Fig. 2

Identification of PLAGL2 as the miR-506-3p target that regulates MYCN expression. a Venn diagram showing the strategy to identify TFs that are predicted to be direct targets of miR-506-3p and are also predicted to regulate MYCN transcription. The numbers of genes identified are shown in parentheses. Tier 1 identifies 225 genes that were down-regulated by ≥40% by miR-506-3p mimic. Tier 2 identifies 11 TFs from the 225 genes. Tier 3 identifies 2 TFs that are predicted to be direct targets of miR-506-3p. b Effect of miR-506-3p overexpression on the mRNA expression of the two TFs in BE(2)-C and Kelly cells. Cells were transfected with 25 nM miR-506-3p mimic or control oligo for 2 days. PLAGL2 and CREB3L2 mRNAs were detected by qPCR. c Effect of knocking down the expression of the two TFs on MYCN expression. Cells were transfected with the indicated siRNAs or control oligo (siControl) at 25 nM for 2 days. MYCN mRNA and N-Myc were detected by qPCR and Western blots, respectively. d Effect of additional siPLAGL2s on N-Myc expression. Cells were transfected with the indicated oligos (25 nM) and protein levels were measured as above. e-f Effect of PLAGL2 knockdown on MYCN mRNA expression in three MYCN non-amplified neuroblastoma cell lines. Cells were transfected with siPLAGL2 or siControl (25 nM) for 2 days, and MYCN mRNA levels were measured as above. e, mRNA expression as measured by qPCR. f, The depletion of PLAGL2 protein expression by the siPLAGL2 was confirmed by Western blots. g Effect of PLAGL2 overexpression on MYCN mRNA expression in SKNSH cells. Cells were transfected with the indicated vectors (PLAGL2 or Control, 0.8 ng/μl) and oligos (miR-506-3p or control oligo, 2.5 nM) for 2 days, and MYCN mRNA and PLAGL2 protein levels were measured as above. h-i Luciferase reporter assay for validating the target site of miR-506-3p in the 3’UTR of PLAGL2. h, Schematic graph of the cloned wildtype (PLAGL2-WT) and mutant (PLAGL2-MU) 3’UTR. The mutated nucleotides are shown in red. i, Validation of the target site by luciferase assay. BE(2)-C cells were co-transfected with the indicated vectors (0.8 ng/μl) and oligos (miR-506-3p mimic or control oligo, 2.5 nM). Two days after transfection, cells were lysed and luciferase activity was measured. *, p < 0.05. Values shown above the bands in the Western blot images are the calculated relative intensities of the corresponding bands