Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells
(A) A series of 5’ deletions of the human miR-200b~200a~429 locus comprising the promoter and potential enhancer were cloned into a firefly luciferase reporter plasmid. (B) The reporter plasmids were transiently transfected along with the Renilla pTK vector into epithelial HMLE cells (white bars) or mesenchymal HMLE cells (black bars). Luciferase activity was assayed approximately 48 hours later using the Dual-Luciferase Reporter Assay System (Promega). Data are expressed as normalized luciferase activity and represent means ± SD of at least four independent experiments. (C) The enhancer region (-5771/-4607 ENH) was cloned in both directions immediately upstream of the minimal miR-200b~200a~429 promoter (-321/+19 PRO) or the Luciferase coding region creating PRO&ENH and ENH as well as PRO&ENH (-) and ENH (-) with ENH oriented in the sense and antisense orientation, respectively. Luciferase activity was assay as described in (B).