Figure 6. HuR binding to a U₈ element within HuRBS is sufficient to prevent the miR-1192-mediated inhibition of HMGB1 translation.

(a) Schematic representation of the HuRBS and the seed element of miR-1192 (miR-1192 BS). (b) Diagram illustrating the bioinformatic approach used to predict miR-1192 as a putative miRNA targeting HMGB1 3′UTR. Shown here are complementarities of miR-1192 with HMGB1 3′UTR. (c) Schematic diagrams of luciferase constructs. (d-g) Effects of HuR, miRNAs, antagomirs, and mimics. Error bars represent S.E.M of three independent experiments. ***P<0.0001, **P<0.001, *P<0.05 (Student’s t test). The amount of reporter RNA expressed in cells was determined by qPCR and used to normalize Renilla luciferase activities for each treatment. (d) Exponentially growing C2C12 cells were treated with siCtr or siHuR and 24 h later luciferase constructs were introduced. Luciferase activity of R Luc construct was considered as 100%. (e) miR-1192 and miR-16 antagomirs were transfected in siHuR-treated C2C12 cells previously transfected either with R luc alone or R luc-3′HMGB1. (f) Upper panel: Schematic representation of the Mimic miR-1192 and Mimic mutant miR-1192. The asterisk (*) indicates the A to C mutation in the Mimic mutant miR-1192. Lower panel: HeLa cells were transfected with Mimic miR-1192 or Mimic mutant miR-1192 and 24 h later luciferase constructs were transfected. The luciferase activity of the mock treated cells was set as reference. (g) Immunoprecipitation experiments were performed using a monoclonal HuR antibody, or IgG as a control, on total cell lysates from exponentially growing C2C12 cells. RNA was isolated from the immunoprecipitate, and quantitative RT-PCR was performed using primers specific to miR-1192, miR-16 and U6 RNAs. The levels of miR-1192 and U6 RNAs in each IP were normalized against the level of miR-16. Error bars represent S.E.M of three independent experiments. t test was used for statistical analysis. (h–i) Immunoprecipitation experiments were performed as in (g) on total cell lysates from C2C12 cells transfected with either R-luc alone, R-luc-3′HMGB1 or R-luc-3′HMGB1-mut-miRBS. (h) Western blot was performed using an HuR antibody. This blot is a representation of three independent experiments. (i) The levels of miR-1192 in each IP were determined and are plotted with the S.E.M. of three independent experiments. ***P<0.0001, **P<0.001 (Student’s t test).