Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells
Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells
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DOI：
10.1016/j.gene.2017.03.006
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发表时间：
2017-05-30
期刊：
影响因子：
3.5
通讯作者：
Limaye, Anil M.
中科院分区：
文献类型：
--
作者：
Manjegowda, Mohan C.;Gupta, Paridhi Singhal;Limaye, Anil M.
GPER1，又称GPR30，是一种新型的七跨膜g蛋白偶联雌激素受体，可介导雌激素在靶细胞和组织中的短期（非基因组）和长期（基因组）效应。在过去的二十年中，大量的工作强调了其治疗或预后的效用。然而，关于乳腺组织中GPER1表达的临床数据尚不明确。TCGA RNAseq数据分析显示，与正常乳腺组织相比，原发性乳腺肿瘤中GPER1 mRNA的平均表达量显著降低。这为GPER1的肿瘤抑制作用提供了支持。然而，减少表达的机制尚不完全清楚。我们通过RT-PCR分析了GPER1 mRNA变体在MCF-7和MDA-MB-231细胞中的表达水平，并通过改进的COBRA测定和亚硫酸盐测序分析了GPER1位点上两个CpG岛的甲基化状态。我们的研究结果表明，与MDA-MB-231细胞相比，MCF-7细胞表达更高水平的GPER1 mRNA变体。改良的COBRA检测显示，上游CpG岛（upCpGi）与两个GPER1变体（GPER1v2和v3）的第一个外显子重叠，而下游CpG岛（dnCpGi）与所有变体共有的编码区重叠，甲基化差异不存在。亚硫酸氢盐测序结果显示，核心upCpGi在MCF-7和MDA-MB-231细胞中都是低甲基化的。然而，在MDA-MB-231细胞中，upCpGi 3'端的8个CpGs被超甲基化。DNA甲基转移酶抑制剂5-氮杂胞苷可诱导MDA-MB-231细胞中GPER1 mRNA变体的表达水平。TCGA乳腺癌数据的表达-甲基化相关分析显示，upCpGi侧区域的CpGs甲基化与GPER1 mRNA表达显著负相关。综上所述，我们的研究结果证明了DNA甲基化在GPER1抑制中的作用，暗示了upCpGi的侧翼区域（shore），并提出了乳腺肿瘤中GPER1沉默的潜在机制。(C) 2017 Elsevier B.V.版权所有
GPER1, also known as GPR30, is a novel seven-transmembrane G-protein coupled estrogen receptor that mediates both short-term (non-genomic) and long-term (genomic) effects of estrogen in target cells and tissues. A substantial body of work over the last two decades has highlighted its therapeutic or prognostic utility. However, the clinical data on the expression of GPER1 in breast tissue is ambiguous. Analysis of TCGA RNAseq data revealed significantly lower mean expression of GPER1 mRNA in primary breast tumors compared to that in normal breast tissues. This provides support to the tumor suppressor role for GPER1. However, the mechanisms underlying the reduced expression are not completely understood. We analyzed the expression levels of GPER1 mRNA variants in MCF-7 and MDA-MB-231 cells by RT-PCR, and the methylation status of two CpG islands in the GPER1 locus by modified COBRA assays and bisulfite sequencing. Our results show that MCF-7 cells express higher levels of GPER1 mRNA variants compared to MDA-MB-231 cells. Modified COBRA assays revealed differential methylation in the upstream CpG island (upCpGi) that overlaps with the first exon of two GPER1 variants (GPER1v2 and v3) but not in the downstream CpG island (dnCpGi) that overlaps with the coding region common to all variants. Bisulfite sequencing results showed that the core upCpGi was hypo-methylated in both MCF-7 and MDA-MB-231 cells. However, eight CpGs in the 3' end of the upCpGi were hyper-methylated in MDA-MB-231 cells. 5-Azacytidine, a DNA methyltransferase inhibitor, induced the expression levels of GPER1 mRNA variants in MDA-MB-231 cells. Expression-methylation correlation analysis of TCGA breast cancer data revealed that methylation of CpGs in the regions flanking the upCpGi significantly correlated negatively with GPER1 mRNA expression. Taken together, our results demonstrate the role of DNA methylation in GPER1 repression, implicate the flanking regions (shore) of the upCpGi, and suggest a potential mechanism of GPER1 silencing in breast tumors. (C) 2017 Elsevier B.V. All rights reserved.