Analysis of Promoter Methylation in Breast Cancer

Promoter methylation was assayed in a number of breast cancer and control normal samples along with the effects of 5'-aza-2'-deoxycytidine on breast cancer cell line transcriptomes. Aberrant promoter hypermethylation is frequently observed in cancer. The potential for this to contribute to tumour development depends on whether the genes affected are repressed because of their methylation. Many aberrantly methylated genes play important roles in development and are bivalently marked in ES cells suggesting that their aberrant methylation may reflect developmental processes. We investigated this possibility by analysing promoter methylation in 19 breast cancer cell lines, 10 normal tissues and 47 primary breast tumours. In order to determine the role of DNA methylation in silencing genes in breast cancer, we also examined the effects of the demethylating agent 5-aza-2?-deoxycytidine on gene expression in 3 breast cancer cell lines and HCT116 cells. Gene expression changes were also assayed in the DNA methyltransferase deficient HCT116 DKO cell line. Our findings implicate aberrant DNA methylation as a marker of cell lineage rather than tumour progression and suggest that, in most cases, it does not cause the repression with which it is associated. A number of human breast cancer cell lines, breast tumours and normal tissues were analysed on Illumina Infinium Methylation27 Beadchips to assay promoter methylation. Selected cell lines were analysed on expression arrays before and after treatment with 5-aza-2'-deoxycytidine.

本研究针对多组乳腺癌样本与正常对照样本开展启动子甲基化（promoter methylation）检测，并同时探究5'-氮杂-2'-脱氧胞苷（5'-aza-2'-deoxycytidine）对乳腺癌细胞系转录组的影响。异常启动子高甲基化在癌症中十分常见，此类异常甲基化能否促进肿瘤发生，取决于受影响的基因是否因甲基化而被沉默。众多异常甲基化基因在发育过程中发挥重要作用，且在胚胎干细胞（ES cells）中呈二价染色质标记状态，这提示其异常甲基化可能反映了发育相关进程。为此，我们通过分析19株乳腺癌细胞系、10份正常组织以及47例原发性乳腺肿瘤的启动子甲基化水平，对这一假说展开验证。为明确DNA甲基化在乳腺癌基因沉默中的作用，我们还检测了去甲基化试剂5'-氮杂-2'-脱氧胞苷（5'-aza-2'-deoxycytidine）对3株乳腺癌细胞系及HCT116细胞的基因表达影响。同时，我们还在DNA甲基转移酶缺陷型HCT116 DKO细胞系中检测了基因表达变化。本研究结果显示，异常DNA甲基化更倾向于作为细胞谱系的标志物，而非肿瘤进展的标志物；且在大多数情况下，异常甲基化并不会与其伴随的基因沉默存在因果关联。我们采用Illumina Infinium Methylation27 Beadchips（Illumina甲基化27K微珠芯片）对多株人乳腺癌细胞系、乳腺肿瘤组织及正常组织开展启动子甲基化检测。针对部分筛选出的细胞系，我们分别在经5'-氮杂-2'-脱氧胞苷处理前后，通过表达芯片检测其基因表达变化。