Epigenetic studies in rhabdomyosarcoma

横纹肌肉瘤的表观遗传学研究

• 批准号: 9153908
• 负责人: Frederic Barr
• 金额: $ 43.38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153908
• 关键词: Algorithms; Biological Assay; Bone Marrow; Categories; Cell Culture Techniques; Cell Line; Childhood; Chromatin; Collection; DNA Methylation; DNA Methyltransferase Inhibitor; Data; Deoxycytidine; Development; Diagnostic; EMILIN1 gene; Epigenetic Process; Event; Family; Genes; Genetic Transcription; Genomic DNA; Goals; Human Genome; Hypermethylation; Malignant Neoplasms; Messenger RNA; Methylation; Modeling; Normal tissue morphology; Nucleic Acid Regulatory Sequences; PAX3 gene; PAX7 gene; Point Mutation; Principal Component Analysis; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; Sampling; Site; Skeletal Muscle; Skeletal bone; Tissue Sample; Tissue-Specific Gene Expression; base; bead chip; bisulfite; cohort; genome wide methylation; genome-wide; inhibitor/antagonist; mRNA Expression; promoter; pyrosequencing; soft tissue; tumor

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.

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 甲基化芯片检测了 20 个融合阳性和 17 个融合阴性 RMS 样本中的 DNA 甲基化。无监督的层次聚类和主成分分析表明，RMS 肿瘤聚类成两组，仅由融合阳性或融合阴性病例组成。我们还在一组 5 个融合阳性和 5 个融合阴性 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 RMS 肿瘤相比，融合阳性肿瘤中显着高甲基化的探针和其他显着低甲基化的探针。为了研究任何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 中的甲基化与两种正常组织（骨骼肌和骨髓）进行了比较。使用两种 RMS 亚型之间差异甲基化的 CpG 位点，层次聚类显示正常组织样本紧密聚集，并与融合阴性 RMS 样本一起分组在一个主分支上，而所有融合阳性样本都在另一主分支上。尽管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，但融合阳性肿瘤中也存在一些差异甲基化位点的甲基化状态与其中一个或两个正常组织非常相似。这些甲基化研究显示两种融合亚型与正常组织在不同基因组上的相似性，我们提出在融合阳性肿瘤中发生“异常”低甲基化和高甲基化事件，而在融合阴性肿瘤中发生其他“异常”高甲基化和低甲基化事件。为了进一步检查甲基化和表达之间的关系，我们用 DNA 甲基转移酶抑制剂 5-aza-2'-deoxycytidine (aza-dC) 处理 6 个融合阳性和 5 个融合阴性 RMS 系。我们重点关注 EMILIN1 基因，并使用焦磷酸测序在 6 个对照处理的融合阳性品系中的 5 个中发现了非常高 (80%) 的 DNA 甲基化水平，而在 5 个对照处理的融合阴性品系中仅发现了 1 个。 aza-dC 处理后，1 个融合阴性系和 5 个融合阳性系中 EMILIN1 启动子甲基化水平显着降低。定量 RT-PCR 分析表明，对于相同的 1 个融合阴性品系和 5 个融合阳性品系，对照处理的 EMILIN1 mRNA 水平非常低，而 aza-dC 处理的 mRNA 水平显着增加。这些发现表明 EMILIN1 启动子甲基化和表达呈负相关，甲基化缺失会导致 EMILIN1 mRNA 表达增加。