PMC

(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.

(A) Wild type p53-inducible H1299 cells were treated with 2.5μg/ml of Ponasterone A (PonA) for 24 hours, and compared with non-induced cells. WE-68 cells were treated with 10nM Nutlin-3a for 16 hours, compared with non-induced cells. Western blots for p53 (and β-actin as a loading control) are shown. (B) Based on microarray profiling, a dendrogram generated by cluster analysis shows the separation of p53 uninduced cells from induced cells, and separation of representative genes activated, repressed or not significantly changed by p53. (C) SNHG1 expression levels were determined by RT-PCR after p53 induction in H1299 and WE-68 cells. (D) ChIP assay, using the anti-p53 antibody DO-1, was performed to determine relative p53 occupancy in p53-induced H1299 cells (+PonA). RT-PCR results show relative p53 occupation at upstream of the SNHG1 promoter, and p53 null H1299 (-PonA) was used as a negative control. ** p<0.01 versus controls for all experiments.

(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.

(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.

(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.