【摘要】 目的探讨人乳腺癌雌激素受体(ER)基因启动子的去甲基化和ER蛋白表达功能恢复的关系。方法用甲基化特异性PCR(MSP)和亚硫酸氢盐基因组测序方法检测乳腺癌细胞ER基因启动子的甲基化状态;用RT-PCR方法从mRNA水平检测ER和孕激素受体(PR)基因的表达;用Western blot方法从蛋白水平探测ER基因的表达;MTT方法检测重新表达ER蛋白的功能。结果在ER阴性乳腺癌细胞系MDA-MB-231中,ER基因启动子甲基化的水平较高,而ER mRNA和ER蛋白均不表达。MDA-MB-231细胞用去甲基化剂5-杂氮-2’-脱氧胞嘧啶(5-AZA-2’-deoxyC)处理,可以恢复该细胞系ER mRNA和ER蛋白的表达,同时伴有ER基因启动子甲基化水平的降低以及PR基因的表达。MDA-MB-231细胞经去甲基化逆转ER表达后,应用三苯氧胺治疗,乳腺癌细胞增殖受到明显抑制(P<0．05)。结论人乳腺癌ER基因的失活与其基因启动子高甲基化关系密切。去甲基化剂5-AZA-2’-deoxyC能较好地降低MDA-MB-231细胞DNA甲基化,并有效激活功能性ER基因的再表达,为ER基因阴性的乳腺癌患者接受内分泌治疗提供了新的途径和理论基础。更多还原

【Abstract】 Objective To investigate the correlation between the lack of estrogen receptor (ER) gene expression and hypermethylation of ER gene, and detect whether re-expressed ER protein is activated. Methods The methylation status of ER gene promoter in the ER-negative breast cancer cells was evaluated by methyiation specific PCR ( MSP) and genomic sequencing. The expression of ER and progesterone receptor (PR) mRNA as well as the production of ER protein were detected by RT-PCR and Western blot method, respectively. MTT assay was used to examine the function of re-expressed ER protein. Results The ER gene promoter was highly methylated, while ER mRNA and ER protein were not expressed in the ER-negative breast cell line MDA-MB-231. The ER-negative breast cells treated with demethylating agent 5-aza-2’-deoxycytidine(5-AZA-2’-deoxyC)restored the expression of ER mRNA and ER protein. Expression of the endogenous ER-responsive PR gene was activated and the methylation of ER gene was simultaneously decreased. After MDA-MB-231 was treated with 5-AZA-2’-deoxyC, the protein of ER was re-expressed and the growth of cells treated with tamoxifen were inhibited significantly ( P < 0. 05 ). Conclusion The inactivation of ER gene has a close relationship with the abnormal methylation of ER gene promoter. 5-AZA-2’-deoxyC may effectively cause demethylation and restore functional expression of ER silenced by aberrant hypermethylation. The result may offer a new measure and theory for breast cancer patients with ER-negative expression to receive endocrine therapies.更多还原