摘要
目的:探讨雌激素受体(ER)β诱导结肠癌细胞自噬的作用机制。方法:将ERβ质粒转染人结肠癌细胞株HCT116(p53野生型)和SW480(p53突变型)后,通过荧光显微镜观察自噬现象;采用蛋白印迹法检测LC3-Ⅱ蛋白、哺乳动物雷帕霉素靶蛋白(mTOR)、AMPK和p70S6K蛋白及相应磷酸化蛋白的表达变化;应用实时定量PCR法检测DRAM1、Sestrin1和Sestrin2的mRNA表达变化。结果:ERβ过表达能促进HCT116和SW480细胞发生自噬;LC3-Ⅱ和p-AMPK蛋白在HCT116细胞中分别上调了2.81倍和1.80倍(P<0.05);在SW480细胞中分别上调了1.70倍和2.48倍(P<0.05)。p-mTOR和p-p70S6K蛋白的表达水平在HCT116细胞中分别下降了29.52%和34.54%,在SW480细胞中分别下降37.89%和41.86%。除了Sestrin1 mRNA在SW480细胞中的表达无变化外,DRAM1和Sestrin2 mRNA的表达量在2种细胞中均显著上调(P<0.05)。结论:ERβ可通过对AMPK/mTORC1轴的调节作用诱导结肠癌细胞发生自噬。
Objective:To investigate the mechanism of estrogen receptor(ER) β on autophagy in colon cancer cells.Methods:After transfection with ERβ plasmid,HCT116(wild-type p53) and SW480(mutant p53),autophagy was observed under fluorescence microscope in human colon cancer cell lines.The protein levels of LC3-Ⅱ,mTOR,AMPK,p70S6K and their related phosphorylated proteins were detected by Western blot.The mRNA expression levels of DRAM1,Sestrin1 and Sestrin2 were measured by real-time PCR.Results:Over-expression of ERβ could induce autophagy in HCT116 and SW480 cells.The protein levels of LC3-Ⅱ and p-AMPK were up-regulated to 2.81-fold and 1.80-fold in HCT116 cells,1.70-fold and 2.48-fold in SW480 cells,respectively(P<0.05).Conversely,the protein levels of p-mTOR and p-p70S6K decreased 29.52% and 34.54% in HCT116 cells,37.89% and 41.86% in SW480 cells,respectively.The mRNA expression levels of DRAM1 and Sestrin2 increased significantly in both two cell lines,whereas the expression of Sestrin1 mRNA had no obvious change in SW480 cells.Conclusions:The modulation of AMPK/mTORC1 axis might play a role in autophagy induced by ERβ in colon cancer cells.
出处
《诊断学理论与实践》
2013年第3期279-283,共5页
Journal of Diagnostics Concepts & Practice
基金
国家自然科学基金面上项目(No.30872973)
上海市重点学科(外科学)(开放课题S30204-k03)
