Omura Noriyuki, Li Chung-Pin, Li Ang, Hong Seung-Mo, Walter Kimberly, Jimeno Antonio, Hidalgo Manuel, Goggins Michael
Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
Cancer Biol Ther. 2008 Jul;7(7):1146-56. doi: 10.4161/cbt.7.7.6208. Epub 2008 Apr 29.
Many genes undergo aberrant methylation in human cancers, and microarray platforms enable more comprehensive profiling of aberrant DNA methylation patterns.
1,010 of 87,922 probes on the 88 K promoter array (606 genes) had a higher signal (log(2) > 2) in the pancreatic cancer line, Panc-1 compared to the non-neoplastic pancreatic duct line, HPDE. Using this cut-off, bisulfite sequencing and/or MSP confirmed differential methylation of all 27 genes (66 probes) predicted to be methylated by the MCA array. More than 1/2 of the genes aberrantly hypermethylated in Panc-1 were not expressed in the pancreatic duct (HPDE) by expression array analysis. Using the 244 K CpG island array, 1,968 CpG islands were differentially methylated in MiaPaca2 compared to normal pancreas. The MCA method was more likely to identify hypermethylation within CpG islands than a cocktail of methylation sensitive restriction enzymes. DNA methylation profiles using 10 ng of DNA were highly correlated with those obtained using 5 ug of DNA (R2 = 0.98). Analysis of 57 pancreatic cancers and 34 normal pancreata using MSP identified MDFI, hsa-miR-9-1, ZNF415, CNTNAP2 and ELOVL4 as methylated in 96%, 89%, 86%, 82% and 68% of the cancers vs. 9%, 15%, 6%, 3% and 97% of normal pancreata, respectively.
We used methylated CpG island amplification (MCA) and Agilent promoter and CpG island microarrays to identify differential DNA methylation patterns in pancreatic cancer vs. normal pancreas. We examined MCA array reproducibility, compared it to methylation profiles obtained using a cocktail of methylation-sensitive restriction enzymes and examined gene expression of methylated genes.
Promoter and CpG island array analysis finds aberrant methylation of hundreds of promoters and CpG islands in pancreatic cancer cells.
许多基因在人类癌症中发生异常甲基化，微阵列平台能够更全面地分析异常DNA甲基化模式。
在88K启动子阵列上的87,922个探针中，有1,010个（606个基因）在胰腺癌系Panc-1中的信号较高（log(2)>2），而在非肿瘤性胰腺导管系HPDE中则不然。使用此临界值，亚硫酸氢盐测序和/或甲基化特异性PCR（MSP）证实了MCA阵列预测甲基化的所有27个基因（66个探针）的差异甲基化。通过表达阵列分析，Panc-1中异常高甲基化的基因超过1/2在胰腺导管（HPDE）中不表达。使用244K CpG岛阵列，与正常胰腺相比，MiaPaca2中有1,968个CpG岛存在差异甲基化。与甲基化敏感限制酶混合物相比，MCA方法更有可能识别CpG岛内的高甲基化。使用10ng DNA的DNA甲基化谱与使用5μg DNA获得的谱高度相关（R2 = 0.98）。使用MSP分析57例胰腺癌和34例正常胰腺，发现MDFI、hsa-miR-9-1、ZNF415、CNTNAP2和ELOVL4在癌症中的甲基化率分别为96%、89%、86%、82%和68%，而在正常胰腺中的甲基化率分别为9%、15%、6%、3%和97%。
我们使用甲基化CpG岛扩增（MCA）以及安捷伦启动子和CpG岛微阵列来识别胰腺癌与正常胰腺之间的差异DNA甲基化模式。我们检查了MCA阵列的可重复性，将其与使用甲基化敏感限制酶混合物获得的甲基化谱进行比较，并检查了甲基化基因的基因表达。
启动子和CpG岛阵列分析发现胰腺癌细胞中数百个启动子和CpG岛存在异常甲基化。
