Hypoxia and transforming growth factor ß1 regulation of long non-coding RNA transcriptomes in human pulmonary fibroblasts.

One of the key characteristics of idiopathic pulmonary fibrosis (IPF) is accumulation of excess fibrous tissue in the lung, which leads to hypoxic conditions. Transforming growth factor (TGF) ß is a major mediator that promotes the differentiation of fibroblasts to myofibroblasts. However, how hypoxia and TGFß together contribute the pathogenesis of IPF is poorly understood. Long non-coding RNAs (lncRNAs) have regulatory effects on certain genes and are involved in many diseases. In this study, we determined the effects of hypoxia and/or TGFß on mRNA and lncRNA transcriptomes in pulmonary fibroblasts. Hypoxia and TGFß1 synergistically increased myofibroblast marker expression. RNA sequencing revealed that hypoxia and TGFß1 treatment resulted in significant changes in 669 lncRNAs and 2,676 mRNAs compared to 150 lncRNAs and 858 mRNAs in TGFß1 alone group and 222 lncRNAs and 785 mRNAs in hypoxia alone group. TGFß1 induced the protein expression of HIF-1α, but not HIF-2α. On the other hand, hypoxia enhanced the TGFß1-induced phosphorylation of Smad3, suggesting a cross-talk between these two signaling pathways. In all, 10 selected lncRNAs (five-up and five-down) in RNA sequencing data were validated using real-time PCR. Two lncRNAs were primarily located in cytoplasm, three in nuclei and five in both nuclei and cytoplasm. The silencing of HIF-1α and Smad3, but not Smad2 and HIF-2α rescued the downregulation of FENDRR by hypoxia and TGFß1. In conclusion, hypoxia and TGFß1 synergistically regulate mRNAs and lncRNAs involved in several cellular processes, which may contribute to the pathogenesis of IPF.

EZH2 enhances the differentiation of fibroblasts into myofibroblasts in idiopathic pulmonary fibrosis.

The accumulation of fibroblasts/myofibroblasts in fibrotic foci is one of the characteristics of idiopathic pulmonary fibrosis (IPF). Enhancer of zeste homolog 2 (EZH2) is the catalytic component of a multiprotein complex, polycomb repressive complex 2, which is involved in the trimethylation of histone H3 at lysine 27. In this study, we investigated the role and mechanisms of EZH2 in the differentiation of fibroblasts into myofibroblasts. We found that EZH2 was upregulated in the lungs of patients with IPF and in mice with bleomycin-induced lung fibrosis. The upregulation of EZH2 occurred in myofibroblasts. The inhibition of EZH2 by its inhibitor 3-deazaneplanocin A (DZNep) or an shRNA reduced the TGF-ß1-induced differentiation of human lung fibroblasts into myofibroblasts, as demonstrated by the expression of the myofibroblast markers α-smooth muscle actin and fibronectin, and contractility. DZNep inhibited Smad2/3 nuclear translocation without affecting Smad2/3 phosphorylation. DZNep treatment attenuated bleomycin-induced pulmonary fibrosis in mice. We conclude that EZH2 induces the differentiation of fibroblasts to myofibroblasts by enhancing Smad2/3 nuclear translocation.

Regulation of myofibroblast differentiation by miR-424 during epithelial-to-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is one of the most common and severe interstitial lung diseases. Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to IPF. MicroRNAs (miRNAs) are small non-coding RNAs of 18-24 nucleotides in length which regulate gene expression. Several studies have implicated miRNAs in EMT; however, specific miRNAs that regulate EMT in IPF have not yet been identified. In this study, we identified 6 up-regulated and 3 down-regulated miRNAs in a human lung epithelial cell EMT model using miRNA microarray and real-time PCR. Overexpression of one of these up-regulated miRNAs, miR-424, increased the expression of α-smooth muscle actin, an indicator of myofibroblast differentiation, but had no effects on the epithelial or mesenchymal cell markers. miR-424 enhanced the activity of the TGF-ß signaling pathway, as demonstrated by a luciferase reporter assay. Further experiments showed that miR-424 decreased the protein expression of Smurf2, a negative regulator of TGF-ß signaling, indicating that miR-424 exerts a forward regulatory loop in the TGF-ß signaling pathway. Our results suggest that miR-424 regulates the myofibroblast differentiation during EMT by potentiating the TGF-ß signaling pathway, likely through Smurf2.

Tumor Spheroids as an in vitro model for determining the therapeutic response to proton beam radiotherapy and thermally sensitive nanocarriers.

Multicellular Tumor Spheroids (MCTS) strongly resemble tumor tissues, which makes them useful tools for radiation biology studies and screening of various chemotherapeutics. The goal of this pilot study was to use MCTS as an in vitro model to determine the response of cells to low temperature-sensitive liposomes (LTSLs) encapsulating doxorubicin (Dox) and proton beam radiotherapy (PBRT). Prior to treatment, MCTS were characterized for morphology and LTSLs were characterized for size, encapsulation efficiency, and ability to thermally release Dox (a model anticancer agent). Two groups of MCTS were treated with LTSL in combination with mild hyperthermia (40-42 °C) or PBRT alone in the presence of appropriate controls. Cytotoxic response was assessed after 48-72 h using an acid phosphatase assay. At 72 h, LTSL in combination with heat significantly reduced the viability of MCTS (15-30%) compared to the control (P < 0.05). A similar cytotoxic response was observed with PBRT treatment. The data suggest that like a monolayer cell culture, MCTS can be used to determine cytotoxic outcomes of thermal and proton therapy.