MicroRNAs (miRNAs) are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. The aim of this study was to explore how miR‐448‐5p affects airway remodeling and transforming growth factor‐β1 (TGF‐β1)‐stimulated epithelial‐mesenchymal transition (EMT) by targeting Sine oculis homeobox homolog 1 (Six1) in asthma. Asthmatic mice models with airway remodeling were induced with ovalbumin solution. MiRNA expression was evaluated using quantitative real‐time polymerase chain reaction. Transfection studies of bronchial epithelial cells were performed to determine the target genes. A luciferase reporter assay system was applied to identify whether Six1 is a target gene of miR‐448‐5p. In the current study, we found that miR‐448‐5p was dramatically decreased in lung tissues of asthmatic mice and TGF‐β1‐stimulated bronchial epithelial cells. In addition, the decreased level of miR‐448‐5p was closely associated with the increased expression of Six1. Overexpression of miR‐448‐5p decreased Six1 expression and, in turn, suppressed TGF‐β1‐mediated EMT and fibrosis. Next, we predicted that Six1 was a potential target gene of miR‐448‐5p and demonstrated that miR‐448‐5p could directly target Six1. An SiRNA targeting Six1 was sufficient to suppress TGF‐β1‐induced EMT and fibrosis in 16HBE cells. Furthermore, the overexpression of Six1 partially reversed the protective effect of miR‐448‐5p on TGF‐β1‐mediated EMT and fibrosis in bronchial epithelial cells. Taken together, the miR‐448‐5p/TGF‐β1/Six1 link may play roles in the progression of EMT and pulmonary fibrosis in asthma.

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<b><i>Background:</i></b> Transforming growth factor-β1 (TGF-β1)-in­duced proliferation of airway smooth muscle cells plays critical roles in the development of airway remodeling<i>.</i> Six1 (sine oculis homeobox homolog 1) has been demonstrated to be involved in airway inflammation and remodeling in asthmatic mice. <b><i>Objectives:</i></b> The aim of this work was to investigate the potential role of miR-204-5p in the proliferation and extracellular matrix (ECM) production of airway smooth muscle cells in asthma. <b><i>Methods:</i></b> Real-time PCR was used to measure the expression of miR-204-5p in asthmatic airway smooth muscle cells. Cell viability and apoptosis were detected to evaluate the effect of miR-204-5p on airway smooth muscle cells. Dual-luciferase reporter experiments were applied to identify the target genes of miR-204-5p. <b><i>Results:</i></b> MiR-204-5p was downregulated notably in asthmatic airway smooth muscle cells as well as cells stimulated with TGF-β1. Overexpression of miR-204-5p markedly suppressed the TGF-β1-induced proliferation of airway smooth muscle cells and the deposition of ECM, whereas the inhibition of miR-204-5p significantly enhanced the proliferation of airway smooth muscle cells and upregulated the level of fibronectin and collagen III. Furthermore, subsequent analyses demonstrated that Six1 was a direct target of miR-204-5p, and Western blot further indicated that miR-204-5p negatively regulated the expression of Six1. Most importantly, the restoration of Six1 expression reversed the inhibitory effect of miR-204-5p on TGF-β1-induced proliferation and ECM production. <b><i>Conclusions:</i></b> MiR-204-5p inhibits TGF-β1-in­duced proliferation and ECM production of airway smooth muscle cells by regulating Six1, identifying a potential therapeutic target for preventing airway remodeling in asthma.

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Introduction: The homeodomain transcription factor sine oculis homeobox homolog 1 (Six1) plays a crucial role in embryogenesis and is not expressed in normal adult tissue but is expressed in many pathological processes, including airway remodelling in asthma. The current study aimed to reveal the effects of Six1 in regulating the airway remodelling and its possible mechanism. Methods: A mouse model of ovalbumin-induced asthma-associated airway wall remodelling and a bronchial epithelial cell (16HBE) model of transforming growth factor β1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) were used to investigate the role of Six1. Then, 16HBE cells were transformed with Six1 expression vectors and treated with a TGFβ1 pathway inhibitor to determine the role of Six1 in EMT. The effect of Six1 and its possible mechanism were assessed by immunohistochemistry, RT-PCR, and Western blot. Results: Six1 expression was elevated in the lungs in an OVA mouse model of allergic asthma and in 16HBE cells treated with TGFβ1. Six1 overexpression promoted an EMT-like phenotype with a decreased protein expression of E-cadherin and increased protein expression of α-smooth muscle actin (α-SMA) as well as fibronectin in 16HBE cells; these effects appeared to promote TGFβ1 and phospho-Smad2 (pSmad2) production, which are the main products of the TGFβ1/Smad signalling pathway, which could be reduced by a TGFβ1 inhibitor. Conclusion: These data reveal that Six1 and TGFβ1 are potentially a part of an autocrine feedback loop that induces EMT, and these factors can be reduced by blocking the TGFβ1/Smad signalling pathway. As such, these factors may represent a promising novel therapeutic target for airway remodelling in asthma.