Figure 4

miR-26b downregulates USP9X expression via directly targeting the USP9X 3′UTR. (A) Sequence alignment of the 3′UTR in Hs(human), Mm(mouse), Rn(rat), and Cf (dog) with miR-26b indicates that miR-26b targets USP9X expression. (B) Representative western blots of GFP expression in Huh7 and Hep3B cells under the indicated experimental conditions. (C) Representative fluorescent microscope images of Smad4 expression in Huh7 and Hep3B cells following treatment with the empty vector, miR-26b inhibitor and miR-26b mimic are shown. (D) Luciferase activity assay of indicated cells transfected with the pGL3-USP9X-3′UTR reporter with increasing amounts (10, 50 ,100 nM) of miR-26b mimic- or miR-26b inhibitor-oligonucleotides. (E) Luciferase assays on Huh7 or Hep3B HCC cells transfected with the pGL3 control reporter, pGL3-USP9X-3′UTR reporter, or pGL3-USP9X-3′UTR-mu reporters and increasing amounts of miR-26b mimic oligonucleotides (10, 50 nM), as indicated. Error bars represent mean 6 SD from three independent experiments. *P < 0.05.