摘要
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LipofectamineTM2000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x珋±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�
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/1 estradiol and 10 nmol/1 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＋ ceils. 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 x^-±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 control, 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 e
出处
《中华乳腺病杂志（电子版）》
CAS
CSCD
2015年第1期21-26,共6页
Chinese Journal of Breast Disease(Electronic Edition)
基金
安徽省自然科学基金资助项目(11040606M180)
