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目的探讨1,25二羟维生素D3靶控的雌激素受体α表达载体(VDRE-Tk-ERα)对雌激素受体阴性的MDA-MB-231乳腺癌细胞周期进程的影响及其机制。方法利用MTT法检测1,25二羟维生素D3联合他莫西芬对转染VDRE-Tk-ERα表达载体的MDA-MB-231细胞增殖能力的影响,通过流式细胞仪法检测1,25二羟维生素D3联合他莫西芬对转染该表达质粒的MDA-MB-231细胞周期相的分布。用RT-PCR、Western blot和免疫沉淀法检测细胞Rb及其磷酸化水平、Cy-clin D1、CDK4的表达以及Cyclin D1/CDK4激酶复合物的结合力变化。结果1,25二羟维生素D3联合他莫西芬能够有效抑制转染该质粒的MDA-MB-231细胞的增殖活性并将其细胞周期进程阻遏于G0/G1期。与此同时,Cyclin D1的表达则显著降低,其与CDK4的结合力亦显著降低。Rb的表达虽无显著变化,但其磷酸化水平显著下降。结论1,25二羟维生素D3可通过靶控MDA-MB-231细胞内外源性雌激素受体α的表达进而对G1/S期相关分子的表达和活性进行调控从而阻止雌激素受体α阴性的乳腺癌细胞的周期进程并恢复对他莫西芬的化疗敏感性。
Objective To investigate the effect and mechanism of VDRE-Tk-ERα targeted by 1,25-dihydroxyvitamin D3 on the estrogen receptor-negative cell cycle of MDA-MB-231 breast cancer cells. Methods The effects of 1,25-dihydroxyvitamin D3 and tamoxifen on the proliferation of MDA-MB-231 cells transfected with VDRE-Tk-ERα expression vector were detected by MTT assay. The cytotoxicity of 1,25 Distribution of hydroxyvitamin D3 combined with tamoxifen on the cell cycle phase of MDA-MB-231 cells transfected with this expression plasmid. The changes of Rb and phosphorylation, Cy-clin D1, CDK4 expression and the binding ability of Cyclin D1 / CDK4 kinase complex were detected by RT-PCR, Western blot and immunoprecipitation. Results 1,25 dihydroxyvitamin D3 combined with tamoxifen could effectively inhibit the proliferation activity of MDA-MB-231 cells transfected with this plasmid and repress the cell cycle progression in G0 / G1 phase. At the same time, the expression of Cyclin D1 was significantly reduced, and its binding capacity with CDK4 was also significantly reduced. Although the expression of Rb did not change significantly, its phosphorylation level decreased significantly. Conclusions 1,25-dihydroxyvitamin D3 can prevent the expression of estrogen receptor alpha in the MDA-MB-231 cells and regulate the expression and activity of G1 / S-related molecules Of breast cancer cell cycle progression and restore chemosensitivity to tamoxifen.