Schematic representations of EpCAM promoter with luciferase gene and miRNA targets vector constructs and expressional studies of the constructs using luciferase assay.

<p>miRNA target sequences two copies or four copies as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083398#pone-0083398-t001" target="_blank">Table 1</a>. The reporter gene luciferase under CMV promoter was cloned in EpCAM-TK construct by removing TK gene and inserting luciferase gene. miRNA targets were cloned into 3′ UTR region of the expression vector containing Gluc gene under EpCAM promoter (A). The luciferase expression was quantified by secreted Gaussia luciferase. The plasmid constructs EGP2-luc, EGP2-luc-2T, EGP2-luc-4T, EGP2-luc-2C, and EGP2-luc-4C were transfected into MIO-M1, Nthy-ori 3-1, Y79, WERI-Rb1 cell lines. After incubation for 48 hrs the results were normalized to un-transfected cells and were expressed in relative light units (B). Luciferase levels are expressed as percentage mean ± S.D (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs EGP-2-luc-2T.