Department of Basic Pathology, Federal University of Parana, Curitiba, Brazil.
BMC Cancer. 2010 Jan 28;10:23. doi: 10.1186/1471-2407-10-23.
CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor alpha (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data.
First, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis.
CXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ER alpha protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220).
This is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.
CXCL12 是一种趋化因子，在许多器官和组织中组成性表达。在原发性乳腺癌中已检测到 CXCL12 启动子超甲基化，这有助于其转移潜能。已经表明，雌激素受体 alpha（ESR1）基因也可以被 DNA 甲基化沉默。在这项研究中，我们使用甲基化特异性 PCR（MSP）分析肿瘤细胞系和原发性乳腺癌样本中 CXCL12 启动子的两个区域和 ESR1 的甲基化状态，并将我们的结果与临床病理数据相关联。
首先，我们通过 RT-PCR 分析乳腺癌细胞系中的 CXCL12 表达。我们还使用 5-氮杂-2'-脱氧胞苷（5-aza-CdR）处理和 DNA 亚硫酸氢盐测序来研究乳腺癌细胞系中 CXCL12 的一个特定区域的启动子甲基化。我们通过甲基化特异性 PCR（MSP）评估原发性肿瘤样本中的 CXCL12 和 ESR1 甲基化。最后，使用 Fisher 确切检验分析这些基因的启动子超甲基化，并使用卡方检验、Kaplan-Meier 生存分析和 Cox 回归分析将其与临床病理数据相关联。
CXCL12 启动子第一区域（岛 2）和第二区域（岛 4）的高甲基化与肿瘤细胞系中基因表达缺失相关。在原发性肿瘤中，14.5%的样本中岛 2 发生高甲基化，54%的样本中岛 4 发生高甲基化。ESR1 启动子在 41%的乳腺癌样本中发生高甲基化。此外，随着 ESR1 甲基化频率的增加，ER alpha 蛋白表达水平降低（p<0.0001）。这项研究还表明，CXCL12 岛 4 和 ESR1 甲基化同时以高频率发生（p=0.0220）。
这是第一项表明巴西女性中 CXCL12 和 ESR1 基因的表观遗传调控同时参与的研究。这两个基因的甲基化状态与组织学上更晚期的疾病、转移和死亡显著相关。因此，这些基因的甲基化模式可以用作预测乳腺癌结局的分子标志物。
