Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels
Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels
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DOI：
10.3892/ijo.2012.1464
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发表时间：
2012-08-01
影响因子：
5.2
通讯作者：
Wu, Ling
中科院分区：
文献类型：
--
作者：
Lian, Zhen-Qiang;Wang, Qi;Wu, Ling
为了筛选用于乳腺癌早期检测的候选甲基化标记物并探讨甲基化与基因表达之间的关系，我们对 10 个癌性乳腺组织和 10 个非癌性乳腺组织中 4162 个基因的 61982 个 CpG 位点进行了甲基化 CpG 岛恢复测定 (MIRA) 结合 CpG 岛阵列。在独立的癌性和非癌性样本中进行直接亚硫酸氢盐测序和组合亚硫酸氢盐限制性分析（COBRA）。通过微阵列分析基因表达并使用 RT-PCR 进行验证。我们在乳腺癌组织中检测到70个显着高甲基化的基因，包括许多新的高甲基化基因，如ITGA4、NFIX、OTX2和FGFI2。直接亚硫酸氢盐测序显示乳腺癌组织中 WTI、PAX6 和 ITGA4 基因的基因内区域以及 OTX2 基因的启动子区域广泛存在甲基化。 COBRA 检测证实乳腺癌组织中 WTI、OTX2 和 PAX6 基因高甲基化。对70个显着高甲基化基因的基因表达进行聚类分析表明，乳腺癌中大多数高甲基化基因在乳腺组织中不表达。 RT-PCR检测证实WTI和PITX2仅在乳腺癌组织中微弱表达，而在大多数非癌性乳腺组织中不表达。 OTX2 和 PAX6 在乳腺癌或非癌组织中均不表达。总之，这些结果将扩大我们对高甲基化基因和甲基化位点的了解，以用于乳腺癌的早期检测，并加深我们对甲基化与基因表达之间关系的理解。 MIRA 方法可以比基于基因表达微阵列的策略更有效地筛选候选甲基化基因以进行进一步的临床验证。
To screen candidate methylation markers for early detection of breast cancer and to explore the relationship between methylation and gene expression, we performed methylated-CpG island recovery assay (MIRA) combined with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous and non-cancerous samples. Gene expression was analyzed by microarrays and validated using RT-PCR. We detected 70 significantly hypermethylated genes in breast cancer tissues, including many novel hypermethylated genes such as ITGA4, NFIX, OTX2 and FGFI2. Direct bisulfite sequencing showed widespread methylation occurring in intragenic regions of the WTI, PAX6 and ITGA4 genes and in the promoter region of the OTX2 gene in breast cancer tissues. COBRA assay confirmed that the WTI, OTX2 and PAX6 genes were hypermethylated in breast cancer tissues. Clustering analysis of the gene expression of 70 significantly hypermethylated genes revealed that most hypermethylated genes in breast cancer were not expressed in breast tissues. RT-PCR assay confirmed that WTI and PITX2 were only weakly expressed in the breast cancer tissues and were not expressed in most non-cancerous breast tissues. OTX2 and PAX6 were not expressed in either breast cancer or non-cancerous tissues. In conclusion, these results will expand our knowledge of hypermethylated genes and methylation sites for early detection of breast cancer and deepen our understanding of the relationship between methylation and gene expression. The MIRA approach can screen candidate methylated genes for further clinical validation more effectively than gene expression microarray-based strategy.