MicroRNA-200 Family Modulation in Distinct Breast Cancer Phenotypes

The epithelial to mesenchymal transition (EMT) contributes to tumor invasion and metastasis in a variety of cancer types. In human breast cancer, gene expression studies have determined that basal-B/claudin-low and metaplastic cancers exhibit EMT-related characteristics, but the molecular mechanisms underlying this observation are unknown. As the family of miR-200 microRNAs has been shown to regulate EMT in normal tissues and cancer, here we evaluated whether the expression of the miR-200 family (miR-200f) and their epigenetic state correlate with EMT features in human breast carcinomas. We analyzed by qRT-PCR the expression of miR-200f members and various EMT-transcriptional inducers in a series of 70 breast cancers comprising an array of phenotypic subtypes: estrogen receptor positive (ER+), HER2 positive (HER2+), and triple negative (TN), including a subset of metaplastic breast carcinomas (MBCs) with sarcomatous (homologous or heterologous) differentiation. No MBCs with squamous differentiation were included. The DNA methylation status of miR-200f loci in tumor samples were inspected using Sequenom MassArray® MALDI-TOF platform. We also used two non-tumorigenic breast basal cell lines that spontaneously undergo EMT to study the modulation of miR-200f expression during EMT in vitro. We demonstrate that miR-200f is strongly decreased in MBCs compared with other cancer types. TN and HER2+ breast cancers also exhibited lower miR-200f expression than ER+ tumors. Significantly, the decreased miR-200f expression found in MBCs is accompanied by an increase in the expression levels of EMT-transcriptional inducers, and hypermethylation of the miR-200c-141 locus. Similar to tumor samples, we demonstrated that downregulation of miR-200f and hypermethylation of the miR-200c-141 locus, together with upregulation of EMT-transcriptional inducers also occur in an in vitro cellular model of spontaneous EMT. Thus, the expression and methylation status of miR-200f could be used as hypothetical biomarkers to assess the occurrence of EMT in breast cancer.上皮间质转化（epithelial to mesenchymal transition, EMT）可参与多种癌症类型的肿瘤侵袭与转移过程。在人类乳腺癌领域，既往基因表达研究已证实，基底B型/密蛋白低表达型乳腺癌及化生性乳腺癌呈现EMT相关特征，但该现象背后的分子机制仍未阐明。鉴于miR-200微RNA家族已被证明可在正常组织与癌症中调控EMT，本研究旨在评估miR-200家族（miR-200f）的表达水平及其表观遗传状态是否与人类乳腺癌的EMT特征存在相关性。我们通过定量实时逆转录聚合酶链反应（quantitative real-time reverse transcription PCR, qRT-PCR），对70例涵盖多种表型亚型的乳腺癌样本中miR-200f成员及多种EMT转录诱导因子的表达情况进行了分析，所纳入的亚型包括雌激素受体阳性（ER+）、HER2阳性（HER2+）与三阴性乳腺癌（TN），其中还包含一组伴肉瘤样（同源或异源）分化的化生性乳腺癌（MBCs），本研究未纳入伴鳞状分化的MBCs。我们采用Sequenom MassArray® MALDI-TOF平台，检测了肿瘤样本中miR-200f基因座的DNA甲基化状态。此外，我们还选用两株可自发发生EMT的非致瘤性乳腺基底细胞系，在体外研究EMT进程中miR-200f表达的调控机制。本研究证实，相较于其他癌症亚型，化生性乳腺癌中miR-200f的表达水平显著下调；三阴性乳腺癌与HER2阳性乳腺癌的miR-200f表达水平同样低于雌激素受体阳性肿瘤。尤为关键的是，化生性乳腺癌中miR-200f表达下调的同时，伴随EMT转录诱导因子表达水平升高以及miR-200c-141基因座的高甲基化。与肿瘤样本的实验结果一致，我们在体外自发EMT细胞模型中同样观察到miR-200f表达下调、miR-200c-141基因座高甲基化以及EMT转录诱导因子上调的现象。综上，miR-200f的表达与甲基化状态或可作为潜在生物标志物，用于评估乳腺癌中EMT的发生情况。