Dataset Information

Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts.

ABSTRACT:

Background

Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-?), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-?, we hypothesized that TGF-? influences miRNA expression in lung fibroblasts, thereby affecting their function.

Materials and methods

We investigated TGF-?1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-?1-induced miRNA expression changes were validated and replicated in an independent set of lung fibroblasts composted of 10 controls and 15 COPD patients using qRT-PCR. Ago2-immunoprecipitation followed by mRNA expression profiling was used to identify the miRNA-targetomes of unstimulated and TGF-?1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-?1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-?1-stimulated fibroblasts.

Results

Twenty-nine miRNAs were significantly differentially expressed after TGF-?1 stimulation (FDR<0.05). The TGF-?1-induced miR-455-3p and miR-21-3p expression changes were validated and replicated, with in addition, lower miR-455-3p levels in COPD (p<0.05). We identified 964 and 945 genes in the miRNA-targetomes of unstimulated and TGF-?1-stimulated lung fibroblasts, respectively. The TGF-? and Wnt pathways were significantly enriched among the Ago2-IP enriched and predicted targets of miR-455-3p and miR-21-3p. The miR-455-3p target genes HN1, NGF, STRADB, DLD and ANO3 and the miR-21-3p target genes HHEX, CHORDC1 and ZBTB49 were consistently more enriched after TGF-?1 stimulation.

Conclusion

Two miRNAs, miR-455-3p and miR-21-3p, were induced by TGF-?1 in lung fibroblasts. The significant Ago2-IP enrichment of targets of these miRNAs related to the TGF-? and/or Wnt pathways (NGF, DLD, HHEX) in TGF-?1-stimulated fibroblasts suggest a role for these miRNAs in lung diseases by affecting lung fibroblast function.

SUBMITTER: Ong J

PROVIDER: S-EPMC5599028 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts.

Ong Jennie J Timens Wim W Rajendran Vijay V Algra Arjan A Spira Avrum A Lenburg Marc E ME Campbell Joshua D JD van den Berge Maarten M Postma Dirkje S DS van den Berg Anke A Kluiver Joost J Brandsma Corry-Anke CA

PloS one 20170914 9

<h4>Background</h4>Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function.<h4>Materials and methods</h4>We investigated TGF- ...[more]

PMID: 28910321

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Project description:Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 in vitro and in vivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

Dataset Information

Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts.

Background

Materials and methods

Results

Conclusion

Publications

Identification of transforming growth factor-beta-regulated microRNAs and the microRNA-targetomes in primary lung fibroblasts.

Similar Datasets

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