5`aza-dC demethylation of three short term cultured glioblastomas

Glioblastoma, the most aggressive and least treatable form of malignant glioma, is the most common human brain tumor. Although many regions of allelic loss occur in glioblastomas, relatively few tumor suppressor genes have been found mutated at such loci. To address the possibility that epigenetic alterations are an alternative means of glioblastoma gene inactivation, we coupled pharmacological manipulation of methylation with gene profiling to identify potential methylation-regulated, tumor-related genes. Triplicates of three short-term cultured glioblastomas were exposed to 5μM 5-aza-dC for 96 hours followed by cRNA hybridization to an oligonucleotide microarray (Affymetrix U133A). We based candidate gene selection on bioinformatics, RT-PCR, bisulfite sequencing, methylation-specific PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two genes identified in this manner, RUNX3 and Testin (TES), were subsequently shown to harbor frequent tumor-specific epigenetic alterations in primary glioblastomas. This overall approach therefore provides a powerful means to identify candidate tumor suppressor genes for subsequent evaluation and may lead to the identification of genes whose epigenetic dysregulation is integral to glioblastoma tumorigenesis. Keywords: expression profile following global demethylation Duplicates of three short term cultured glioblastoma cell lines (internal IDs: GLI56;GLI60;GLI72) were either exposed to 5umol 5`aza-dC for 96h or left untreated. Total RNA was extracted of treated and untreated cells after 96h and hybridized to U133A chip. Gene expression profiles of treated and untreated cells were compared. Upregulation of gene expression in the treated (demethylated) samples was interpreted as potentially being regulated by methylation - pointing towards hypermethylation in the related cell line. Identification of novel tumor suppressor genes, regulated by methylation, was the overall goal.

胶质母细胞瘤（Glioblastoma）作为恶性胶质瘤中侵袭性最强、治疗预后最差的亚型，是人类最常见的脑恶性肿瘤。尽管胶质母细胞瘤中存在多处等位基因缺失区域，但在这些位点上发生突变的抑癌基因（tumor suppressor gene）却相对较少。为探究表观遗传改变是胶质母细胞瘤基因失活的另一潜在机制这一假说，我们将甲基化药物干预与基因表达谱分析相结合，以筛选受甲基化调控的潜在肿瘤相关基因。我们将3株短期培养的胶质母细胞瘤样本设置3个生物学重复，用5μM的5-氮杂-2'-脱氧胞苷（5-aza-dC）处理96小时，随后提取cRNA并与寡核苷酸微阵列（Affymetrix U133A）进行杂交。我们通过生物信息学分析、逆转录聚合酶链反应（RT-PCR）、亚硫酸氢盐测序、甲基化特异性PCR以及基质辅助激光解吸电离飞行时间质谱来筛选候选基因。通过该方法筛选得到的两个基因RUNX3与Testin（TES），后续被证实在原发性胶质母细胞瘤中存在频繁的肿瘤特异性表观遗传改变。因此，本研究采用的整体策略为筛选可用于后续验证的候选抑癌基因提供了高效手段，或可鉴定出表观遗传失调在胶质母细胞瘤发生发展中发挥关键作用的相关基因。
关键词：全基因组去甲基化后的基因表达谱
本研究设置3株短期培养的胶质母细胞瘤细胞系（内部编号：GLI56、GLI60、GLI72），各设置2个生物学重复，分别用5μmol的5-氮杂-2'-脱氧胞苷处理96小时，同时设置未处理对照组。处理96小时后，分别提取处理组与对照组细胞的总RNA，随后与U133A芯片进行杂交。对处理组与对照组细胞的基因表达谱进行比较分析。处理组（去甲基化组）样本中基因表达上调，被认为是受甲基化调控的潜在标志，提示对应细胞系中该基因存在高甲基化状态。本研究的整体目标为筛选受甲基化调控的新型抑癌基因。