Expression Of EZH2 Protein In Tamoxifen-resistant Breast Cancer MCF-7 Cells And Its Influence On Cell Proliferation

Objective: To investigate the expression of EZH2 protein 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: 1. The expression of EZH2 in MCF-7/Tam+ and MCF-7/Tam- cells were detected by Western blotting. 2. 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 blotting. 3. si RNA with targeted inhibition of EZH2(si RNA-EZH2) was transfected into MCF-7/Tam+ using Lipofectamine 2000. The real-time PCR and Western blotting were used to respectively detect the m RNA and protein expression of EZH2 in order to confirm the validity of si RNA. 4. si RNA-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 ability(optical density at 570 nm wavelength). Measurement data results were expressed in x±s, and t test was used to compare the means of two groups. Results:Results: 1. The relative expression of EZH2 protein in MCF-7/Tam+ and MCF-7/Tam-were 2.237±0.091、1.870±0.114,respectively.(t=8.582, P=0.013). 2. Compared with the negative control group( EZH2 relative expression :0.480±0.017), the relative expression of EZH2 was 0.657±0.015 in MCF-7/Tamafter being treated by 10 nmol/l estradiol for 24 h, and the relative expression of EZH2 was 0.423±0.012 after being treated by 10 nmol/l 4-OHT for 24 h(F=175.032, P=0.000). 3. After transfecting si RNA- EZH2 into MCF-7/Tam+ cells, the expression of EZH2 m RNA was significantly decreased by 69%(t = 14.06, P = 14.06) and the expression of EZH2 protein was significantly decreased by 42%(t = 15.366, P = 15.366). 4. The cell count assay showed that compared with the si RNA-control group, the cell number of MCF-7/Tam- cells transfected with si RNA-EZH2 declined sharply on day 3,4,5(F=77.515, P=0.001);compared with the si RNA-control group, the cell number of MCF-7/Tam+ cells transfected with si RNA-EZH2 declined sharply on day 3,4,5(F=702.347, P=0.000). 5. MTT results showed that compared with control group, the optical density at the wavelength of 570 nm was reduced by 43%(t=9.676, P=0.011)and by 77%(t=65.069, P=0.000)in EZH2-si RNA-transfected MCF-7/Tam- and MCF-7/Tam+ cells, respectively.Conclusions: The expression of EZH2 protein was higher in tamoxifen- resistant breast cancer MCF-7 cells(MCF-7/Tam+) than that in tamoxifen- sensitive breast cancer MCF-7 cells(MCF-7/Tam-). To confirm the effectiveness of si RNA,si RNA which targeted inhibition of EZH2(si RNA-EZH2) was transfected into MCF-7/Tam+, EZH2 expression were declined in m RNA and protein levels, respectively. Cell proliferation capacity decreased significantly in MCF- 7 / Tam- and MCF- 7 / Tam + cells which transfected with si RNA EZH2.