Figure 5

MicroRNA-185 targets DNMT1 mRNA at the 3'-UTR. (A) Schema of the firefly luciferase reporter constructs for the DNMT1, indicating the interaction sites between miR-185 and the 3'-UTRs of the DNMT1. (B) Luciferase activities. U251 cells were co-transfected with firefly luciferase constructs containing the DNMT1 wild-type or mutated 3-UTRs and miR-185 or scrambled oligonucleotides, respectively. The firefly luciferase activities were measured and normalized to β-galactosidase activity. Data are expressed as mean ± SD of relative levels of luciferase activity of each group of cells from three independent experiments. (C) Quantitative RT-PCR analysis of DNMT1 expression. Glioma cells were transfected with miR-185, si-DNMT1 or controls for 48 h. The levels of DMNT1 mRNA transcripts were determined by Real time PCR. Data are expressed as mean ± SD of each group of cells from three independent experiments. (D) Western blot analysis of DNMT1 protein. The levels of DNMT1 expression in the transfected cells described above were characterized by Western blot assays. Data shown are representative images of each group of cells from three separate experiments. (E) The levels of DNMT1 mRNA transcripts in normal brain tissue and primary gliomas. (F) The levels of miR-185 expression in normal brain tissue and primary gliomas. Data are expressed as mean ± SD of each group of samples from three separate experiments. Then indicates the sample size of each group. *p< 0.05 vs. control.