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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2019, Vol. 13 ›› Issue (03) : 137 -144. doi: 10.3877/cma.j.issn.1674-0807.2019.03.002

所属专题： 文献；

论著

大黄素联合survivin短发夹RNA对人乳腺癌细胞株MCF-7增殖的影响
祖聪1, 秦光远1, 郑新宇2,†()   
  1. 1. 110001　沈阳，中国医科大学附属第一医院肿瘤研究所第一研究室
    2. 110001　沈阳，中国医科大学附属第一医院肿瘤研究所第一研究室；110001　沈阳，中国医科大学附属第一医院乳腺外科
  • 收稿日期:2017-10-30 出版日期:2019-06-01
  • 通信作者: 郑新宇
  • 基金资助:
    国家自然科学基金资助项目(81272920)

Effect of emodin combined with survivin shRNA on proliferation of human breast cancer MCF-7 cells

Cong Zu1, Guangyuan Qin1, Xinyu Zheng2,†()   

  1. 1. Laboratory No.1 of Cancer Institute, First Affiliated Hospital of China Medical University, Shenyang 110001, China
    2. Laboratory No.1 of Cancer Institute, First Affiliated Hospital of China Medical University, Shenyang 110001, China; Department of Breast Surgery, First Affiliated Hospital of China Medical University, Shenyang 110001, China
  • Received:2017-10-30 Published:2019-06-01
  • Corresponding author: Xinyu Zheng
  • About author:
    Corresponding author: Zheng Xinyu, Email:
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引用本文：

祖聪, 秦光远, 郑新宇. 大黄素联合survivin短发夹RNA对人乳腺癌细胞株MCF-7增殖的影响[J]. 中华乳腺病杂志(电子版), 2019, 13(03): 137-144.

Cong Zu, Guangyuan Qin, Xinyu Zheng. Effect of emodin combined with survivin shRNA on proliferation of human breast cancer MCF-7 cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2019, 13(03): 137-144.

目的

探讨大黄素与survivin短发夹RNA(shRNA)联合应用对人乳腺癌细胞株MCF-7增殖的影响。

方法

先用MTT法检测不同浓度(0、10、20、40、80 μmol/L)大黄素对人乳腺癌细胞株MCF-7生长抑制的影响，为后续联合用药实验筛选出适宜浓度(40 μmol/L)的大黄素。然后，将人乳腺癌细胞株MCF-7分为以下5组：空白对照组，细胞未做任何处理；阴性对照组，即NC shRNA组，加入空载质粒；大黄素处理组，用40 μmol/L大黄素处理细胞；survivin shRNA处理组，转染survivin shRNA质粒；联合处理组，用40 μmol/L大黄素与survivin shRNA联合处理细胞。利用MTT法、流式细胞术分别检测各组细胞的增殖(用吸光度值表示)和凋亡能力，用real-time PCR及蛋白质印迹法(Western blot法)分别检测细胞中survivin的mRNA和蛋白表达。由于细胞增殖活性检测结果显示空白对照组与阴性对照组差别不显著，故流式细胞术中不再设置阴性对照组。多组细胞间凋亡率及survivin的mRNA和蛋白表达量比较采用单因素方差分析，吸光度值比较采用重复测量的方差分析，组间两两比较采用LSD法。

结果

MTT法显示，不同浓度(0、10、20、40、80 μmol/L)大黄素作用24、48、72 h后，各组细胞间吸光度值比较，差异有统计学意义(F＝1 873.565, P<0.001)，并且，40 μmol/L大黄素对细胞增殖的抑制率为(53.6±1.2)%(>50.0%)，因此选择该浓度进行后续实验。用不同方法处理细胞24、48、72 h后，空白对照组、阴性对照组、大黄素处理组、survivin shRNA处理组及联合处理组细胞的吸光度值比较，差异有统计学意义(F＝1 686.953, P<0.001)，不同时间点间细胞吸光度值的差异也有统计学意义(F＝288.790, P<0.001)，分组与时间点存在交互作用(F＝67.916, P<0.001)。流式细胞检测结果显示：空白对照组、大黄素处理组、survivin shRNA处理组和联合处理组细胞凋亡率分别为(0.57±0.10)%、(5.96±0.56)%、(9.00±0.73)%和(18.33±0.98)%，4组比较，差异有统计学意义(F＝114.848, P<0.001)；大黄素处理组细胞凋亡率明显高于空白对照组(P<0.001)；联合处理组细胞凋亡率明显高于其他各组(P均<0.001)。real-time PCR结果显示：空白对照组、阴性对照组、大黄素处理组、survivin shRNA处理组和联合处理组的mRNA表达量分别为1.000±0.000、0.968±0.033、0.774±0.049、0.483±0.026和0.364±0.012，5组比较，差异也有统计学意义(F＝284.199, P<0.001)，进一步两两比较发现，联合处理组survivin mRNA表达量低于大黄素处理组(P<0.001)和survivin shRNA处理组(P＝0.001)。Western blot结果显示：空白对照组、阴性对照组、大黄素处理组、survivin shRNA处理组和联合处理组的survivin蛋白表达量分别为1.000±0.000、0.987±0.031、0.696±0.050、0.534±0.065和0.312±0.039，5组比较，差异有统计学意义(F＝142.311, P<0.001)，进一步两两比较显示，联合处理组survivin蛋白表达量明显低于大黄素处理组和survivin shRNA处理组(P均<0.001)。

结论

大黄素与survivin shRNA两者联合应用可明显抑制人乳腺癌细胞株MCF-7的增殖。

关键词: 乳腺肿瘤, 大黄素, Survivin, shRNA, 细胞增殖
Objective

To investigate the effect of emodin combined with survivin shRNA on the proliferation of human breast cancer MCF-7 cells.

Methods

The human breast cancer MCF-7 cells were cultured and treated with different concentrations of emodin (0, 10, 20, 40, 80 μmol/L). The effect of emodin on the proliferation of human breast cancer MCF-7 cells was determined by MTT assay to select suitable concentration (40 μmol/L) for subsequent experiments. Then the human breast cancer MCF-7 cells were divided into five groups: blank control group, negative control group (NC shRNA group, treated by empty plasmid), emodin treatment group (treated by 40 μmol/L emodin), survivin shRNA treatment group (transfected with survivin shRNA plasmid) and combined treatment group (treated by 40 μmol/L emodin plus survivin shRNA). The proliferation (optical density) and apoptosis of human breast cancer MCF-7 cells in each group were analyzed by MTT assay and flow cytometry, respectively. Because the result of MTT assay showed no significant difference in cell proliferation activity between blank control group and negative control group, no negative control group was established for the flow cytometry. Survivin mRNA and protein expressions were also assessed using real-time PCR and Western blot analysis, respectively. The apoptosis rate and survivin mRNA and protein expression were compared among multiple groups by one-way analysis of variance. The optical density was compared by repeated measurement analysis of variance. Pairwise comparison was performed using LSD method.

Results

The result of MTT assay showed that after 24, 48, and 72 h treatment of emodin at different concentrations (0, 10, 20, 40, 80 μmol/L), the optical density of each group was significantly different (F=1 873.565, P<0.001). The inhibitory rate of 40 μmol/L emodin on cell proliferation was (53.6±1.2)% (>50.0%), so this concentration of emodin was used for the following experiments. After the cells were treated with differeht methods for 24, 48, and 72 h, the optical density presented a significant difference among blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group (F=1 686.953, P<0.001), the optical density was significantly different at different time points (F=288.790, P<0.001), and there was an interaction between grouping and time points (F=67.916, P<0.001). The results of flow cytometry showed that the apoptosis rate was (0.57±0.10)%, (5.96±0.56)%, (9.00±0.73)% and (18.33±0.98)% in blank control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=114.848, P<0.001). The apoptosis rate of emodin treatment group was significantly higher than that of blank control group (P<0.001); the apoptosis rate of combined treatment group was significantly higher than that of any other group (P<0.001). The results of real-time PCR showed that survivin mRNA expression was 1.000±0.000, 0.968±0.033, 0.774±0.049, 0.483±0.026 and 0.364±0.012 in blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=284.199, P<0.001). Pairwise comparison showed that survivin mRNA expression in combined treatment group was significantly lower than that in emodin treatment group (P<0.001) or survivin shRNA treatment group (P=0.001). Western blot analysis showed that survivin protein expression was 1.000±0.000, 0.987±0.031, 0.696±0.050, 0.534±0.065 and 0.312±0.039 in blank control group, negative control group, emodin treatment group, survivin shRNA treatment group and combined treatment group, respectively, indicating a significant difference (F=142.311, P<0.001). Pairwise comparison showed that survivin protein expression in combined treatment group was significantly lower than that in emodin treatment group or survivin shRNA treatment group (both P<0.001).

Conclusion

The combination of emodin and survivin shRNA can inhibit the proliferation of human breast cancer MCF-7 cells.

Key words: Breast neoplasms, Emodin, Survivin, shRNA, Cell proliferation
表1 不同浓度大黄素分别处理人乳腺癌细胞株MCF-7后的吸光度值比较(±s)
组别 实验次数 24 h 48 h 72 h
0 μmol/La 3 0.392±0.009 0.584±0.007 0.606±0.003
10 μmol/L 3 0.371±0.012 0.541±0.006 0.528±0.004
20 μmol/L 3 0.345±0.009 0.497±0.004 0.429±0.005
40 μmol/Lb 3 0.269±0.023 0.313±0.003 0.287±0.007
80 μmol/L 3 0.239±0.015 0.189±0.003 0.188±0.004
表2 各组人乳腺癌细胞株MCF-7吸光度值比较(±s)
组别 实验次数 24 h 48 h 72 h
空白对照组 3 0.392±0.009 0.584±0.007 0.606±0.003
阴性对照组(NC shRNA组) 3 0.375±0.011 0.559±0.002 0.567±0.025
大黄素处理组a 3 0.269±0.023 0.313±0.003 0.287±0.007
survivin shRNA处理组a 3 0.213±0.024 0.258±0.003 0.249±0.006
联合处理组b 3 0.159±0.004 0.168±0.002 0.136±0.006
图1 采用流式细胞术检测各组人乳腺癌细胞株MCF-7的凋亡率　a～d图分别所示空白对照组、大黄素处理组(用40 μmol/L大黄素处理细胞)、survivin shRNA处理组和联合处理组(40 μmol/L大黄素与survivin shRNA联合处理细胞)的细胞凋亡率
图2 采用RT-PCR检测各组人乳腺癌细胞株MCF-7中survivin mRNA的表达
表3 各组人乳腺癌细胞株MCF-7中survivin的mRNA及蛋白表达(±s)
组别 实验次数 survivin mRNA survivin蛋白
空白对照组 3 1.000±0.000 1.000±0.000
阴性对照组(NC shRNA组) 3 0.968±0.033 0.987±0.031
大黄素处理组 3 0.774±0.049a 0.696±0.050a
survivin shRNA处理组 3 0.483±0.026a 0.534±0.065a
联合处理组 3 0.364±0.012b 0.312±0.039c
F值 ? 284.199 142.311
P值 ? <0.001 <0.001
图3 采用Western blot检测各组人乳腺癌细胞株MCF-7中survivin蛋白的表达
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