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中华乳腺病杂志(电子版) ›› 2015, Vol. 09 ›› Issue (01) : 23 -29. doi: 10.3877/cma. j. issn.1674-0807.2015.01.006

论著

EZH2 基因在他莫昔芬耐药乳腺癌MCF-7细胞系的表达及其对细胞增殖的影响
骆广涛1, 施航2, 王本忠1,()   
  1. 1.230032 合肥,安徽医科大学第一附属医院乳腺外科
    2.315000 浙江省宁波市中医院肿瘤科
  • 收稿日期:2014-10-30 出版日期:2015-02-01
  • 通信作者: 王本忠
  • 基金资助:
    安徽省自然科学基金资助项目(11040606M180)

Expression of EZH2 gene in tamoxifen-resistant breast cancer MCF-7 cells and its influence on cell proliferation

Guangtao Luo1, Hang Shi2, Benzhong Wang1,()   

  1. 1.Department of Breast Surgery, First Affiliated Hospital, Anhui Medical University, Hefei 230032, China
    2.Department of Oncology,Traditional Chinese Medicine Hospital of Ningbo City,Ningbo 31500,China
  • Received:2014-10-30 Published:2015-02-01
  • Corresponding author: Benzhong Wang
引用本文:

骆广涛, 施航, 王本忠. EZH2 基因在他莫昔芬耐药乳腺癌MCF-7细胞系的表达及其对细胞增殖的影响[J/OL]. 中华乳腺病杂志(电子版), 2015, 09(01): 23-29.

Guangtao Luo, Hang Shi, Benzhong Wang. Expression of EZH2 gene in tamoxifen-resistant breast cancer MCF-7 cells and its influence on cell proliferation[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2015, 09(01): 23-29.

目的

检测EZH2 基因在他莫昔芬耐药乳腺癌MCF-7 细胞(MCF-7/Tam+细胞)及他莫昔芬敏感乳腺癌MCF-7 细胞(MCF-7/Tam-细胞)的表达,并探讨EZH2 对两种乳腺癌细胞系增殖的影响。

方法

首先通过Western blot 法在MCF-7/Tam+细胞和MCF-7/Tam-细胞中检测EZH2 蛋白表达差异,并采用10 nmol/L 的雌二醇和10 nmol/L 4-羟他莫昔芬(4-OHT)分别处理MCF-7/Tam-细胞24 h 后检测EZH2 蛋白的变化。 使用LipofectamineTM 2000 将靶向抑制EZH2 的siRNA(siRNA-EZH2)转染至MCF-7/Tam+细胞,运用实时PCR 检测EZH2 mRNA 的相对表达量,Western blot 法检测EZH2 蛋白表达量,以验证siRNA 的有效性。 最后将siRNA-EZH2 转染至MCF-7/Tam-和MCF-7/Tam+细胞,采用细胞计数检测细胞数,用MTT 法检测细胞增殖情况(570 nm 波长处吸光度值)。 计量资料用±s 表示,两组均数比较用t 检验,细胞计数比较采用重复测量的方差分析。

结果

EZH2 蛋白在MCF-7/Tam+细胞中相对表达量为2.237±0.091,高于在MCF-7/Tam-细胞中的相对表达量(1.870±0.114)(t=8.582,P=0.013)。 经10 nmol/L 的雌二醇诱导MCF-7/Tam-细胞24 h 后EZH2 蛋白相对表达量为0.657±0.015,阴性对照组EZH2 相对表达量为0.480±0.017,而相同浓度的4-羟基他莫昔芬处理MCF-7/Tam-细胞24 h 后EZH2蛋白相对表达量为0.423±0.012,雌二醇+他莫昔芬处理组为0.483±0.006,4 组差异有统计学意义(F=175.032,P=0.000)。 将siRNA-EZH2 转染至MCF-7/Tam+细胞后,转染siRNA-EZH2 组与空质粒转染组(siRNA-Control 组)EZH2 mRNA 相对表达量分别为0.310±0.062、1.007±0.136,差异有统计学意义(t=14.061,P=0.005);转染siRNA-EZH2 组与siRNA-Control 组EZH2 蛋白的相对表达量分别为3.783±0.045、6.500±0.327,差异有统计学意义(t=15.366,P=0.004)。 细胞计数结果显示,转染siRNA-EZH2的MCF-7/Tam-细胞组(Tam-EZH2 组)的细胞数(单位: 1×105)在第3、4、5 天分别为7.540±0.790、10.243±1.360、14.270±1. 992,Tam-Control 组细胞数在第3、4、5 天分别为12. 113±1. 666、17. 853±1.900、25.670±2.318,组间差异有统计学意义(F=77.515, P=0.001);转染siRNA-EZH2 的MCF-7/Tam+细胞组(Tam+EZH2 组)细胞数在第3、4、5 天分别为6.373±0.863、7.680±1.077、8.813±1.165,Tam+Control 组细胞数在第3、4、5 天分别为12.553±2.878、19.780±2.400、31.617±3.081,组间差异有统计学意义(F=702.347, P=0.000)。 MTT 结果显示Tam-EZH2 组及Tam-Control 组细胞在570 nm 波长处的吸光度值分别为0.643±0.085 及1.120±0.135,差异有统计学意义(t=9.676, P=0.011);Tam+EZH2 组及Tam+Control 组在570 nm 波长处的吸光度值分别为0.313±0.075 及1.350±0.101,差异有统计学意义(t=65.069, P=0.000)。

结论

EZH2 在MCF-7/Tam+细胞中高表达,并能促进细胞增殖。

Objective

To investigate the expression of EZH2 gene in tamoxifen-resistant breast cancer MCF-7 cells (MCF-7/Tam+) and tamoxifen-sensitive breast cancer MCF-7 cells(MCF-7/Tam-), and explore the effects of EZH2 on the proliferation of the two breast cancer cell lines.

Methods

Firstly, the expressions of EZH2 in MCF-7/Tam+ and MCF-7/Tam- cells were detected by Western blot. After MCF-7/Tam- cells being treated by 10 nmol/l estradiol and 10 nmol/l 4-hydroxy tamoxifen(4-OHT) for 24 h, the expression of EZH2 protein was detected by Western blot. siRNA with targeted inhibition of EZH2 (siRNA-EZH2) was transfected into MCF-7/Tam+ using LipofectamineTM 2000. The real-time PCR and Western blot were used to detect the mRNA and protein expression of EZH2 respectively in order to confirm the validity of siRNA.Finally, siRNA-EZH2 was transfected into MCF-7/Tam- and MCF-7/Tam+ cells. Cell count assay was used to measure the cell number and MTT to detect the cell proliferation(optical density at 570 nm wavelength).Measurement data were expressed as ±s, and t test was used to compare the means.The cell count data were compared by repeated measurement analysis of variance.

Results

The relative expression of EZH2 protein in MCF-7/Tam+ was 2.237±0.091,significantly higher than 1.870±0.114 in MCF-7/Tam-(t=8. 582,P=0.013).The relative expression of EZH2 protein was 0.657±0.015 in MCF-7/Tam- cells after being treated by 10 nmol/L estradiol for 24 h,0.480±0.017 in negative controls,and 0.423±0.012 in MCF-7/Tam- cells after being treated by 10 nmol/L 4-OHT for 24 h,0.483±0.006 in MCF-7/Tam-cells treated by estradiol+4-OHT,the difference was statistically significant(F=175.032,P=0.000).After transfecting siRNA-EZH2 into MCF-7/Tam+ cells,the relative expression of EZH2 mRNA in siRNA-EZH2 group and siRNA-control group were 0.310±0.062 and 1.007±0.136,the difference was statistically significant (t=14.061,P=0.005);the relative expression of EZH2 protein in siRNA-EZH2 group and siRNA-control group were 3.783±0.045 and 6.500±0.327 respectively,the difference was statistically significant (t=15.366,P=0.004). The cell count assay showed that on days 3,4,5,the cell number (unit: 1×105)was 7.540±0.790,10.243±1.360,14.270±1.992 in MCF-7/Tam- cells transfected with siRNA-EZH2,12.113±1.666,17.853±1.900,25.670±2.318 in Tam-control group,and the difference was statistically significant between two groups(F=77.515,P=0.001);on days 3,4,5,the cell number was 6.373±0.863,7.680±1.077,8.813±1.165 in MCF-7/Tam+ cells transfected with siRNA-EZH2,and 12.553±2.878,19.780±2.400,31.617±3.081 in Tam+control group,and the difference was statistically significant between two groups(F=702.347,P=0.000).MTT results showed that the optical density at the wavelength of 570 nm was 0.643±0.085 and 1.120±0.135 in MCF-7/Tam+ cells transfected with siRNA-EZH2 and Tam+control group (t=9.676,P=0.011),0.313±0.075 and 1.350±0.101 in MCF-7/Tam- cells transfected with siRNA-EZH2 and Tam-control group(t=65.069,P=0.000),the differences were statistically significant.

Conclusion

EZH2 is highly expressed in tamoxifen-resistant breast cancer MCF-7cells, which can promote cell proliferation.

图1 EZH2 蛋白在MCF-7/Tam+和MCF-7/Tam-细胞中的表达
图2 雌二醇和4-羟基他莫西芬处理MCF-7/Tam-细胞24 h 后EZH2 蛋白表达 注: NC 为阴性对照组;E2 为雌二醇处理组;Tam 为4-羟基他莫西芬处理组;E2+Tam 为雌二醇加4-羟基他莫西芬处理组
图3 转染siRNA-EZH2 组与对照组细胞中EZH2 mRNA的表达
图4 转染siRNA-EZH2 组与对照组细胞EZH2 蛋白表达
表1 siRNA-EZH2 转染MCF-7 /Tam-细胞后细胞数的变化
表2 siRNA-EZH2 转染MCF-7 /Tam+细胞后细胞数的变化
图5 siRNA-EZH2 转染MCF-7/Tam-细胞后细胞数的变化
图6 siRNA-EZH2 转染MCF-7/Tam+细胞后细胞数的变化
图7 siRNA-EZH2 转染MCF-7/Tam-和MCF-7/Tam+细胞后的吸光度值比较 注: a与Tam-对照组细胞相比, t = 9.676,P = 0.010;b 与Tam+对照组细胞相比,t=65.069,P=0.000
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