie1, Maoshan Chen3, Jingdong Li2, Ajedichiga Aduah Emmanuel2, Minghao Wang4, Shishan Deng4, Tianyong Xing1, Xiaobo Zhao1,†()   

  1. 1.Department of Thyroid and Breast Surgery
    2.Institute of Hepatobiliarypancreatic-intestinal Diseases, 5Department of Anatomy, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan Province, China
    3.Department of Breast and Thyroid Surgery,Suining Central Hospital, Suining 629000, Sichuan Province, China
    4.Breast Disease Center, Southwest Hospital,Third Military Medical University, Chongqing 400016,China
  • Received:2015-11-10 Published:2016-12-01
  • Corresponding author: Xiaobo Zhao
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侯令密, 谢少利, 陈茂山, 李敬东, Emmanuel Ajedichiga Aduah, 王明浩, 邓世山, 幸天勇, 赵小波. E6-AP 基因在乳腺癌MDA-MB-231 细胞中调节膜联蛋白A2 的表达[J/OL]. 中华乳腺病杂志(电子版), 2016, 10(06): 326-332.

Lingmi Hou, Shaoli Xie, Maoshan Chen, Jingdong Li, Ajedichiga Aduah Emmanuel, Minghao Wang, Shishan Deng, Tianyong Xing, Xiaobo Zhao. E6-AP gene regulates Annexin A2 expression in breast cancer MDA-MB-231 cells[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2016, 10(06): 326-332.

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-APsiRNA1 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。

关键词: 乳腺肿瘤, 细胞增殖, 细胞凋亡, 肿瘤侵润

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
图1 转染后同一视野下观察E6-AP-siRNA1 组乳腺癌MDA-MB-231 细胞(×50) 注:a 图为普通光学显微镜观察; b 图为荧光显微镜观察
图2 RT-PCR 检测E6-AP 及Annexin A2 的mRNA 表达水平 注:1 为E6-AP-siRNA1 组;2 为E6-AP-siRNA2 组;3 为E6-AP-siRNA3组;4 为阴性对照组;5 为脂质体组;6 为空白对照组
图3 Western blot 检测E6-AP 及Annexin A2 的蛋白表达 注:1 为空白对照组;2 为阴性对照组;3 为E6-AP-siRNA1 组;Mr 代表相对分子质量
吸光度值的比较(±s)
组别 转染时间
24h 48h 72h 96h
空白对照组 0.448±0.027 0.494±0.013 0.607±0.024 0.717±0.020
阴性对照组 0.476±0.044 0.496±0.020 0.602±0.009 0.704±0.020
E6-AP-siRNA1组 0.390±0.028 0.434±0.026 0.480±0.029 0.544±0.016
图4 流式细胞仪检测MDA-MB-231 细胞凋亡 注:a 图为E6-AP-siRNA1 组,b 图为阴性对照组,c 图为空白对照组;PI 为碘化丙啶,Annexin V-FITC 为膜联蛋白5-异硫氰酸荧光素
图5 Transwell 检测乳腺癌MDA-MB-231 细胞的侵袭能力(×100) 注:a、b、c 图分别为空白对照组、阴性对照组、E6-AP-siRNA1 组
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