人乳腺癌细胞雌激素受体基因的去甲基化及其表达

Demethylation of estrogen receptor gene and its re-expression in estrogen receptor-negative breast cancer cells

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。

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 methylation 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.