sno-miR-28 is over-expressed in breast tumours.

<p>(A) The feed-forward loop between p53, sno-miR-28, and TAF9B is shown as proposed. (B) sno-miR-28, SNORD28, and SNORD25 expression levels were determined using TaqMan assay in breast tumours compared with paired normal adjacent tissues, while SNHG1 expression levels were determined by RT-PCR. In this part of figure, RNA expression levels are shown as the ratio relative to normal tissue expression; e.g., 1 represents equal expression to normal tissues. (C) MCF10A cell proliferation is shown after overexpression of sno-miR-28 compared with negative control RNA (ncRNA). (D) Colony formation assay in MCF10A cells after expression of sno-miR-28 or a negative control RNA (ncRNA). Representative images are included on the left, and relative quantitation of the graph is on the right. ** p<0.01 and * p≤0.1 versus controls for all experiments.</p

sno-miR-28 alters p53 protein stability through TAF9B and forms a feed-forward loop between p53, sno-miR-28, and TAF9B.

<p>(A) Western blots are shown to determine the protein levels of p53 and p21 after overexpression of sno-miR-28 and/or induction of p53 by Ponasterone A (PonA) in inducible H1299 cell line, compared with cells transfected with negative control RNA (ncRNA) and/or uninduced H1299 cells. (B) p53 mRNA levels in MCF10A and PonA-treated H1299 cells after overexpression of sno-miR-28 are shown by RT-PCR, compared with scrambled negative control (ncRNA). (C) The mRNA expression levels of p53 downstream targets are shown after overexpression of sno-miR-28 (or negative control) in H1299 cells upon p53 activation. (D) sno-miR-28 (or negative control) was either expressed (mimic) or inhibited (LNA) in MCF10A cells and the protein expression of p53 was determined by Western blot. (E) The mRNA expression levels of p53 downstream targets are shown after overexpression of sno-miR-28 in MCF10A cell line, compared with ncRNA. (F,G) TAF9B was knocked using an siRNA in H1299 cells compared with a negative control. Successful knockdown is shown at both the protein (F) and mRNA (G) levels. (H) CDKN1A expression was determined by RT-PCR after TAF9B was knockdown. ** p<0.01 versus controls for all experiments. β-actin is included as a loading control for all Western blots. “-”mark in sno-miR-28 mimic and anti-sno-miR-28 LNA experiments represent negative control transfections using a scrambled ncRNA.</p

snoRNAs repressed by p53 in H1299 and WE-68 cells.

<p>Affymetrix gene expression profiling identified six snoRNAs that were repressed in common in H1299 and WE-68 cell lines as wild-type p53 was induced. Statistical significance of these snoRNAs is characterized by p-values that compare p53 induced (H1299 treated with PonA and WE-68 treated with Nutlin) versus uninduced cells, with their host genes listed.</p><p>snoRNAs repressed by p53 in H1299 and WE-68 cells.</p

sno-miR-28 functions as a miRNA.

<p>(A) Proposed sno-miR-28 binding site within the TAF9B 3’UTR. The seed-recognition site is marked in bold; hypothesized duplexes formed by the interaction of TAF9B and sno-miR-28 are illustrated, and the predicted free energy of the hybrid is indicated. Conservation of the seed region across 4 species is also indicated. (B, C) sno-miR-28 (or negative control RNA, ncRNA) was overexpressed in H1299 cells. TAF9B mRNA and protein levels were determined by RT-PCR and Western blot, respectively. (D) Using a dual-luciferase reporter system, H1299 cells were co-transfected with sno-miR-28 mimics (or negative control RNA), and psiCHECK2 luciferase reporter plasmids with either wild type (WT) or mutated TAF9B 3’-UTR (MUT) cloned at downstream of the Renilla luciferase gene (Luc). Relative luciferase activities are shown. (E, F) sno-miR-28 was either overexpressed (mimics) or inhibited (LNA) in MCF10A cells. TAF9B mRNA levels were determined by RT-PCR (E), and protein expression was determined by Western blot (F). ** p<0.01 versus controls for all experiments, and β-actin was included as a loading control for all Western blots.</p

p53 repressed snoRNAs are processed into miRNAs.

<p>(A) SNHG1 is processed into snoRNAs including SNORD25 and SNORD28. The top panel shows RNA deep-sequencing and HITS-CLIP (high throughput sequencing of crosslinked and immunoprecipitated RNA) results at the SNHG1 genomic loci. Predicted stem-loop folding of SNORD25 and SNORD28 are shown. The regions marked in bold are processed into sno-miRNAs which can bind to Argonaute proteins, which was confirmed by RNA deep-sequencing and HITS-CLIP results. The solid lines between chains represent hydrogen bonds between adenine (A)-uracil (U) pairs and guanine (G)-cytosine (C) pairs, whereas dashed lines represent G-U pairing. (B) RNA-seq and HITS-CLIP mapping reads across the SNORD28 region is shown indicating precise binding of sno-miR-28 to AGO (C) p53 was induced by PonA treatment in inducible H1299 cells, and the expression levels of SNORD25, SNORD28 and sno-miR-28 were determined using TaqMan assay and RT-PCR. Expression levels of SNORD25, SNORD28 and sno-miR-28 were shown in induced or uninduced cells. (D) Isogenic HCT116 -/-p53 and HCT116 +/+p53 cell lines were used to investigate the relation of SNHG1 and sno-miR-28 expression levels with p53. Left: p53 protein expression in the HCT116 isogenic cell lines was shown by Western blot and β-actin was used as a loading control. Right: SNHG1 and sno-miR-28 expression was determined by RT-PCR as shown. ** p<0.01 versus controls for all experiments.</p

Gene expression analysis of <i>miR-200b eRNA</i>, miR-200b~200a~429 and EMT-affiliated genes.

<p>(A) Real-time PCR of <i>miR-200b </i><i>eRNA</i>, EMT markers and miR-200 genes in a panel of breast epithelial and mesenchymal cell lines. mRNA (top panel) is normalized to GAPDH while miRNA (bottom panel) is normalized to U6 snRNA. Comparative quantitation was used to determine expression profiles of the genes in the cell line panel. Expression of E-cadherin and <i>miR-200b </i><i>eRNA</i> in HMLE cells is set to a value of 1, whereas Zeb-1 is expressed relative to mesHMLE cells having a value of 1. Error bars represent mean ± SD of three independent experiments. (B) Relative expression levels of <i>miR-200b </i><i>eRNA</i> during EMT time course of HMLE cells treated with TGF-β1 for up to 18 days. The realtime PCR data is normalized to GAPDH. Expression of <i>miR-200b </i><i>eRNA</i> is set to a value of 1 in HMLE cells (C) Relative concentration of <i>miR-200b </i><i>eRNA</i>, U6, HOTAIR, GAPDH and βActin RNA transcripts in the nucleus and cytoplasm of HMLE cells. Absolute quantitation was used to determine the expression level of each gene in the cytoplasmic and nuclear fractions. Error bars represent mean ± SD of three independent experiments.</p

Over-expression of <i>miR-200b eRNA</i> has no effect on miR-200b~200a~429 promoter activity in epithelial and mesenchymal HMLE cells.

<p>Schematic representation of the miR-200b~200a~429 luciferase reporter constructs, PRO (-321/+19), LOCUS (-5771/+19), PRO&ENH (-321/+19 to -5711/4607) and ENH (-5711/-4607). The miR-200b~200a~429 reporters, pcDNA3.1, pcDNA3.1 Zeb1, pcDNA3.1 Zeb2 or the pcDNA3.1 <i>miR-200b </i><i>eRNA</i> plasmids, and the Renilla vector were co-transfected into epithelial HMLE (white bars) or mesenchymal HMLE (black bars) cells. Transiently transfected cells were incubated for approximately 24-48 hours. Data are normalized by Renilla luciferase activity and represent means ± SD of at least three independent experiments.</p

Identification of an upstream enhancer region that increases the transcription of the miR-200b~200a~429 promoter in epithelial breast cancer cells.

<p>(A) A series of 5’ deletions of the human miR-200b~200a~429 locus comprising the promoter and potential enhancer were cloned into a firefly luciferase reporter plasmid. (B) The reporter plasmids were transiently transfected along with the Renilla pTK vector into epithelial HMLE cells (white bars) or mesenchymal HMLE cells (black bars). Luciferase activity was assayed approximately 48 hours later using the Dual-Luciferase Reporter Assay System (Promega). Data are expressed as normalized luciferase activity and represent means ± SD of at least four independent experiments. (C) The enhancer region (-5771/-4607 ENH) was cloned in both directions immediately upstream of the minimal miR-200b~200a~429 promoter (-321/+19 PRO) or the Luciferase coding region creating PRO&ENH and ENH as well as PRO&ENH (-) and ENH (-) with ENH oriented in the sense and antisense orientation, respectively. Luciferase activity was assay as described in (B).</p

An active chromatin domain is located upstream of the miR-200b~200a~429 locus.

<p>Normalized ChIP-seq signal profiles were generated for H3K4me1, H3K4me3, H3K9/14ac, H3K27ac and H3K27me3 at the miR-200b~200a~429 locus (chr1:1,090,000-1,105,000) in (A) epithelial HMLE cells and (B) mesenchymal HMLE that have undergone EMT. The x-axis shows the distance in kilobases (kb) relative to the TSS (designated 0), the chromosomal coordinate marking the TSS is indicated and a schematic diagram of the primary miR-200b~200a~429 transcript is positioned boxes indicate the mature miRNA hairpin transcripts. The y-axis shows the sequencing coverage per million reads for each histone modification normalized to the Input control sample. (C) CpG methylation analysis of the miR-200b~200a~429 locus in epithelial HMLE cells (left panel) and in mesenchymal HMLE cells following 46 days of TGF-β1 (right panel) treatment using Illumina HM450K methylation array [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075517#B36" target="_blank">36</a>]. An arrow marks the TSS. The x-axis indicates the distance in kb from the TSS designated 0. The y-axis shows the % CpG methylation occurring at each genomic region.</p

<i>miR-200b eRNA</i> is transcribed from the upstream intergenic enhancer region of miR-200b~200a~429.

<p>(A) Schematic representation of the miR-200b~200a~429 locus. Black box indicates the position of the potential enhancer. A black arrow marks the TSS direction of the primary miR-200b~200a~429 transcript. Grey boxes indicated the mature miR-200b, miR-200a and miR-429 genes. Black bars indicate the positions (in kilobases, kb) of the PCR primers used for qRT-PCR. (B) Expression levels of HOTAIR, <i>miR-200b </i><i>eRNA</i> and the primary miR-200b~200a~429 transcript as determined by qRT-PCR in epithelial and mesenchymal HMLE cells using random hexamer primed cDNA synthesized from total RNA. The x-axis shows the distance from the miR-200b~200a~429 TSS in kb. Data represents mean ± SD of three independent experiments. (C) Schematic representation of the enhancer region located relative to the miR-200b~200a~429 TSS. Boxes indicate the locations of PCR amplicons used to detect the <i>miR-200b </i><i>eRNA</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075517#pone-0075517-g003" target="_blank">Figure 3B</a>. RACE PCR primers and their start locations relative to the miR-200b~200a~429 are indicated. 5’ and 3’ RACE-seq analysis of the <i>miR-200b </i><i>eRNA</i> with cDNA prepared from total RNA of HMLE, mesHMLE, MDA-MB-468 and MDA-MB-231 cells as described in the Materials and Methods. 5’ and 3’ ends of the <i>miR-200b </i><i>eRNA</i> transcript are mapped as % total reads for each cell line with extreme 5’ and 3’ ends indicated by colored arrows below. A consensus <i>miR-200b </i><i>eRNA</i> transcript is indicated.</p