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目的研究六氟双酚A(bisphenol AF,BPAF)诱导T47D乳腺癌细胞中细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)激活的分子机制,探讨雌激素受体α(estrogen receptorα,ERα)在BPAF诱导ERK激活过程中的作用。方法通过蛋白免疫印迹实验(Western Blot)检测ERK磷酸化水平,研究BPAF对ERK磷酸化的诱导作用。运用表达ERαshRNA的慢病毒感染细胞,下调表达ERα,研究ERα在BPAF激活ERK过程中的作用。结果Western Blot结果显示,BPAF在浓度为50 nmol/L时即可诱导ERK磷酸化水平显著升高(P<0.05),并且随着剂量的升高呈现出剂量-效应关系。当BPAF浓度达到1μmol/L时ERK磷酸化水平达到最高(P<0.05)。BPAF诱导ERK激活时间效应实验中,BPAF处理细胞5~60 min时,ERK磷酸化水平均显著升高(P<0.05),在BPAF处理细胞15 min时,ERK磷酸化水平达到最高。在运用表达ERαshRNA的慢病毒感染细胞后,ERα的表达水平显著降低(P<0.05)。与正常细胞相比,在下调表达ERα的细胞中,BPAF对ERK磷酸化的诱导作用显著降低(P<0.05)。结论 BPAF能够诱导ERK磷酸化,并且呈现剂量-效应关系。ERα在BPAF诱导的ERK激活过程中起到重要作用。
Objective To investigate the molecular mechanism of extracellular regulated protein kinase (ERK) activation induced by bisphenol AF (BPAF) in T47D breast cancer cells and to explore the role of estrogen receptorα (ERα) in BPAF induces ERK activation. Methods The phosphorylation of ERK was detected by Western Blot and the induction of phosphorylation of ERK by BPAF was studied. The cells were infected with lentivirus expressing ERαshRNA and the expression of ERα was down-regulated to investigate the role of ERα in the activation of ERK by BPAF. Results The results of Western Blot showed that the phosphorylation level of ERK induced by BPAF was significantly increased at a concentration of 50 nmol / L (P <0.05), and showed a dose-response relationship with increasing dose. The level of ERK phosphorylation reached the peak when BPAF concentration reached 1μmol / L (P <0.05). BPAF-induced ERK activation time effect experiment, BPAR treatment of cells 5 ~ 60 min, ERK phosphorylation levels were significantly increased (P <0.05), BPAMP-treated cells at 15 min, ERK phosphorylation level reached the highest. After infected with lentivirus expressing ERαshRNA, the expression of ERα was significantly decreased (P <0.05). Compared with normal cells, the induction of ERK phosphorylation by BPAF was significantly decreased (P <0.05) in ERα-overexpressing cells. Conclusions BPAF can induce ERK phosphorylation and show a dose-response relationship. ERα plays an important role in BPAF-induced ERK activation.