Epigenetic studies in rhabdomyosarcoma

该项目的直接目标是研究融合阳性和融合阴性横纹肌肉瘤（RMS）肿瘤之间DNA甲基化的差异。在之前的研究中，我们使用Illumina甲基化阵列检测了20个融合阳性和17个融合阴性RMS样本的DNA甲基化。无监督分层聚类和主成分分析表明，RMS肿瘤聚为两组，仅由融合阳性或融合阴性病例组成。我们还在5个融合阳性和5个融合阴性的RMS细胞系中发现了类似的聚类。一项监督分析发现，与融合阴性的RMS肿瘤相比，融合阳性的探针显著高甲基化，其他探针显著低甲基化。为了研究甲基化差异是否代表融合阳性或融合阴性RMS中的“异常”事件，我们将RMS中的甲基化与骨骼肌和骨髓这两种正常组织进行了比较。利用两种RMS亚型之间甲基化差异的CpG位点，分层聚类显示正常组织样本紧密聚集，并且与融合阴性RMS样本聚集在一个主分支上，而所有融合阳性样本都在另一个主分支上。虽然在融合阴性肿瘤和正常组织中，许多差异甲基化位点的甲基化状态相似，但在融合阳性肿瘤中，也有一些差异甲基化位点的甲基化状态与正常组织中的一个或两个非常相似。这些甲基化研究表明，不同基因组的融合亚型与正常组织相似，我们提出融合阳性肿瘤中存在“异常”的低甲基化和高甲基化事件，而融合阴性肿瘤中存在其他“异常”的高甲基化和低甲基化事件。为了进一步研究甲基化与表达之间的关系，我们用DNA甲基转移酶抑制剂5-aza-2'-脱氧胞苷（aza-dC）处理了6个融合阳性和5个融合阴性的RMS细胞系。我们专注于EMILIN1基因，并使用焦磷酸测序发现6个对照处理的融合阳性细胞系中有5个DNA甲基化水平非常高（80%），而5个对照处理的融合阴性细胞系中只有1个。aza-dC处理后，1个融合阴性和5个融合阳性细胞系的EMILIN1启动子甲基化水平显著降低。定量RT-PCR分析显示，对照处理的EMILIN1 mRNA水平极低，而aza-dC处理的EMILIN1 mRNA水平显著升高。这些发现表明，EMILIN1启动子甲基化与表达呈负相关，甲基化缺失导致EMILIN1 mRNA表达增加。

The immediate goal of this project was to investigate differences in DNA methylation between fusion-positive and fusion-negative rhabdomyosarcoma (RMS) tumors. In previous studies, we used Illumina methylation arrays to examine DNA methylation in 20 fusion-positive and 17 fusion-negative RMS samples. Unsupervised hierarchical clustering and principal component analyses showed that the RMS tumors clustered into two groups consisting exclusively of fusion-positive or fusion-negative cases. We also found a similar clustering in a panel of 5 fusion-positive and 5 fusion-negative RMS cell lines. A supervised analysis identified probes that were significantly hypermethylated and other probes that were significantly hypomethylated in fusion-positive compared to fusion-negative RMS tumors. To investigate whether any methylation difference represents an "aberrant" event in fusion-positive or fusion-negative RMS, we compared methylation in RMS with two normal tissues, skeletal muscle and bone marrow. Using the CpG sites that are differentially methylated between the two RMS subtypes, hierarchical clustering revealed that the normal tissue samples were tightly clustered, and were grouped together on one main branch with the fusion-negative RMS samples while all fusion-positive samples were on the other main branch. Though the methylation status of numerous differentially methylated sites is similar in fusion-negative tumors and the normal tissues, there are also differentially methylated sites for which the methylation status in fusion-positive tumors closely resembles either or both normal tissues. These methylation studies show similarities of both fusion subtypes to normal tissues for different sets of genes, and we propose that there are "aberrant" hypo- and hypermethylation events occurring in fusion-positive tumors, and other "aberrant" hyper- and hypomethylation events occurring in fusion-negative tumors. To further examine the relationship between methylation and expression, we treated 6 fusion-positive and 5 fusion-negative RMS lines with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (aza-dC). We focused on the EMILIN1 gene and used pyrosequencing to find very high (80%) DNA methylation levels in 5 of 6 control-treated fusion-positive lines and only 1 of 5 control-treated fusion-negative lines. After aza-dC treatment, there was a significant decrease in EMILIN1 promoter methylation levels in the 1 fusion negative and 5 fusion-positive lines. Quantitative RT-PCR analysis showed that there are very low EMILIN1 mRNA levels with control treatment and significantly increased mRNA levels with aza-dC treatment of the same 1 fusion negative and 5 fusion-positive lines. These findings indicate that EMILIN1 promoter methylation and expression are inversely correlated, and that loss of methylation results in increased EMILIN1 mRNA expression.