Abstract
BackgroundLung 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.Materials and methodsWe investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays. TGF-β1-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-β1-stimulated primary lung fibroblasts (n = 2). The genes affected by TGF-β1-modulated miRNAs were identified by comparing the miRNA targetomes of unstimulated and TGF-β1-stimulated fibroblasts.ResultsTwenty-nine miRNAs were significantly differentially expressed after TGF-β1 stimulation (FDR<0.05). The TGF-β1-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-β1-stimulated lung fibroblasts, respectively. The TGF-β 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-β1 stimulation.ConclusionTwo miRNAs, miR-455-3p and miR-21-3p, were induced by TGF-β1 in lung fibroblasts. The significant Ago2-IP enrichment of targets of these miRNAs related to the TGF-β and/or Wnt pathways (NGF, DLD, HHEX) in TGF-β1-stimulated fibroblasts suggest a role for these miRNAs in lung diseases by affecting lung fibroblast function.
Highlights
Lung fibroblasts play a key role in extracellular matrix (ECM) homeostasis and maintenance of the normal lung architecture and are crucial players in lung damage and repair
Of the top 1,500 most IP-enriched probes in the TGF-β1-stimulated lung fibroblasts of the two control subjects, we identified 1,083 probes overlapping between the control subjects, which corresponded to 945 unique genes, and these were identified as the miRNA-targetome of the TGF-β1-stimulated cells (Fig 5B)
We investigated the effect of TGF-β1 on miRNA expression in primary lung fibroblasts and used the miRNA-targetomes of these cells to identify transcripts that are likely to be affected by the TGF-β1-dependent modulation of miRNA expression levels
Summary
Lung fibroblasts play a key role in extracellular matrix (ECM) homeostasis and maintenance of the normal lung architecture and are crucial players in lung damage and repair. They have been postulated as important players in lung diseases with disturbed ECM homeostasis and aberrant repair such as chronic obstructive pulmonary disease (COPD). Transforming growth factor beta (TGF-β), an important cytokine in tissue repair and remodeling, stimulates ECM production by fibroblasts mainly through signaling via the downstream SMAD proteins [1,2,3]. Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are crucial in lung tissue repair and remodeling. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function
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