Abstract
BackgroundExcessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis.ResultsmiR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model.ConclusionsmiR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-016-0044-2) contains supplementary material, which is available to authorized users.
Highlights
Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis
Results miR-9-5p is induced by TGF-β1 and reduces type II transforming growth factor-β (TGF-β) receptor (TGFBR2) expression in human dermal fibroblasts To study the possible implication of miR-9-5p in skin fibrosis, HDFs were treated with 5 ng/ml TGF-β1 for the indicated times and its expression was analyzed by quantitative reverse transcription-polymerase chain reaction
To assess if miR-9-5p-related reduction in TGFBR2 expression was associated with a decrease in the TGF-βinduced pro-fibrogenic transformation of fibroblasts, HDFs were transfected with 40-nM precursor of miR-95p and incubated with 5 ng/ml TGF-β1 for different times
Summary
Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. Myofibroblasts are commonly identified by the expression of alpha-smooth muscle actin (α-SMA), and they are characterized by a pronounced rough endoplasmic reticulum, stress fibers, and a large nucleus. They mainly derive from resident fibroblasts, they can be recruited from skin-derived precursors and bone marrow fibrocytes. Epithelial and endothelial to mesenchymal transition (EMT and EndoMT, respectively), vascular smooth muscle cells, and pericytes may contribute to fibroblast accumulation [6]
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