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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 (01) : 10 -13. doi: 10.3877/cma.j.issn.1674-0807.2016.01.003

论著

Rac1 蛋白调控乳腺癌MDA-MB-231 细胞分泌血管内皮生长因子的研究
马骥1, 赵庆丽1,†(), 赵易1, 刘颖1   
  1. 1.730050 解放军兰州军区兰州总医院乳腺科
  • 收稿日期:2015-08-25 出版日期:2016-02-01
  • 通信作者: 赵庆丽
  • 基金资助:
    国家青年科学基金资助项目(81202085)甘肃省科技计划资助项目(1506RJDA296)兰州市科技计划资助项目(2014-1-39)

Regulatory effect of Rac1 protein on vascular endothelial growth factor secreted in breast cancer MDAMB-231 cells

Ji Ma1, Qingli Zhao1,†(), Yi Zhao1, Ying Liu1   

  1. 1.Department of Breast Surgery, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
  • Received:2015-08-25 Published:2016-02-01
  • Corresponding author: Qingli Zhao
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马骥, 赵庆丽, 赵易, 刘颖. Rac1 蛋白调控乳腺癌MDA-MB-231 细胞分泌血管内皮生长因子的研究[J/OL]. 中华乳腺病杂志(电子版), 2016, 10(01): 10-13.

Ji Ma, Qingli Zhao, Yi Zhao, Ying Liu. Regulatory effect of Rac1 protein on vascular endothelial growth factor secreted in breast cancer MDAMB-231 cells[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2016, 10(01): 10-13.

目的

探讨Rac1 蛋白对乳腺癌MDA-MB-231 细胞分泌血管内皮生成因子(VEGF)的调控作用。

方法

在乳腺癌MDA-MB-231 细胞中分别转染Rac1 正显性真核表达质粒(V12Rac1 组)及空白对照质粒(空白对照组)和Rac1 小干扰RNA(siRac1 组)及对照小干扰RNA (沉默对照组)后,通过ELISA 分别检测转染后24、48、72 h 由MDA-MB-231 细胞分泌的VEGF 蛋白水平;通过Western blot 检测各组血管生成相关因子p53 和VEGF 表达的变化。 两组Western blot 定量分析数据比较采用t 检验,VEGF 分泌数据采用重复测量的方差分析。

结果

ELISA 分析结果显示:当MDA-MB-231 细胞中高表达Rac1 时,细胞分泌的VEGF 水平随着时间的延长而逐渐升高,与空白对照组相比组间差异有统计学意义(组间比较:F=837.122,P ＜0.001;不同时间点比较:F=57 806.374,P ＜0.001;交互作用:F=7 663.095,P＜0.001);当MDA-MB-231 细胞中低表达Rac1 时,细胞分泌的VEGF 水平随着时间的延长逐渐升高,但低于沉默对照组,差异有统计学意义(组间比较:F=511.891,P＜0.001;不同时间点比较,F=268.078,P＜0.001;交互作用:F=120.708,P=0.001)。 Western blot 结果显示,当MDA-MB-231 细胞高表达Rac1,与空白对照组相比,p53 蛋白表达降低(t=-6.392, P=0.003),VEGF 蛋白表达升高(t=7.497, P=0.002);当MDA-MB-231 细胞中低表达Rac1,与沉默对照组相比,p53 蛋白表达增加(t=5.307, P=0.006),VEGF 蛋白表达降低(t=-7.395, P=0.002)。

结论

在乳腺癌MDA-MB-231 细胞中,Rac1 表达高低可以引起细胞分泌VEGF 的水平变化,Rac1 可能通过抑制p53 表达而增加VEGF 表达。

关键词: 乳腺肿瘤, 血管内皮生长因子类, 肿瘤抑制蛋白质p53, Rac1 蛋白质,人

Objective

To investigate the regulatory effect of Rac1, one member of Rho protein family, on vascular endothelial growth factor (VEGF) in breast cancer MDA-MB-231 cells.

Methods

MDAMB-231 cells were transfected by Rac1 constitutively-active mutant plasmid pCEFL-GST-V12Rac1 (V12Rac1 group), blank plasmid pCEFL-GST-neo (blank control group), small interference Rac1 RNA (siRac1 group)and small interference RNA of control (si-control group) respectively. VEGF expression levels in MDA-MB-231 cells were determined by ELISA assay at different time points(24, 48, 72 h after transfection). Western blot assay was performed to detect the relative expressions of angiogenesis-related factors p53 and VEGF.VEGF expression levels were compared by repeated measurement analysis of variance and t test was used to compare the quantitative data of Western blot.

Results

ELISA assays showed that after up-regulating Rac1 expression in MDA-MB-231 cells, VEGF level was gradually increased with time, indicating a significant difference compared with blank control group (group comparison: F=837.122, P ＜0.001; comparison of different time points:F=57 806.374,P＜0.001;interaction of grouping and time:F=7 663.095,P＜0.001).After down-regulating Rac1,VEGF level was significantly lower than that in si-control group (group comparison:F=511.891,P＜0.001; comparison of different time points: F=268.078,P＜0.001; interaction of grouping and time: F=120.708,P=0.001). Western blot assay showed that in MDA-MB-231 cells, the increased Rac1 expression could inhibit p53 expression (t=-6.392, P=0.003) and enhance VEGF expression (t=7.497, P=0.002) compared with blank control group, while decreased Rac1 expression could elevate p53 expression (t=5.307, P=0.006) and inhibit VEGF expression (t=-7.395, P=0.002) compared with sicontrol group.

Conclusion

Rac1 expression could affect VEGF expression in breast cancer MDA-MB-231 cells and Rac1 may promote VEGF expression through inhibiting p53 expression.

Key words: Breast neoplasms, Vascular endothelial growth factors, Tumor suppressor protein p53, RAC1 protein, human
表1 ELISA 检测V12Rac1 对MDA-MB-231 细胞分泌VEGF 的影响
组别 实验次数 VEGF蛋白表达水平(pg/ml,xˉ±s)
24 h 48 h 72 h
空白对照组 3 102.21±2.01 135.31±3.60 168.09±2.62
V12Rac1组 3 113.54±4.53 173.11±3.55 255.02±4.55
表2 ELISA 检测siRac1 对MDA-MB-231 细胞分泌VEGF 的影响
组别 实验次数 VEGF蛋白表达水平(pg/ml,xˉ±s)
24 h 48 h 72 h
沉默对照组 3 102.04±3.11 136.55±3.72 168.62±4.35
siRac1组 3 93.11±3.20 104.23±4.00 107.15±3.83
图1 Western blot 检测过表达Rac1 对p53 和VEGF表达的影响
表3 siRac1 组及沉默对照组Western blot 实验定量分析结果()
组别 实验次数 p53a VEGFa
沉默对照组 3 1.407±0.077 1.163±0.140
siRac1组 3 2.623±0.389 0.483±0.075
t值 5.307 -7.395
P值 0.006 0.002
表4 V12Rac1 组及空白对照组Western blot 实验定量分析结果()
组别 实验次数 p53a VEGFa
空白对照组 3 2.043±1.114 0.643±0.130
V12Rac1组 3 1.425±0.123 1.323±0.089
t值 -6.392 7.497
P值 0.003 0.002
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