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

doi:10.3877/cma.j.issn.1674-0807.2019.03.002

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. 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. 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: xyzheng@cmu.edu.cn

全文 ( ) 下载PDF ( ) 导出引用

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/OL]. 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/OL]. 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/L^a	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/L^b	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/L^a	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/L^b	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

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±0.049^a	0.696±0.050^a
survivin shRNA处理组	3	0.483±0.026^a	0.534±0.065^a
联合处理组	3	0.364±0.012^b	0.312±0.039^c
F值	?	284.199	142.311
P值	?	<0.001	<0.001

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±0.049^a	0.696±0.050^a
survivin shRNA处理组	3	0.483±0.026^a	0.534±0.065^a
联合处理组	3	0.364±0.012^b	0.312±0.039^c
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

[1]	薛静，王浩. 乳腺癌免疫治疗的研究进展[J/CD]. 中华乳腺病杂志(电子版)，2018, 12(1):43-49.
[2]	李明卉，夏添松，王水. 乳腺癌常用化疗药物的作用机制及血液学不良反应的研究进展[J/CD]. 中华乳腺病杂志(电子版)，2017, 11(3):186-190.
[3]	Khongkow P, Gomes AR, Gong C, et al. Paclitaxel targets FOXM1 to regulate KIF20A in mitotic catastrophe and breast cancer paclitaxel resistance [J]. Oncogene，2016, 35(8):990-1002.
[4]	马哿，王佳，夏添松，等. 淋巴细胞趋化因子对乳腺癌MCF-7细胞的表柔比星药物敏感性及侵袭转移能力的影响[J/CD]. 中华乳腺病杂志(电子版)，2017, 11(6):354-360.
[5]	Yang F, Yuan P, Hao YQ, et al. Emodin enhances osteogenesis and inhibits adipogenesis[J]. BMC Complement Altern Med，2014, 14: 74.
[6]	Xing JY, Song GP, Deng JP, et al. Antitumor effects and mechanism of novel emodin rhamnoside derivatives against human cancer cells in vitro[J]. PLoS One，2015, 10(12):e0144781.
[7]	Thacker PC, Karunagaran D. Curcumin and emodin down-regulate TGF-β signaling pathway in human cervical cancer cells[J]. PLoS One，2015, 10(3):e0120045.
[8]	Li XX, Dong Y, Wang W, et al. Emodin as an effective agent in targeting cancer stem-like side population cells of gallbladder carcinoma[J]. Stem Cells Dev，2013, 22(4): 554-566.
[9]	Pan FP, Zhou HK, Bu HQ, et al. Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK[J]. Oncol Rep，2016, 35(4):1941-1949.
[10]	Garg H, Suri P, Gupta JC, et al. Survivin：a unique target for tumor therapy[J]. Cancer Cell Int，2016, 16: 49.
[11]	Khan Z, Khan AA, Yadav H, et al. Survivin, a molecular target for therapeutic interventions in squamous cell carcinoma[J]. Cell Mol Biol Lett，2017, 22: 8.
[12]	刘翠翠，王光学，赵倩，等. 长链非编码RNA调控乳腺癌增殖、转移、耐药性及乳腺癌干细胞[J/CD]. 中华乳腺病杂志(电子版)，2016, 10(5):310-315.
[13]	孟智超，李淑玲，肖建英．大黄素抑制卵巢癌SKOV3细胞增殖和抑制survivin及XIAP的蛋白表达[J]. 基础医学与临床，2014, 34(9): 1255-1259.
[14]	薛晖，李天人，贺安宁，等. Survivin shRNA与大黄素联合应用抗人卵巢癌细胞株增殖的实验研究[J]. 现代肿瘤医学，2014, 22(11):2557-2561.
[15]	Liu W, Tang L, Ye L, et al. Species and gender differences affect the metabolism of emodin via glucuronidation[J]. AAPS J，2010, 12(3):424-436.
[16]	Huang FJ, Hsuuw YD, Chan WH. Characterization of apoptosis induced by emodin and related regulatory mechanisms in human neuroblastoma cells[J]. Int J Mol Sci，2013, 14(10):20 139-20 156.
[17]	廖子君，宋永春，陈晓泉，等．大黄素对人肺腺癌A-549细胞增殖周期影响的实验研究[J]. 中华肿瘤防治杂志，2007, 14(5):342-344.
[18]	Zu C, Zhang MD, Xue H, et al. Emodin induces apoptosis of human breast cancer cells by modulating the expression of apoptosis-related genes[J]. Oncol Lett，2015, 10(5): 2919-2924.
[19]	周颉，傅建民，石剑，等．大黄素逆转乳腺癌细胞多药耐药及其对ERCC1蛋白表达的影响[J]. 中华肿瘤防治杂志，2010, 17(1):27-29.
[20]	焦冰洋，王闯，刘悦，等．大黄素通过下调Prx V蛋白水平诱导AGS胃癌细胞凋亡[J]. 黑龙江八一农垦大学学报，2018, 30(1):24-28.
[21]	Mattheolabakis G, Ling DD, Ahmad G, et al. Enhanced anti-tumor efficacy of lipid-modified platinum derivatives in combination with surviving silencing siRNA in resistant non-small cell lung cancer[J]. Pharm Res，2016, 33(12): 2943-2953.
[22]	He XJ, Zhang Q, Ma LP, et al. Aberrant alternative polyadenylation is responsible for survivin up-regulation in ovarian cancer[J]. Chin Med J (Engl)，2016, 129(10): 1140-1146.
[23]	Véquaud E, Desplanques G, Jézéquel P, et al. survivin contributes to DNA repair by homologous recombination in breast cancer cells[J]. Breast Cancer Res Treat, 2016，155(1): 53-63.
[24]	Chen X, Duan N, Zhang CG, et al. Survivin and tumorigenesis: molecular mechanisms and therapeutic strategies[J]. J Cancer，2016, 7(3):314-323.
[25]	Kelly RJ, Lopez-Chavez A, Citrin D, et al. Impacting tumor cell-fate by targeting the inhibitor of apoptosis protein survivin [J]. Mol Cancer，2011, 10:35.

[1]	李洋, 蔡金玉, 党晓智, 常婉英, 巨艳, 高毅, 宋宏萍. 基于深度学习的乳腺超声应变弹性图像生成模型的应用研究[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(06): 563-570.
[2]	河北省抗癌协会乳腺癌专业委员会护理协作组. 乳腺癌中心静脉通路护理管理专家共识[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 321-329.
[3]	刘晨鹭, 刘洁, 张帆, 严彩英, 陈倩, 陈双庆. 增强MRI 影像组学特征生境分析在预测乳腺癌HER-2 表达状态中的应用[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 339-345.
[4]	张晓宇, 殷雨来, 张银旭. 阿帕替尼联合新辅助化疗对三阴性乳腺癌的疗效及预后分析[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 346-352.
[5]	邱琳, 刘锦辉, 组木热提·吐尔洪, 马悦心, 冷晓玲. 超声影像组学对致密型乳腺背景中非肿块型乳腺癌的诊断价值[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 353-360.
[6]	程燕妮, 樊菁, 肖瑶, 舒瑞, 明昊, 党晓智, 宋宏萍. 乳腺组织定位标记夹的应用与进展[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 361-365.
[7]	涂盛楠, 胡芬, 张娟, 蔡海峰, 杨俊泉. 天然植物提取物在乳腺癌治疗中的应用[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 366-370.
[8]	朱文婷, 顾鹏, 孙星. 非酒精性脂肪性肝病对乳腺癌发生发展及治疗的影响[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 371-375.
[9]	周荷妹, 金杰, 叶建东, 夏之一, 王进进, 丁宁. 罕见成人肋骨郎格汉斯细胞组织细胞增生症被误诊为乳腺癌术后骨转移一例[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 380-383.
[10]	高杰红, 黎平平, 齐婧, 代引海. ETFA和CD34在乳腺癌中的表达及与临床病理参数和预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 64-67.
[11]	韩萌萌, 冯雪园, 马宁. 乳腺癌改良根治术后桡神经损伤1例[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 117-118.
[12]	张志兆, 王睿, 郜苹苹, 王成方, 王成, 齐晓伟. DNMT3B与乳腺癌预后的关系及其生物学机制[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 624-629.
[13]	王玲艳, 高春晖, 冯雪园, 崔鑫淼, 刘欢, 赵文明, 张金库. 循环肿瘤细胞在乳腺癌新辅助及术后辅助治疗中的应用[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 630-633.
[14]	赵林娟, 吕婕, 王文胜, 马德茂, 侯涛. 超声引导下染色剂标记切缘的梭柱型和圆柱型保乳区段切除术的效果研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 634-637.
[15]	祝炜安, 林华慧, 吴建杰, 黄炯煅, 吴婷婷, 赖文杰. RDM1通过CDK4促进前列腺癌细胞进展的研究[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(06): 618-625.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

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

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

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