Nojima D, Li L C, Dharia A, Perinchery G, Ribeiro-Filho L, Yen T S, Dahiya R
Department of Urology, University of California-San Francisco and Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Cancer. 2001 Oct 15;92(8):2076-83. doi: 10.1002/1097-0142(20011015)92:8<2076::aid-cncr1548>3.0.co;2-a.
The down-regulation of the estrogen receptor-beta (ERbeta) gene is associated with several malignancies, including prostate carcinoma. The purpose of the current study was to investigate the mechanisms of ERbeta inactivation through the analysis of CpG methylation of the promoter region of ERbeta gene.
ERbeta protein expression was examined by immunohistochemistry in 23 cases of human prostate carcinoma and 40 cases of benign prostatic hyperplasia (BPH). DNA was extracted from these tissues and processed for sodium bisulfite genomic sequencing. The percentage of methylation of CpG sites in the promoter region of ERbeta (-376 to -117), which contains 19 CpG sites, was determined from genomic sequencing data. The prostate carcinoma cell lines DU145 and ND1 were treated with the demethylating agent 5-AZAC and ERbeta mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction.
In BPH tissues, ERbeta protein expression was found mainly in epithelial cells. ERbeta protein expression was lacking in 83% of prostate carcinoma samples (19 of 23 samples) whereas all cases of BPH (40 of 40) demonstrated expression of ERbeta protein. The mechanism of inactivation of the ERbeta gene in prostate carcinoma was CpG methylation because the degree of methylation at all CpG sites within the promoter region between -376 and -117 was higher in prostate carcinoma samples compared with BPH tissues. Nine of 19 CpG sites within the promoter region of ERbeta displayed significant differences in methylation between prostate carcinoma and BPH samples. The prostate carcinoma cell lines appeared to lack ERbeta expression. However, 5-AZAC treatment restored ERbeta expression in those cell lines, suggesting that methylation inactivates the ERbeta gene in prostate carcinoma.
The results of the current study demonstrate, for what we believe to be the first time, that the inactivation of the ERbeta gene in prostate carcinoma occurs through CpG methylation of the promoter region of this gene.
雌激素受体β（ERβ）基因的下调与包括前列腺癌在内的多种恶性肿瘤相关。本研究的目的是通过分析ERβ基因启动子区域的CpG甲基化来探讨ERβ失活的机制。
采用免疫组织化学法检测23例人前列腺癌组织和40例良性前列腺增生（BPH）组织中ERβ蛋白的表达。从这些组织中提取DNA并进行亚硫酸氢盐基因组测序。根据基因组测序数据确定ERβ启动子区域（-376至-117）中包含19个CpG位点的CpG位点甲基化百分比。用去甲基化剂5-氮杂胞苷处理前列腺癌细胞系DU145和ND1，并通过逆转录聚合酶链反应分析ERβ mRNA的表达。
在BPH组织中，ERβ蛋白表达主要见于上皮细胞。83%的前列腺癌样本（23个样本中的19个）缺乏ERβ蛋白表达，而所有BPH病例（40个样本中的40个）均显示ERβ蛋白表达。前列腺癌中ERβ基因失活的机制是CpG甲基化，因为与BPH组织相比，前列腺癌样本中启动子区域-376至-117之间所有CpG位点的甲基化程度更高。ERβ启动子区域19个CpG位点中的9个在前列腺癌和BPH样本之间的甲基化存在显著差异。前列腺癌细胞系似乎缺乏ERβ表达。然而，5-氮杂胞苷处理可恢复这些细胞系中的ERβ表达，表明甲基化使前列腺癌中的ERβ基因失活。
本研究结果首次证明，前列腺癌中ERβ基因的失活是通过该基因启动子区域的CpG甲基化发生的。
