Salivary cortisol concentrations in the environmentally enriched and control groups.

<p>Salivary samples were collected 0, 6, and 12 weeks after the beginning of the treatment, in the morning (1000 h) or in the afternoon (1530 h). ***intergroup comparison, Mann–Whitney test, P≤0.001; ⁺⁺intragroup comparison, Wilcoxon tests, P≤0.01.</p

Illustration of the top gene network upregulated in the environmentally enriched (A) and control (B) groups.

<p>These data were generated by the Ingenuity Pathway Analysis (IPA) software using the 400 genes differentially expressed at <i>P</i><0.005. The genes marked with a red symbol are from the list of differentially expressed genes, whereas the genes marked with a white symbol are intermediary genes of the network added by the software.</p

TELiS and oPOSSUM analyses of the genes differentially expressed between the environmentally enriched (EE) and the control groups.

<p>In TELiS analyses, the ratio of transcription factor-binding motif (TFBM) representation (control to EE horses) is shown. In the oPOSSUM analysis, significant over-represented transcription factors in either control or EE animals is shown using the resulting z-score. Differences in transcription factors with a z-score >10 and a Fisher score <0.01 are highly significant.</p

Schematic representation of the protocol.

<p>Each horse (10 months old) was subjected to either the environmental enrichment (EE; <i>n</i> = 10) or control treatment (<i>n</i> = 9) for 12 consecutive weeks.</p

“Seed-Milarity” Confers to hsa-miR-210 and hsa-miR-147b Similar Functional Activity

<div><p>Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5′-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (<em>i.e.</em> lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a “minimal” 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.</p> </div

Biological consequences of miR-210, miR-147a and miR-147b overexpression on A549 cells proliferation and viability.

<p>A549 cells were transfected with 10 nM pre-miR-210, pre-miR-147a, pre-miR-147b or pre-miR-Neg and analyzed for several proliferation (A-E) and viability (F-G) parameters. A) Confluent cell monolayer was wound and filmed for 55h under light time laps microscope. Curves represent wound beds closure quantified by measuring the wound bed surface at the indicated times after injury using the Image J software. Values are expressed in percentage of the initial surface and correspond to the mean ± SD of 3 microscope fields. B) Effect of miR-210 and miR-147 family on A549 cell proliferation. Exponentially growing A549 cells were transfected and counted each day during 4 days with blue Trypan. Data show mean ± SD values of trypan blue negative (left panel) and trypan-blue positive cell number (right panel) from 2 independent experiments performed in triplicate. C) Cells were stained with propidium iodide and analyzed by flow cytometry. The G0/G1 (1), S (2) and G2/M (3) fractions were quantified in each condition. D) Quantification of each of these 3 fractions (G0/G1, S and G2/M) from 3 independent experiments. E) Expression of Cyclin D, Cyclin A, Cyclin E, CDK4, CDK6, pRB (6 molecules involved in G1 phase progression), p27Kip1 (inhibitor of G1 phase progression) and Cyclin B (involved in G2/M phase) were assessed by Western blot. Hsp60 corresponds to the loading control. F) Caspase 3/7 assay was performed at 3, 4 and 5 days after transfection. Data are mean ± SD values of 3 independent experiments performed in triplicate. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919.s004" target="_blank">Figure S4A</a>. G) Expression of active caspase-3 (cleaved) and PARP, a substrate of caspase-3 was analyzed by Western blot. HSP60 corresponds to the loading control. Normalized densitometric quantification are shown for each lane. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919.s004" target="_blank">Figure S4C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919.s004" target="_blank">Figure S4D</a> (*<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005).</p

Table summarizing the data of microarrays and luciferase assays concerning the subset of 15 transcripts.

*<p>corresponds to previously validated targets from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919-Puissegur1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919-Giannakakis1" target="_blank">[50]</a>.</p

Enrichment of miR-210 and miR-147 family members-predicted targets in the different subsets of modulated transcripts following transfection by each miRNA candidate. A

<p>) Graphs adapted from our webtool miRontop <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919-LeBrigand1" target="_blank">[33]</a> showing the significance of the enrichment (represented as –log10 (adjPVal) according to the fold enrichment in experiments of overexpression of miR-210, miR-147a and miR-147b. Enrichment was calculated according to TargetScan, microCosm or a 2–8 seed search in 3′UTR. On each panel, miR-210, miR-147a and miR-147b are highlighted as blue, green and red dots, respectively. <b>B</b>) Venn diagram summarizing the predicted common targets of miR-210 and miR-147 family members using the combination of transcriptomic data and different bioinformatics prediction tools: TargetScan and a search of a 6 nt complementary seed 2–7 sequences in 3′UTR. Cut offs used for the selection of down-regulated transcripts: log2Average>8; logFC<-0.5 and Adj.pVal<0.05.</p

Targets validation using luciferase assays.

<p>A) Direct targeting of the 15 candidate transcripts by miR-210, miR-147a and miR-47b was analyzed <i>in vitro</i>. A549 cells were co-transfected with pre-miR-210, pre-miR-147a, pre-miR-147b or pre-miR-Neg and different pSI-Check-2™ constructs containing the 3′UTR of interest described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919.s011" target="_blank">Table S5</a>. Cells were harvested two days after transfection and luciferase activities were analyzed. B) Venn diagram summarizing the predicted (using the microarray approach) and validated (after luciferase assay) common targets of miR-210 and miR-147 family members. (*<i>p</i><0.05, **<i>p</i><0.005, ***<i>p</i><0.0005).</p

<i>In silico</i> prediction of miR-210 and miR-147 family targets.

<p>A) Alignment of miR-210, miR-147a and miR-147b mature sequences. The seed region is indicated in grey. The 1nt-substitution in miR-147a seed is underlined. B) Venn diagram summarizing the predictive common targets of miR-210 and miR-147 family members using different bioinformatics prediction tools: TargetScan, microCosm and a search of a 7 nt complementary seed 2–8 sequences in 3′UTR using our laboratory-made tool “MicroCible” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044919#pone.0044919-Pottier1" target="_blank">[25]</a> (<a href="http://www.microarray.fr:8080/merge/index" target="_blank">http://www.microarray.fr:8080/merge/index</a> follow the link to microRNA and Bioinformatic tools).</p