Fig. 5. MiR-29 functions as a decoy for HuR.

(A) Model of miR-29 as a decoy against an RNA-destabilizing factor that contributes to A20 mRNA turnover. (B) Sequences of precursor and mature miR-29. Predicted HuR binding sites are in blue. The seed sequence and HuR binding site are underlined in mature miR-29. (C) Western blot for HuR in human xenograft sarcoma tumors compared with control murine muscle tissue, human differentiated osteoblasts, or murine fibroblasts. (D) H&E staining and immunohistochemistry for A20 and HuR in formalin-fixed paraffin-embedded osteosarcoma. Images are representative of four samples, stained in duplicate; scale bars, 4 μm. (E) Western blotting and qRT-PCR for A20 in lysates from Rh30 cells transfected with control or HuR siRNA. (F) Immunoprecipitation for HuR or IgG followed by qRT-PCR for miR-29 or miR-181 in Rh30 cell lysates. (G) RMSA with a ³²P-labeled miR-29 probe and extracts from Rh30 cells transfected with HuR (lanes 2 to 5), miR-29 (lane 1), HuR siRNA (lane 3), unlabeled miR-29 (lane 4, cold competitor), or miR-29 containing a mutated HuR binding site (lane 5, HuR mut). (H) Top: PCR fragments were amplified from the full-length (FL) A20 3′UTR or deletion constructs (Δ1 to Δ6) (table S1). Bottom: Western blotting for HuR in ultraviolet–cross-linked lysates from Rh30 cells transfected with or without miR-29 and the indicated fragment. Data are representative of two experiments. (I) A20 3′UTR luciferase reporter activity in Rh30 cells cotransfected as indicated. (J) Immunoprecipitation for HuR and qRT-PCR for miR-29 in lysates from U2OS cells transfected with plasmids expressing WT or mutated (mut 1 to mut 3, fig. S9A) precursor miR-29. (K) Western blotting for A20 in U2OS lysates transfected with WT or mutated m i R - 2 9. Blots are representative of at least two independent repeats, and data are means ± SEM from three independent experiments; *P < 0.05, **P < 0.01.