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. 2008 May 21;3(5):e2236. doi: 10.1371/journal.pone.0002236

Figure 1. MYCN transactivates the miRNA 17-5p-92 cluster in neuroblastoma by directly binding to its promoter.

Figure 1

(A) Western blot of MYCN in different neuroblastoma cell lines. A representative experiment is shown. (B) miRNA qRT-PCR analysis of miRNAs pertaining to the miRNA 17-5p-92 cluster in different neuroblastoma cell lines. The level of each miRNA is normalized to its expression in SK-N-AS cells (set as 1). Mean±s.d. (n = 3). (C) Schematic representation of the genomic region encompassing the miRNA 17-5p-92 cluster. Five putative MYCN binding sites (included in fragments 1, 2, 3, 4, 5) are indicated. (D) Left panel. Representative Western blot of MYCN in Tet-21/N cells untreated (0 h) or treated with doxycycline (Dox) for 2 or 24 h. ERp57 was used for normalization. Right panel. Chromatin immunoprecipitation with an anti-MYCN or a control anti-IgG antibodies on lysates from Tet-21/N cells untreated or treated with doxycycline for the indicated times. Control amplifications were carried out on either chromatin before immunoprecipitation (Input) or immunoprecipitated chromatin with oligonucleotides amplifying the β-actin gene (Actin). (E) Promoter assay with the pGL4Prom17M construct containing a 3731 bp fragment of the miRNA 17-5p-92 cluster promoter upstream the luciferase gene (indicated in C). SH-EP cells were transfected with pGL4 vector (Empty) or pGL4Prom17M (Prom17M) in combination with pcDNA3 (pcDNA) or piRV-neoSV-MYCN and luciferase activity was measured 72 h post-transfection. The bars represent the normalized luciferase activity (mean±s.d., n = 5). (F) miRNA qRT-PCR analysis of miRNAs pertaining to the miRNA 17-5p-92 cluster in Tet-21/N untreated (− Dox) or treated with doxycycline for 96 h (+ Dox). The level of each miRNA is reported as percentage of its expression in untreated cells (set as 100%). Mean±s.d. (n = 3). * P<0.05, ** P<0.01, *** P<0.001.