A Long Noncoding RNA links TGF-β Signaling in Lung Fibrosis

Pulmonary fibrosis is an increasing cause of morbidity and mortality worldwide with limited therapeutic options. Idiopathic pulmonary fibrosis (IPF) is a particularly severe form of lung fibrosis, with no known etiology and a median survival of 2.5–3.5 years after diagnosis (1). The pathogenesis of IPF is complex and involves loss of epithelial integrity and excessive fibroblast activation (1, 2).

The TGF-β (transforming growth factor β) signaling pathway plays a central role in the initiation and progression of tissue fibrosis (3). Strategies to target the TGF-β signaling pathway have been extensively investigated in preclinical settings (4) and in clinical trials for patients with IPF. Owing to the pleiotropic nature of TGF-β, directly blocking TGF-β signaling may have adverse effects. Alternative strategies, such as partial inhibition of TGF-β using αvβ6 integrin antibodies, have been investigated (5).

In addition to protein-coding RNAs, many noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long ncRNAs (lncRNAs), have been recently described. miRNAs are short (∼22 nt in length), single-stranded ncRNAs that inhibit the production of target proteins or induce the degradation of mRNAs, thereby suppressing target gene expression. Dysregulation of miRNAs has been shown in the lungs of patients with IPF (6), as well as in animal models of lung fibrosis (7). The roles of miRNAs in lung fibrosis have been studied in humans and in mice (8, 9). lncRNAs are RNA transcripts that are more than 200 nt long and may play a role in gene transcriptional regulation, post-transcriptional regulation, and epigenetic regulation in development and diseases (10).

In this issue of the Journal, Savary and colleagues (pp. 184–198) report that the lncRNA DNM3OS (DNM3 opposite strand/antisense RNA) serves as an miRNA reservoir in TGF-β signaling (11). Using RNA sequencing and small RNA sequencing in a human lung fibroblast cell line (MRC-5) stimulated with TGF-β1, the authors found that the lncRNA DNM3OS was one of the most strongly induced lncRNAs. Fluorescent in situ hybridization targeting RNA molecules showed that the primary expression of DNM3OS was restricted to the fibrotic area of bleomycin-injured mouse lungs, specifically in Acta2⁺ myofibroblasts.

DNM3OS and the miR-199a/miR-214 cluster are located on chromosome 1 at 1q24.3 on the opposite strand of the DNM3 gene (encoding dynamin 3). However, DNM3 mRNA (and its encoded miR-3120) was not modulated by TGF-β, suggesting that DNM3OS may not function as a cis-acting RNA mediating its host gene, but rather as a trans-acting lncRNA mediating TGF-β signaling components at distinct locations. lncRNAs can regulate gene expression and biological processes by giving rise to miRNAs. In this study, the lncRNA DNM3OS was shown to be a precursor that gives rise to three mature miRNAs (miR-199a-5p, miR-199a-3p, and miR-214-3p) in lung fibroblasts. With relevance to human disease, the study showed that DNM3OS and these three mature miRNAs were all upregulated in IPF fibroblasts and regulated by TGF-β.

The study further showed mechanistically that these three mature miRNAs regulate distinct TGF-β signaling activities. miR-199a-5p promoted lung fibrosis through a CAV1-dependent mechanism in lung fibroblasts in vitro and in a mouse model in vivo. miR-199-3p targets FGF7 and HGF, possibly in concert with TGF-β–induced suppression of HGF/FGF7 production. On the other hand, miR-214-3p targets GSK-3β and COX-2 in the β-catenin pathway, suggesting that DNM3OS may mediate the crosstalk between the TGF-β and Wnt profibrotic pathways.

This study further demonstrates the translational possibility of targeting lncRNAs as a therapeutic strategy for IPF. Loss-of-function experiments with gapmers (antisense oligonucleotides) showed that DNM3OS was a critical downstream effector of TGF-β signaling in lung fibroblasts in vitro, as well as in bleomycin-induced lung fibrosis in mice in vivo. The miRNAs miR-199a2 and miR-214 showed activity in response to TGF-β1 in mouse and human fibroblast lines in vitro and in mouse models in vivo. Two antisense oligonucleotides (antimiRs) against miR-199a-3p, miR-199a-5p and miR-214-3p, impacted fibrogenesis in cell lines and in bleomycin models in mice, respectively. These in vivo experiments have profound significance in that they suggest the ability of RNAse-H activating gapmers to silence the miRNA cluster in an in vivo setting. Thus, antisense oligonucleotide–based therapeutic strategies targeting either mature miRNAs or polycistronic miRNA precursor transcripts may represent an effective approach for the treatment of IPF.

Although this was a comprehensive study involving extensive datasets, several points should be considered. First, the expression and function of DNM3OS in other cell types in the lung should be carefully examined. For example, a recent study using single-cell RNA sequencing identified that lncRNA MEG3 was expressed in airway epithelial cells from healthy donors and IPF basal-like epithelial cells (12). Epithelial apoptosis pathways are activated in the lungs of patients with lung injury, in part by activation of the TGF-β signaling pathway. Deletion of TGFβR2 in epithelial cells protects mice from lung fibrosis (13). Impaired alveolar type 2 cell renewal may contribute to a dysregulated lung-injury response in IPF (14). Therefore, the role of these lncRNAs in epithelial cells should be further investigated. Second, the necessity of DNM3OS for TGF-β signaling can be established with the lncRNA knockout in mice. Moreover, it is hard to imagine that one lncRNA works alone in a complex disease such as IPF. An lncRNA circuit may exist to regulate the fibrogenic pathways. It was previously reported that embryonic stem cell pluripotency is mediated by several dozens of lncRNAs with diverse mechanisms (15). Third, the induction of cleavage of the target lncRNA DNM3OS with gapmers or antisense oligonucleotides should be examined biochemically, genetically, and pharmacologically. For example, gapmer-RNase H complex formation, RNase H1/H2 involvement, cleavage efficacy, and access to the injury area are worth careful investigation. Nevertheless, this study shows that gapmer-based therapy against the lncRNA DNM3OS may represent a new therapeutic approach to treat patients with IPF.