Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología (INCan), Universidad Nacional Autónoma de México (UNAM), México, DF, México.
BMC Cancer. 2012 Jan 25;12:40. doi: 10.1186/1471-2407-12-40.
In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. In the present study, we evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. In addition, we analyzed the association of CTCF and covalent histone modifications at the miR-125b1 locus.
To assess the DNA methylation status of the miR-125b1, genomic DNA was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. The miR-125b1 gene expression was analyzed by qRT-PCR using U6 as a control for constitutive gene expression. CTCF repressive histone marks abundance was evaluated by chromatin immunoprecipitation assays.
The disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors.
A reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.
在癌细胞中，转录基因沉默与遗传和表观遗传缺陷有关。DNA 甲基化模式和共价组蛋白标记的破坏与癌症的发展有关。直到最近，miRNA（miRNA）基因沉默还没有得到很好的理解。特别是，miR-125b1 被认为是一种具有肿瘤抑制活性的 miRNA，并且已经在各种人类癌症中被证明是失调的。在本研究中，我们评估了 miR-125b1 启动子近端 CpG 岛的 DNA 甲基化在癌细胞系以及正常组织和妇科肿瘤样本中的情况。此外，我们分析了 CTCF 和 miR-125b1 基因座上的共价组蛋白修饰的关联。
为了评估 miR-125b1 的 DNA 甲基化状态，将基因组 DNA 用亚硫酸氢盐转化，然后用修饰的引物进行 PCR 扩增并测序。使用 U6 作为组成型基因表达的对照，通过 qRT-PCR 分析 miR-125b1 的基因表达。通过染色质免疫沉淀试验评估 CTCF 抑制性组蛋白标记的丰度。
乳腺癌细胞中 CTCF 的破坏与抑制性组蛋白标记如 H3K9me3 和 H3K27me3 的掺入以及异常的 DNA 甲基化模式相关。为了确定 miR-125b1 的 CpG 岛上的 DNA 甲基化对该基因表达的影响，我们进行了 qRT-PCR 测定。与对照相比，我们观察到癌细胞中 miR-125b1 的表达显著降低，表明 CpG 岛上的 DNA 甲基化可能降低 miR-125b1 的表达。这些效应在其他妇科癌症中也观察到，包括卵巢和宫颈癌。
在癌症中，miR-125b1 的表达减少与甲基化、抑制性组蛋白标记和启动子区域的 CTCF 结合丢失相关。
