Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening.
Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening.
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DOI：
10.3892/ijo.2016.3532
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发表时间：
2016-07
影响因子：
5.2
通讯作者：
Ha PK
中科院分区：
文献类型：
--
作者：
Ling S;Rettig EM;Tan M;Chang X;Wang Z;Brait M;Bishop JA;Fertig EJ;Considine M;Wick MJ;Ha PK
涎腺腺样囊性癌（ACC）是一种罕见的头颈部恶性肿瘤，目前尚无可用于预测ACC化疗疗效或预后的分子生物学标志物，其癌基因和抑癌基因（TSGs）的基因表达通过DNA启动子甲基化调控可能在ACC的发生发展中起作用。5-氮杂-2 ′-脱氧胞苷（5-AZA）用于揭示ACC小鼠异种移植模型中假定的TSG沉默。将新鲜异种移植物传代，一式三份植入每天用5-AZA处理28天的小鼠中。然后使用Illumina Infinium HumanMethylation 27 BeadChip阵列评估这些异种移植物的全基因组DNA甲基化模式。通过亚硫酸氢盐测序（BS-seq）在6个ACC原发性肿瘤和6个正常对照唾液腺组织的单独群组中进行32个候选基因的验证。在所有6个对照组织中，HCN 2基因启动子均出现高甲基化，但在所有6个ACC肿瘤组织中均出现低甲基化。在一个较大的原发性肿瘤队列（n=32）中进行了BS-seq检测区域中HCN 2启动子甲基化水平的定量验证，证实与正常样本相比，ACC中存在显著的HCN 2低甲基化（n=10; P=0.04）。对ACC组织微阵列进行HCN 2免疫组织化学染色。正常组织中HCN 2染色强度和H评分均显著强于ACC组织，但阳性染色细胞百分比无显著差异。通过我们新的筛选和测序方法，我们确定了几个甲基化的候选基因。这些基因中最重要的HCN 2实际上在肿瘤中是低甲基化的。然而，启动子甲基化状态似乎不是正常和ACC组织中HCN 2表达的主要决定因素。HCN 2低甲基化是ACC的生物标志物，可能在ACC的发生发展中起重要作用。
Salivary gland adenoid cystic carcinoma (ACC) is a rare head and neck malignancy without molecular biomarkers that can be used to predict the chemotherapeutic response or prognosis of ACC. The regulation of gene expression of oncogenes and tumor suppressor genes (TSGs) through DNA promoter methylation may play a role in the carcinogenesis of ACC. To identify differentially methylated genes in ACC, a global demethylating agent, 5-aza-2′-deoxycytidine (5-AZA) was utilized to unmask putative TSG silencing in ACC xenograft models in mice. Fresh xenografts were passaged, implanted in triplicate in mice that were treated with 5-AZA daily for 28 days. These xenografts were then evaluated for genome-wide DNA methylation patterns using the Illumina Infinium HumanMethylation27 BeadChip array. Validation of the 32 candidate genes was performed by bisulfite sequencing (BS-seq) in a separate cohort of 6 ACC primary tumors and 6 normal control salivary gland tissues. Hypermethylation was identified in the HCN2 gene promoter in all 6 control tissues, but hypomethylation was found in all 6 ACC tumor tissues. Quantitative validation of HCN2 promoter methylation level in the region detected by BS-seq was performed in a larger cohort of primary tumors (n=32) confirming significant HCN2 hypomethylation in ACCs compared with normal samples (n=10; P=0.04). HCN2 immunohistochemical staining was performed on an ACC tissue microarray. HCN2 staining intensity and H-score, but not percentage of the positively stained cells, were significantly stronger in normal tissues than those of ACC tissues. With our novel screening and sequencing methods, we identified several gene candidates that were methylated. The most significant of these genes, HCN2, was actually hypomethylated in tumors. However, promoter methylation status does not appear to be a major determinant of HCN2 expression in normal and ACC tissues. HCN2 hypomethylation is a biomarker of ACC and may play an important role in the carcinogenesis of ACC.