Figure 6

mir-200b/a was responsible for Restin-mediated downregulation of ZEB1. RT-PCR was performed to detect mir-200a and mir-200b levels in Restin overexpressed MDA-MB-231 cells (A) and Restin knockdown MCF-7 cells (B). Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to Control or si-Con group. (C) mir-200b/a/429 promoter activity was determined in HEK293 cells by luciferase reporter assay. HEK293 cells were seeded ontn 24-well plates and transfected with mir-200b/a/429 promoter plasmids and different dose of Restin overexpression lentivirus. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of lentivirus (−), ^# p < 0.05 relative to their Control group. (D) ZEB1 3’UTR (WT and mutant) activity was determined by luciferase reporter assay upon Restin overexpression. WT and mutant ZEB1 3’UTR with deletion of mir-200 binding sites were transfected into HEK293 cells. 24 h later, different dose of Restin overexpression lentivirus were added. The firefly luciferase activity was normalized to that of the renilla luciferase. Values were expressed as means ± S.M. of at least three independent experiments. * p < 0.05 relative to cells without addition of Restin lentivirus.