Fig. 2
Identification of Gpr158 as a direct target of miR-449a. a Venn diagram with eight candidate genes emanating from 101 in silico putative targets and 1000 down-regulated genes in experimental PNETs compared with gliomas by analysis of exon expression array. b Candidate gene expression level is validated by RT-qPCR in Rb/p53 (orange error bars), Rb/p53ant (red bars) and Pten/p53 cells (grey bars). Most differentially expressed Ccnd1 and Gpr158 are further analysed, as their expression is similar in Rb/p53ant and Pten/p53 cells, but significantly higher than in Rb/p53 cells. c IHC staining shows that Gpr158 expression is minimal in miR-449 highly expressing PNETs, but strong in miR-449 low expressing gliomas. Scale bar 50 µm. d Schematic illustration of Ago2 and biotin double pull-down assay for assessment of miRNA-mRNA binding. Commercial synthetic miR-449a mimics are transfected into neural stem cells, and Ago2 immunoprecipitation is carried out to confirm that miRNA-mRNA binding is RISC dependent. Fraction 1 represents the input RNA, fraction 2 the Ago2 depleted fraction, i.e, miRNA and mRNA unbound to Ago2. Fraction 3 represents miRNA449a-mRNA complex bound to Ago2, representing the degradation complex RISC. These fractions were then tested for the enrichment of Gpr158 and Ccnd1 transcripts: e Enrichment of Ccnd1 and Gpr158 is measured after pull-down using RT-qPCR. The x axis shows the fraction as described in (d). There is a highly significant enrichment in fraction 3 (Ago2-dependent miR-449a –Gpr158 complex) indicating direct interaction. f miR-449a binding sequence in the 3′ UTR of Gpr158. A mutation of the 3’UTR of Gpr158 generated in the site complementary to the seed region of miR-449a. *Indicates the mutant nucleotides. g miR-449a directly targets Gpr158 by interacting with its 3′ UTR. Relative luciferase activity (normalized to control) of BTSCs transfected with pMIR-Gpr158-3′ UTR-wt or pMIR-Gpr158-3′ UTR-mut, and co-transfected with miRNA negative control or miR-449a mimics. This suggests a significant miR-449a mediated downregulation of Gpr158, which is not seen in the mutant control. All figures: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 (Student’s t-test). Each bar represents mean ± sd
