Department of Pediatrics, University of Chicago, 900 Ease 57th Street, KCBD Rm, 5100 Chicago, IL 60637, USA.
BMC Cancer. 2010 Jun 14;10:286. doi: 10.1186/1471-2407-10-286.
Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype.
Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity.
Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the THBS-1 promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar.
Our results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.
已证实神经母细胞瘤（NB）患儿中存在表观遗传学异常和 CpG 岛甲基化表型与不良预后相关。本研究分析了七个与成人癌症中存在表观遗传学改变且在调节血管生成、肿瘤生长和细胞凋亡中发挥重要作用的癌症相关基因（THBS-1、CASP8、HIN-1、TIG-1、BLU、SPARC 和 HIC-1），以探讨表观遗传学改变在决定 NB 表型中的作用。
使用两种 NB 细胞系（LA1-55n 和 LA1-5s），它们在软琼脂中形成集落和裸鼠肿瘤形成的能力不同。对 LA1-5s、LA1-55n 和用 5-Aza-dC 处理的 LA1-55n NB 细胞系中的七个基因进行定量 RNA 表达分析。使用甲基化特异性 PCR 检测 THBS-1、HIN-1、TIG-1 和 CASP8 启动子周围的甲基化状态。使用染色质免疫沉淀分析检测 THBS-1 启动子上的组蛋白修饰。使用荧光素酶测定法测定 THBS-1 启动子活性。使用细胞增殖测定法检测 5-Aza-dC 对 NB 细胞生长的影响。使用软琼脂测定法测定肿瘤发生能力。
与 LA1-5s 细胞相比，LA1-55n 细胞的 THBS-1、HIN-1、TIG-1 和 CASP8 启动子的甲基化值更高。与启动子甲基化状态一致，在 LA1-55n 细胞中检测到的基因表达水平较低。在 LA1-55n 细胞中鉴定到与抑制性染色质状态相关的组蛋白标记（H3K9Me3、H3K27Me3 和 H3K4Me3），但在 LA1-5s 细胞中没有。相反，与活性染色质状态相关的三种组蛋白标记（乙酰化 H3、乙酰化 H4 和 H3K4Me3）存在于 LA1-5s 细胞的 THBS-1 启动子区域，而 LA1-55n 细胞中不存在，这表明可接近的染色质结构对 THBS-1 表达很重要。我们还表明，LA1-55n 细胞用 5-Aza-dC 处理会改变 THBS-1 启动子的 DNA 甲基化状态和组蛋白编码，改变细胞形态，并抑制其在软琼脂中形成集落的能力。
我们的结果表明，表观遗传学异常导致 NB 表型，并且可以通过用 5-Aza-dC 逆转表观遗传学改变来抑制致瘤特性。
