Lung microRNA deregulation associated with impaired alveolarization in rats after intrauterine growth restriction

<div><p>Intrauterine growth restriction (IUGR) was recently described as an independent risk factor of bronchopulmonary dysplasia, the main respiratory sequelae of preterm birth. We previously showed impaired alveolarization in rat pups born with IUGR induced by a low-protein diet (LPD) during gestation. We conducted a genome-wide analysis of gene expression and found the involvement of several pathways such as cell adhesion. Here, we describe our unbiased microRNA (miRNA) profiling by microarray assay and validation by qPCR at postnatal days 10 and 21 (P10 and P21) in lungs of rat pups with LPD-induced lung-alveolarization disorder after IUGR. We identified 13 miRNAs with more than two-fold differential expression between control lungs and LPD-induced IUGR lungs. Validated and predicted target genes of these miRNAs were related to “tissue repair” at P10 and “cellular communication regulation” at P21. We predicted the deregulation of several genes associated with these pathways. Especially, E2F3, a transcription factor involved in cell cycle control, was expressed in developing alveoli, and its mRNA and protein levels were significantly increased at P21 after IUGR. Hence, IUGR affects the expression of selected miRNAs during lung alveolarization. These results provide a basis for deciphering the mechanistic contributions of IUGR to impaired alveolarization.</p></div

miRNA expression profiling in rat lungs at postnatal day 10 (P10), or during, and at the end (P21) of alveolarization after low-protein diet (LPD)-induced intrauterine growth restriction (IUGR).

<p>(A) Unsupervised hierarchical cluster analysis of miRNA expression in postnatal developing lungs after a control-diet gestation clearly distinguishes P21 from P10 samples. (B) Supervised hierarchical cluster analysis, heatmap representation (left panel) and volcano plot (right panel) of miRNAs differentially expressed at P10 after LPD-induced IUGR <i>vs</i>. control-diet gestation (<i>p</i> <0.05). (C) Supervised hierarchical cluster analysis, heatmap representation (left panel) and volcano plot (right panel) of miRNAs differentially expressed at P21 after LPD-induced IUGR <i>vs</i>. control-diet gestation (<i>p</i> <0.05). MiRNAs with significantly different expression between LPD-induced IUGR and control-diet gestation (fold-change ≥ 2; <i>p</i> < 0.05) are highlighted in red; miRNAs from total lungs of 5 animals/group were analyzed. We used qRT-PCR to validate this miRNA expression profile for the 13 differentially expressed miRNAs. We used the same rat pup samples than in the microarray experiments. The microarray and real-time PCR data were highly correlated, with r² = 0.669 (<i>p</i> = 0.0003), thus inferring high reliability of the microarray data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190445#pone.0190445.g002" target="_blank">Fig 2</a>). Again the correlation was greater for P10 than P21 data. At P10, the differential expression of the three miRNAs was statistically confirmed, with miR128-3p and miR34c-5p downregulated and miR434-3p upregulated. At P21, the differential expression of 5 of 10 of the miRNAs was statistically confirmed, with miR378a-5p, miR127-3p, and miR184 downregulated and miR30e-5p and miR449a-5p upregulated.</p

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

qRT-PCR analysis of validated mRNA targets of the miRNAs differentially expressed in lungs at P10 and P21 after LPD-induced IUGR.

<p>Five genes were selected from among the target genes according to their described function and quantified by qRT-PCR at P10 and P21. The relative expression was normalized to that of housekeeping genes (GAPDH and MMP16 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190445#pone.0190445.ref016" target="_blank">16</a>]). Data are mean ± SEM of 5 animals/group. *, <i>p</i> < 0.05; two-tailed Mann-Whitney test.</p

Gene ontology enrichment analysis of validated and predicted targets of miRNAs differentially expressed in rat lungs at P10 and P21 after LPD-induced IUGR.

<p>Comparison of the significantly deregulated genes at P10 and P21 with the validated and predicted targets of the deregulated miRNAs according to TarBase and Mirtarbase gave a new enriched list of 17 and 159 target genes at P10 and P21, respectively. A and B: The top significant pathways (<i>p</i> <0.01) linked to these genes at P10 and P21, respectively, were annotated according to Genomatix GePS. The vertical axis corresponds to enrichment score, which equals [-log10(<i>p</i> value)] and represents the significance level of pathways. C and D: Venn diagram showing the extent, at p10 and P21, respectively, of overlap between the predicted targets genes and genes identified in the literature as being implicated in these GO pathways.</p

qRT-PCR analysis of differentially expressed miRNAs in rat lungs at P10 and P21 after LPD-induced IUGR.

<p>We confirmed the differential induction or repression detected by the global approach in 3 of 3 miRNAs at P10 after LPD-induced IUGR <i>vs</i>. control-diet gestation and 5 of 10 miRNAs at P21. The relative expression was normalized to that of miR-10a-3p and miR-124-3p, the two miRNAs showing the least variability across the samples according to Normfinder. Values are mean ± SEM for 5 animals/group. *, <i>p</i> < 0.05; two-tailed Mann-Whitney test. Insert: linear correlation between the fold-change of miRNA expression measured by qRT-PCR or microarray hybridization at the two times; Pearson r² correlation value and <i>p</i> value are depicted.</p

Experimentally validated targets (source: Tarbase and Mirtarbase) for the 13 significantly modulated miRNAs, sorted by Fold change in expression, during alveolarization in rats with low-protein diet-induced intrauterine growth restriction.

<p>Experimentally validated targets (source: Tarbase and Mirtarbase) for the 13 significantly modulated miRNAs, sorted by Fold change in expression, during alveolarization in rats with low-protein diet-induced intrauterine growth restriction.</p

Table S2 from Association of <i>FOXD1</i> variants with adverse pregnancy outcomes in mice and humans

Primer sequence

Table S1 from Association of <i>FOXD1</i> variants with adverse pregnancy outcomes in mice and humans

Number of FKHD binding sites in the promoters of modified gene

Table S2 from Association of <i>FOXD1</i> variants with adverse pregnancy outcomes in mice and humans

Primer sequence