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目的探讨维生素E调节前列腺癌细胞DNA复制的新分子机制及信传导通路。方法利用基因转染、免疫印迹、实时定量聚合酶链反应(PCR)、免疫沉淀等方法研究不同浓度、不同时间琥珀酸维生素E(VES)对前列腺癌细胞内的微小染色体维持蛋白4(MCM4)的mRNA及蛋白表达水平的影响。结果VES可抑制前列腺癌MCM4 mRNA的表达,并存在时效与量效关系,20μmoL/L VES处理LNCaP细胞36 h,MCM4的mRNA表达水平较对照组下降24.5%(P<0.05),作用96 h下降达60.3%;随着VES浓度的增加,MCM4 mRNA的表达水平受抑制的程度逐步加深。Western blot表明VES可抑制MCM4蛋白的表达。过表达E2F1可拮抗VES对MCM4的抑制作用。VES抑制Rb磷酸化,增加Rb与E2F1的结合程度。BrdU摄入试验表明20μmol/L与40μmol/L VES处理LNCaP细胞24h后,癌细胞内DNA合成较对照组下降63.4%与71.6%(P<0.05);48 h后分别达74.8%与81.5%(P<0.05)。结论我们识别了维生素E抑制前列腺癌细胞DNA复制的一种新机制是下调MCM4的表达,E2F1-Rb蛋白参与这一信号传导通路。
Objective To investigate the new molecular mechanism and signaling pathway mediated by vitamin E in DNA replication of prostate cancer cells. Methods The effects of vitamin E succinate (VES) at different concentrations and times on the expression of MCM4 in prostate cancer cells were studied by gene transfection, western blotting, real-time quantitative PCR and immunoprecipitation. MRNA and protein expression levels. Results VES inhibited the expression of MCM4 mRNA in prostate cancer cells, and there was a relationship between the effect of aging and dose-effect. The mRNA expression of MCM4 in LNCaP cells treated with 20μmoL / L VES for 36 h decreased 24.5% (P <0.05) Up to 60.3%. With the increase of VES concentration, the expression of MCM4 mRNA was inhibited to a lesser extent. Western blot showed that VES inhibited the expression of MCM4 protein. Overexpression of E2F1 antagonized the inhibitory effect of VES on MCM4. VES inhibits Rb phosphorylation and increases the binding of Rb to E2F1. BrdU uptake assay showed that the DNA synthesis in cancer cells decreased by 63.4% and 71.6% (P <0.05) compared with the control group after treating with 20μmol / L and 40μmol / L VES for 24 h, respectively, and reached 74.8% and 81.5% P <0.05). Conclusions We identified a new mechanism by which vitamin E inhibits DNA replication in prostate cancer cells by down-regulating the expression of MCM4, which is involved in this signaling pathway.