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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 investigate the expression of estrogen receptor β (ERβ) in human colon cancer cell line HCT116 and its relationship with mTOR gene. Methods: HCT116 cells were transfected with ERβ plasmid (with or without ERβ agonist), siRNA mTOR gene and 5-aza-dC, and the expression of mTOR gene was detected by real-time quantitative PCR The mRNA expressions of mTOR, ERβ and cyclinD1 in cells were detected by Western blotting. The protein expressions of p-mTOR, mTOR, ERβ and cyclinD1 were detected by Western blot. Results: HCT116 cells transfected with ERβ plasmid could significantly downregulate the expression of p-mTOR and cyclinD1, but there was no change in the expression of mTOR protein, regardless of the presence of ERβ agonist. After siRNA interference with mTOR gene, the expression of ERβ increased significantly and the mRNA and protein expression of cyclinD1 decreased. 5-aza-dC could significantly promote the expression of ERβ in HCT116 cells. The expression of mTOR gene at mRNA and protein levels was not significantly different, but the protein level of p-mTOR decreased, and both the mRNA and protein levels of cyclinD1 decline. CONCLUSION: ERβ and mTOR have negative mutual regulation. The methylation of ERβ promoter also exists in HCT116 cells. This provides a preliminary experimental evidence for the combined use of ERβ and its specific agonists and mTOR inhibitors in the prevention and treatment of colon cancer.