5AZA-induced Gene Expression in Human Breast Cancer Cell Lines and Benign Primary Mammary Epithelial Cell Cultures

The goal of this experiment was to identify genes that were expressed at higher levels in benign human mammary epithelial cells than in breast cancer cell lines and that were induced by 5AZA treatment in breast cancer cell lines. Six breast cancer cell lines were selected for demethylation studies based on known tumor suppressor gene expression regulation by promoter region hypermethylation: HCC1569 (CCND2), HCC1954 (SCGB3A1, APC, RASSF1A), MCF-7 (RAR-beta2), MDA-MB-231 (ESR1), UACC3199 (BRCA1), and BT-549 (hypermethylator phenotype). Other than MCF10A we specifically avoided immortalized benign human mammary epithelial cell lines for this experiment as these cells frequently show tumor suppressor gene methylation (e.g. p16) and gene expression profiles that are intermediate between normal breast epithelial cells and breast cancer. Instead, we opted to test six first-passage benign human mammary epithelial cell cultures (HME) generated in serum-free media from small fragments of normal breast tissue obtained from young women undergoing fibroadenoma excision. The 5AZA dose (0.5 microM) was selected based on evaluation of growth curves and induction of BNC1, SERPINB, and TKTL1 gene expression measured by RT-PCR in benign and malignant cells. The breast cancer cell lines, HME cultures, and MC10A cells were treated with 0.5 microM 5AZA (Sigma-Aldrich, St. Louis, MO) in DMSO or DMSO alone for six days after which the cells were harvested, and RNA prepared using the Illumina TotalPrep kit (AMIL1791, Life Technologies, Grand Island, NY). Whole genome expression was assessed using the Illumina HumanWG-6-v3 chip Gene expression was evaluated in 6 breast cancer cell lines, 6 primary breast epithelial cell cultures, and MCF10A cells after 6 days in DMSO or DMSO plus 0.5 microM 5AZA.

本实验旨在鉴定在良性人乳腺上皮细胞中表达水平高于乳腺癌细胞系，且可在乳腺癌细胞系中经5AZA处理诱导表达的基因。基于启动子区域高甲基化调控的已知抑癌基因表达特征，我们筛选出6株乳腺癌细胞系用于去甲基化研究：HCC1569（关联CCND2）、HCC1954（关联SCGB3A1、APC、RASSF1A）、MCF-7（关联RAR-beta2）、MDA-MB-231（关联ESR1）、UACC3199（关联BRCA1）以及BT-549（呈现高甲基化表型）。本实验特意避开了除MCF10A外的永生化良性人乳腺上皮细胞系，因为此类细胞常出现抑癌基因（如p16）的甲基化修饰，且基因表达谱介于正常乳腺上皮细胞与乳腺癌细胞之间。因此，我们最终选取了6株初代传代的良性人乳腺上皮细胞培养物（HME），这些细胞源自接受纤维腺瘤切除术的年轻女性的正常乳腺组织小块，采用无血清培养基培养获得。本实验选用的5AZA处理浓度为0.5μM，该浓度是通过评估生长曲线，以及采用逆转录聚合酶链反应（RT-PCR）检测良、恶性细胞中BNC1、SERPINB、TKTL1基因的诱导表达水平后确定的。将乳腺癌细胞系、HME培养物及MCF10A细胞分别用含0.5μM 5AZA（Sigma-Aldrich，美国密苏里州圣路易斯市）的二甲基亚砜（DMSO）溶液，或仅含DMSO的对照溶液处理6天；随后收集细胞，采用Illumina TotalPrep试剂盒（货号AMIL1791，美国Life Technologies公司，纽约州格兰德艾兰市）提取总RNA。采用Illumina HumanWG-6-v3芯片对全基因组表达谱进行检测。本研究共对6株乳腺癌细胞系、6株原代乳腺上皮细胞培养物及MCF10A细胞开展基因表达分析，所有样本均经含或不含0.5μM 5AZA的DMSO溶液处理6天。