Application of sensitive EMT/MET reporter to identification of target genes for breast cancer stem cells.

为了鉴定能诱导EMT或MET的cDNA/shRNA，我们建立了一个报告系统，其中vimentin或E-cadherin启动子在HMLE上皮细胞或MDA-MB-231间充质乳腺癌细胞系中驱动杀稻瘟菌素抗性基因和GFP。 用cDNA或shRNA文库感染细胞并进行杀稻瘟菌素选择。在EMT筛选中，Snail和Twist 2被鉴定为EMT诱导剂，并且鉴定了两个基因，线粒体复合物I的成员和信号转导中的hub基因，以支持间充质细胞的软无集落形成。在MET筛选中，鉴定了一种有效的MET诱导shRNA（称为shP 1）。使用来自SUM 159间充质乳腺癌细胞系的单克隆双报告细胞克隆测试shP 1的软琼脂集落形成。有趣的是，存在两组对shP 1具有不同响应的报告基因克隆。在一组报告克隆中，集落形成没有减少，而在另一个克隆中，shP 1减少了集落形成。

In order to identify cDNA/shRNA that can induce EMT or MET, we established a reporter system where vimentin or E-cadherin promoter drives blasticidine-resistant gene and GFP in HMLE epithelial or MDA-MB-231 mesenchymal breast cancer cell line. The cells were infected with cDNA or shRNA library and subjected to blasticidine selection. In EMT screening, Snail and Twist2 were identified as EMT inducer, and two genes, members of mitochondrial complex I, and a hub gene in signal transduction were identified to support soft aga-colonogenecity of mesenchymal cells. In MET screen, a potent MET-inducing shRNA (called shP1) was identified. The shP1 was tested for soft agar colony formation using monoclonal dual reporter cell clones from SUM159 mesenchymal breast cancer cell line. Interestingly, there are two sets of reporter clones with different response to shP1. Colony formation did not decreased in one set of reporter clones, while in the other clone shP1 reduced colony formation.