Figure 5

HAb18G is a potential target of miR-146a. A. Multiple species sequence alignment of the HAb18G/CD147 3′UTR including the putative miR-146a target site sequence (upper). The pGL3- HAb18G reporter gene has the full length of HAb18G 3’-UTR cloned into pGL3 vector. The pGL3- HAb18G -MUT vector has the four miR-146a binding sites mutated and confirmed by sequencing. SMMC-7721 were transfected with pGL3- HAb18G or pGL3-HAb18G -MUT, respectively, together with miR-146a mimics or mimic negative control. The four predicted binding sites of miR-146a were shown in the HAb18G 3′-UTR region. B. The relative luciferase activities were shown from three independent experiments. Error bars represent ± SD. C. Western blotting analysis of CD147 in SMMC-7721 cells transfected with miR-146a or control miRNA (miR-Ctrl) (left panel). Huh-7 and HepG2 cells were transfected with antago miR-146a or negative control (NC) (middle and right panel). D. Western blotting analysis of CD147 in SMMC-7721-miR-146a cells transfected with CD147 without 3′-UTR region (CD147 Re). E. In vitro invasion assay of SMMC-7721 cells transfected with CD147 siRNA for 24 h. The invading cell numbers on each filter were counted and data were plotted in fold change by defining the number from control cells a 100%. Error bars represent ± SD. F. Western blotting analysis of APC, NF-κB p65 and VEGF in SMMC-7721 cells transfected with HAb18G siRNA or scramble control. G-H. NF-κB p65 staining of SMMC-7721 cells transfected with miR-146a, CD147 siRNA or miR-Ctrl (G), or Huh-7 cells transfected with antagomiR-146a and NC (H). Cell nuclei were stained with DAPI. Scale bars represent 50 μm.