Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

International audienceBACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury

RNA-mediated paternal heredity of diet-induced obesity and metabolic disorders

International audienceThe paternal heredity of obesity and diabetes induced by a high-fat and/or high-sugar diet (Western-like diet) has been demonstrated through epidemiological analysis of human cohorts and experimental analysis, but the nature of the hereditary vector inducing this newly acquired phenotype is not yet well defined. Here, we show that microinjection of either testis or sperm RNA of male mice fed a Western-like diet into naive one-cell embryos leads to the establishment of the Western-like diet-induced metabolic phenotype in the resulting progenies, whereas RNAs prepared from healthy controls did not. Among multiple sequence differences between the testis transcriptomes of the sick and healthy fathers, we noted that several microRNAs had increased expression, which was of interest because this class of noncoding RNA is known to be involved in epigenetic control of gene expression. When microinjected into naive one-cell embryos, one of these small RNA, i.e., the microRNA miR19b, induced metabolic alterations that are similar to the diet-induced phenotype. Furthermore, this pathological phenotype was inherited by the offspring after crosses with healthy partners. Our results indicate that acquired food-induced trait inheritance might be enacted by RNA signalling

Culture of rabbit caecum organoids by reconstituting the intestinal stem cell niche in vitro with pharmacological inhibitors or L-WRN conditioned medium

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM used at low concentration represent a suitable model to study differentiated rabbit epithelium

Gut microbiota derived metabolites contribute to intestinal barrier maturation at the suckling-to-weaning transition

In suckling mammals, the onset of solid food ingestion is coincident with the maturation of the gut barrier. This ontogenic process is driven by the colonization of the intestine by the microbiota. However, the mechanisms underlying the microbial regulation of the intestinal development in early life are not fully understood. Here, we studied the co-maturation of the microbiota (composition and metabolic activity) and of the gut barrier at the suckling-to-weaning transition by using a combination of experiments in vivo (suckling rabbit model), ex vivo (Ussing chambers) and in vitro (epithelial cell lines and organoids). The microbiota composition, its metabolic activity, para-cellular epithelial permeability and the gene expression of key components of the gut barrier shifted sharply at the onset of solid food ingestion in vivo, despite milk was still predominant in the diet at that time. We found that cecal content sterile supernatant (i.e. containing a mixture of metabolites) obtained after the onset of solid food ingestion accelerated the formation of the epithelial barrier in Caco-2 cells in vitro and our results suggested that these effects were driven by the bacterial metabolite butyrate. Moreover, the treatment of organoids with cecal content sterile supernatant partially replicated in vitro the effects of solid food ingestion on the epithelial barrier in vivo. Altogether, our results show that the metabolites produced by the microbiota at the onset of solid food ingestion contribute to the maturation of the gut barrier at the suckling-to-weaning transition. Targeting the gut microbiota metabolic activity during this key developmental window might therefore be a promising strategy to promote intestinal homeostasis

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

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