Project description:To identify the target gene repertoire of miRNAs (i.e. the miRNA-targetome) of unstimulated and TGF-β1-stimulated primary parenchymal lung fibroblasts, Ago2-IP was performed followed by mRNA expression profiling.
Project description:mRNA expression profiling after Ago2-immunoprecipitation (IP) in unstimulated and TGF-β1-stimulated primary parenchymal lung fibroblasts of two control subjects
Project description:Here, we showed that baicalein suppressed transforming growth factor β1 (TGF β1)-stimulated the production of type I collagen in lung fibroblast MRC-5 cells. By applying SILAC-based proteomic technology, 158 proteins were identified as baicalein-modulated proteins in TGF β1-stimulated the accumulation of type I collagen in MRC-5 cells. Our proteomic and biochemical analysis demonstrated that baicalein could decrease the expression levels of connective tissue growth factor (CTGF) in TGF β1-stimulated MRC-5 cells. In addition, CTGF overexpression elevated the levels of type I collagen in baicalein-treated fibroblasts. Moreover, our results demonstrated that baicalein-downregulated CTGF expression might be related with the decrease of Smad2 phosphorylation, but not SP1.
Project description:Fibroblasts represent key effector cells responsible for fibrogenesis during wound healing and pathological fibrosis, including idiopathic pulmonary fibrosis (IPF), the most rapidly progressive and fatal fibrotic lung disease. Single-cell RNA sequencing of fibrotic lung tissue has identified a novel population of high collagen-producing fibroblasts not present in non-diseased control lung, characterised by high expression of the marker, collagen triple helix repeat containing 1 (CTHRC1). The cardinal pro-fibrotic mediator TGF-β1 has been widely implicated in promoting fibrogenesis in multiple fibrotic conditions. In addition to the canonical Smad signalling pathway, TGF-β1-induced collagen I production is under critical regulatory control by the mTORC1/4E-BP1 signalling hub. Using pharmacological inhibition (dual ATP-competitive mTOR inhibitors and rapamycin) in combination with gene-editing approaches, we now demonstrate that the role of the mTORC1 axis extends to the regulation of over a third of all TGF-β1 regulated matrisome genes. We further show that the global transcriptome of TGF-β1-stimulated fibroblasts matches that of CTHRC1+ pathological fibroblast population in the IPF lung. In contrast, the TGF-β1 induced transcriptome of fibroblasts in which mTORC1-signalling is disrupted (by RPTOR gene editing using CRISPR-Cas9) does not map to any known fibroblast population. These data define, for the first time, a critical role for both TGF-β1 and the mTORC1 signalling hub in determining the transcriptional identity of CTHRC1+ pathological fibroblasts and provide strong scientific support for targeting mTORC1 as a therapeutic strategy in IPF and potentially other fibrotic conditions associated with dysregulated TGF-β1 profibrotic signalling.
Project description:Lung fibroblasts play an important role in extracellular matrix homeostasis and this process is mainly regulated by transforming growth factor-beta (TGF-β). Hence, lung fibroblasts are postulated to play a crucial role in aberrant lung tissue repair and remodeling, which is a main factor in lung diseases like COPD. In this study, the effect of TGF-β1 on the miRNA expression in parenchymal lung fibroblasts is investigated.
Project description:To elucidate how TGF-β1 regulates translation, we treated human lung fibroblasts (HLF) with TGF-β1 and used RNA-seq to determine the effect of TGF-β1 on total RNAs, and mRNA polysome/monosome ratios.
