人结肠癌细胞株HCT116中雌激素受体β与mTOR基因相互作用的初步研究

Study on interaction between estrogen receptor β and mTOR gene in human colon cancer cell line HCT116

目的:探讨雌激素受体β(ERβ)在人结肠癌细胞株HCT116中的表达及其与mTOR基因的相互关系。方法:分别采用ERβ质粒转染(联合或不联合ERβ激动剂)、siRNA干扰mTOR基因、5-氮脱氧胞苷(5-aza-dC)处理HCT116细胞株;通过实时定量PCR法检测各处理组细胞中之mTOR、ERβ和cyclinD1的mRNA表达;蛋白印迹法检测p-mTOR、mTOR、ERβ和cyclinD1的蛋白表达。结果:HCT116细胞株转染ERβ质粒后,无论是否存在ERβ激动剂,都可明显下调p-mTOR和cyclinD1的蛋白表达水平,但是mTOR蛋白的表达却无变化。siRNA干扰细胞的mTOR基因后,ERβ表达明显增加而cyclinD1的mRNA和蛋白表达水平均下降。5-aza-dC处理后,能明显促进HCT116细胞株中ERβ的表达,mTOR基因在mRNA和蛋白水平的表达都无明显差异,但p-mTOR的蛋白水平降低,cyclinD1的mRNA和蛋白表达水平都下降。结论:ERβ和mTOR之间具有负相互调节作用;HCT116细胞中亦存在ERβ启动子甲基化的现象。这为ERβ及其特异性激动剂和mTOR抑制剂的联合应用防治结肠肿瘤提供了初步的实验依据。

Objective To explore the expression of estrogen receptor β（ERβ） and its relationship with mTOR gene in human colon cancer cell line HCT116.Methods The ERβ plasmid was transfected into HCT116 cells with or without the agonist（Gesintein）.SiRNA directed to mTOR or 5-aza-dC were used to treated the HCT116 cells respectively.The mRNA expression of mTOR,ERβ and cyclinD1 were quantitated by real-time PCR.The protein of phosphorylated-mTOR（p-mTOR）,mTOR,ERβ and cyclinD1 were detected by Western blotting.Results The protein level of p-mTOR and cyclinD1 were significantly inhibited in HCT116 cells transfected by ERβ plasmid with or without the agonist,while the total protein level of mTOR remained unchanged.The mRNA and protein level of ERβ all increased while those of cyclinD1 decreased after the treatment of HCT116 cells with siRNA directed to mTOR.After treatment with 5-aza-dC,the expression of ERβ was significantly increased,while the mTOR expression remained unchanged at the mRNA and protein level;however,the protein level of p-mTOR was decreased.The expression of cyclinD1 was decreased both at the mRNA and protein level.Conclusions ERβ conversely interacted with mTOR;The promoter of ERβ also has DNA methylation in HCT116 cells,which provided primary experimental evidence of combination of ERβ with its specific agonist and mTOR inhibitor in the prevention and treatment of colon cancer.