miR-200a directly targets the human p53 gene.

<p>A. Schematic representation of miR-200a: p53 3′UTR. Top: seed sequence base paring between miR-200a and the 3′ UTR of p53 mRNA. Bottom: p53 constructs with the wild type miR-200a binding site (WT) or a mutated miR-200 binding site (Mut) in the 3′ UTR. B. A reporter assay to determine whether the p53 3′ UTR is targeted by miR-200a. Y axis denotes relative luminescent units (luc/Rluc) in H1299 cells expressing WT or Mut p53 3′ UTR constructs and miR-200a. C. Western blotting analyses of H1299 cell extracts. H1299 cells were transfected with miR-200a and WT or Mut p53 3′ UTR constructs. D. Apoptosis assay of H1299 cells transfected as in C. E. Cell cycle analysis of H1299 cells transfected as in C. The Y axis denotes events (the number of cells) and the X axis denotes the emitted fluorescent light of the DNA dye (PI), that is, DNA content. The values like “60.1±1.0” indicate the percentages of cells in the G1 phase with standard error of the mean. *<i>P</i>≤0.05 with n = 3.</p

Boxplots showing logarithmic values of NF-κB-induced luciferase expression for microRNAs grouped according to cluster.

<p>Annotated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048474#pone-0048474-g002" target="_blank">Figure 2</a>.</p

Boxplots showing logarithmic values of p53-induced luciferase expression for microRNAs grouped according to cluster.

<p>Annotated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048474#pone-0048474-g002" target="_blank">Figure 2</a>.</p

A Systematic Screen Reveals MicroRNA Clusters That Significantly Regulate Four Major Signaling Pathways

<div><p>MicroRNAs (miRNAs) are encoded in the genome as individual miRNA genes or as gene clusters transcribed as polycistronic units. About 50% of all miRNAs are estimated to be co-expressed with neighboring miRNAs. Recent studies have begun to illuminate the importance of the clustering of miRNAs from an evolutionary, as well as a functional standpoint. Many miRNA clusters coordinately regulate multiple members of cellular signaling pathways or protein interaction networks. This cooperative method of targeting could produce effects on an overall process that are much more dramatic than the smaller effects often associated with regulation by an individual miRNA. In this study, we screened 366 human miRNA minigenes to determine their effects on the major signaling pathways culminating in AP-1, NF-κB, c-Myc, or p53 transcriptional activity. By stratifying these data into miRNA clusters, this systematic screen provides experimental evidence for the combined effects of clustered miRNAs on these signaling pathways. We also verify p53 as a direct target of miR-200a. This study is the first to provide a panoramic view of miRNA clusters' effects on cellular pathways.</p> </div

Schematic of luciferase-based microRNA screen.

<p>293T cells were co-transfected with: 1) a vector containing a luciferase gene under control of regulatory elements recognized by AP-1, NF-κB, or p53 (in c-Myc screen, the E2F2-luc construct was used), 2) a member of our microRNA library, and 3) a Renilla luciferase vector for normalization of luciferase values. Following transfection, cells were analyzed by luciferase assay to measure the effects of miRNA regulation of TF-driven luciferase expression. TREs: Transcription response elements, mCMV: minimal CMV promoter, TF: Transcription factor, luc: luciferase, Rluc: Renilla luciferase, UTR: Untranslated region.</p

Boxplots showing logarithmic values of c-Myc-induced luciferase expression for microRNAs grouped according to cluster.

<p>Annotated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048474#pone-0048474-g002" target="_blank">Figure 2</a>.</p

Top microRNA clusters that significantly modulate reporter expression.

*<p>miR-650 was included even as it is not in a cluster.</p

Boxplots showing logarithmic values of luciferase expression for microRNAs grouped according to cluster.

<p>Clusters that yielded values significantly different from the overall mean are marked with a pink diamond and annotated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048474#pone-0048474-t001" target="_blank">Table 1</a>. MicroRNA clusters that caused significant up-regulation of AP-1-driven luciferase gene expression are highlighted in red. MicroRNA clusters that down-regulated this expression are marked in green.</p