Long non‐coding RNAs: Promising new targets in pulmonary fibrosis

Abstract Pulmonary fibrosis is characterized by progressive and irreversible scarring in the lungs with poor prognosis and treatment. It is caused by various factors, including environmental and occupational exposures, and some rheumatic immune diseases. Even the rapid global spread of the COVID‐19 pandemic can also cause pulmonary fibrosis with a high probability. Functions attributed to long non‐coding RNAs (lncRNAs) make them highly attractive diagnostic and therapeutic targets in fibroproliferative diseases. Therefore, an understanding of the specific mechanisms by which lncRNAs regulate pulmonary fibrotic pathogenesis is urgently needed to identify new possibilities for therapy. In this review, we focus on the molecular mechanisms and implications of lncRNAs targeted protein‐coding and non‐coding genes during pulmonary fibrogenesis, and systematically analyze the communication of lncRNAs with various types of RNAs, including microRNA, circular RNA and mRNA. Finally, we propose the potential approach of lncRNA‐based diagnosis and therapy for pulmonary fibrosis. We hope that understanding these interactions between protein‐coding and non‐coding genes will contribute to the development of lncRNA‐based clinical applications for pulmonary fibrosis.


| INTRODUCTION
Tissue fibrosis is a hallmark of chronic diseases in many organs, disturbing their architecture and resulting in dysfunction and failure. 1,2 Almost 45% of deaths are attributed to chronic fibroproliferative diseases, including pulmonary fibrosis. 3 In addition to idiopathic pulmonary fibrosis (IPF) of unknown cause, progressive pulmonary fibrosis is the result of various factors, including environmental and occupational exposure, such as asbestos, mineral dust and radiation damage. Some rheumatic immune diseases, including systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome and dermatomyositis, can be accompanied by pulmonary fibrosis. Sudden public health events, such as the severe acute respiratory syndrome coronavirus, H5N1-virus and the rapid global spread of the COVID-19 pandemic, can also cause pulmonary fibrosis in many patients. 4,5 At present, the longterm pulmonary consequences of COVID-19 remain speculative and should not be assumed without appropriate prospective study. However, from the data of previous coronavirus infections such as severe acute respiratory syndrome and Middle East respiratory syndrome, it is suggested that there could be substantial fibrotic consequences Songzi Zhang and Hongbin Chen contributed equally to this work. following the COVID-19 pandemic, and that even rare complications will have major health effects at the population level. 6,7 Despite significant progress in understanding disease pathogenesis, treatments for lung fibrosis are limited. 8,9 Two drugs, namely nintedanib and pirfenidone, are currently approved by the Food and Drug Administration for IPF treatment. Nintedanib is an intracellular tyrosine kinase inhibitor and pirfenidone is an anti-fibrotic molecule that appears to work by reducing the proliferation and differentiation of fibroblasts. However, these drugs do not improve symptoms and are poorly tolerated. 10,11 Lung transplantation is the only treatment to improve life quality and the survival of pulmonary fibrosis patients. 12 However, only a small proportion of patients can benefit from lung transplantation given that the demand outstrips the supply for organ transplants and immune rejection is still a serious challenge. As a result, a complete understanding of the molecular mechanisms implicated in pulmonary fibrosis pathogensis is urgently required to enable the development of new treatments.
Long non-coding RNA (lncRNA) is defined as a regulatory noncoding RNA of more than 200 nucleotides, including intergenic transcripts and enhancer RNA with sense or antisense orientation. 13,14 lncRNAs regulate numerous biological processes through different mechanisms in the nucleus and cytoplasm and are highly cell/ tissue-specific. 15,16 In this review, we focus on the molecular mechanisms and implications of lncRNAs during pulmonary fibrogenesis.
Meanwhile, how lncRNAs cross-talk with various types of RNAs, including microRNA (miRNA), circular RNA (circRNA) and mRNA, is also discussed. Finally, we propose the potential approach of lncRNA-based diagnosis and therapy for pulmonary fibrosis.

| LNCRNA DIRECTLY TARGETS PROTEIN-CODING GENES TO REGULATE PULMONARY FIBROSIS
The act of transcription or the DNA element within the lncRNA locus are more likely to be the source of regulatory activity than the actual lncRNA itself. 17,18 Using a genome-scale CRISPR-Cas9 activation screen of 10,000 lncRNA transcriptional start sites, Joung et al. 19 reported that the majority of candidate loci appear to regulate nearby genes. In addition, lncRNA can form a RNA-protein complex with its binding protein to exert a positive or negative function. 20 In pulmonary fibrosis, lncRNAs participate in the pathologic development of fibrosis by distinct mechanisms in different cell types, such as epithelial cells, fibroblasts and macrophages, etc.

| lncRNA in epithelial cells
The initial phase of pulmonary fibrosis is usually considered to be a consequence of repetitive micro-injuries to the alveolar epithelium, leading to changes in the cell microenvironment, fibroblast activation and deposition of matrix components. 21,22 Gokey et al. 23 have identified 21 altered lncRNAs using single-cell RNA sequencing and MEG3 was found to be the most increased lncRNA in IPF epithelial cells. lncMEG3 positively correlates with basal cell markers TP63, KRT5,   KRT17, KRT14 and ITGB4, and negatively correlates with normal   alveolar type II cell markers ABCA3, SFTPC and SFTPB. Further studies demonstrated that the lncMEG3 influences abnormal epithelial cell   differentiation and increased epithelial cell migration by regulating   numerous genes, including TP63, STAT3, KRT14, YAP1, AXL, TP53, EZH2 and transforming growth factor (TGF)β. 23 However, the initial molecular mechanism and the close communication between lung epithelial cells and other type cells for fibrogenesis still remains unclear. Recently, Fukushima et al. 24 demonstrated that alveolar epithelium cells apoptosis is sufficient to cause fibrosis by dysregulating the expression of nuclear exosome targeting complex component Rbm7-lncNEAT1 axis, triggering the epithelium apoptosis in Rbm7-deficient mice, bleomycin-induced fibrosis mice, nonhematopoietic (CD45 − ) cells and RBM7 −/− HEK293 cells. Further mechanistic studies revealed that highly expressed Rbm7 induces the degradation of lncNEAT1 paraspeckles and the mislocalization of BRCA1 that disperses from lncNEAT1 paraspekles, leading to impaired DNA repair and triggering apoptosis. The dying alveolar epithelium releases chemokines that recruit atypical monocytes to drive lung fibrosis. 24 On the whole, injured alveolar epithelium cells cause changes in not only TGFβ and chemokines, but also other microenvironment factors, such as hypoxia and reactive oxygen species (ROS).
The role of lncRNA telomeric repeat-containing RNA (TERRA) was also investigated in H 2 O 2 -treated alveolar epithelial cells, bleomycintreated mice lung fibrosis and IPF patients. 25 Functionally, lncTERRA accelerates the fibrotic process by promoting alveolar epithelium apoptosis. Mechanistically, lncTERRA forms a stable complex with the telomere repeat factor TRF2 and telomere DNA repeats to accelerate IPF pathogenesis through telomeric and mitochondrial pathways.
During lung fibrogenesis, lncTERRA prevents telomere elongation by inhibiting telomerase activity and reverse transcriptase, and this condition inhibits cell cycle progression. Proliferating cell nuclear antigen, cyclin D1 and cyclin E are the regulatory factors of telomerase activity. RNA interference on lncTERRA enhances the expression of these factors, and this improvement promotes cell growth. The mitochondrion is also identified as the lncTERRA regulator of the IPF process.
RNA interference on lncTERRA can ameliorate the functions of mitochondria by improving mitochondrial morphology, the electron transport chain and mitochondrial membrane potential. The findings demonstrate that lncTERRA contributes to fibrotic process by causing telomere attrition and mitochondrial dysfunction, in which mitochondria-associated genes (Bcl-2 family, cytochrome c, caspase-9 and caspase-3), oxidative stress-associated genes or products (ROS, superoxide dismutase and catalase) and senescence regulatory genes (P53 and p53-upregulated modulator of apoptosis) are all involved and play different important roles. 25 Targets and therapeutic interventions with respect to the dysfunction of organelles have been developed. Nandrolone decanoate-targeted abnormal telomere shortening is currently in a clinical trial and targets several diseases, including IPF (NCT02055456). 2 MitoQ, which is a mitochondrially targeted antioxidant, is currently used in middle-aged and older adults (NCT02597023). 26 lncRNAs provide new strategies for intervening in the functioning of these organelles. lncAP003419.16 is highly expressed in TGFβ1-treated alveolar epithelial cells and IPF patients, 27 in which lncAP003419.16 facilitates pulmonary fibrosis via the mammalian target of rapamycin (mTOR) signaling pathway dependent on its adjacent gene ribosomal protein S6 kinase B-2 (RPS6KB2). Given that the mTOR signaling pathway and RPS6KB2 are involved in the process of aging and IPF, 28,29 it was proposed that lncAP003419.16 may predict an increased risk of agingassociated IPF. Ageing is a crucial risk factor for pulmonary fibrosis independent of genetics and environment because most patients are older than 60 years. 30,31 Numerous lncRNAs influence the molecular processes that underlie age-associated phenotypes. 32,33 However, additional credible evidence is needed to confirm that lncAP003419.16 predicts the risk of IPF because of the limited number of 20 patients investigated so far. In addition, the A549 cell model is easily questioned. A549 is often regarded as a normal representative of type II human alveolar epithelial cells, 34,35 although it is still a human lung adenocarcinoma cancer cell line. The characterization of fibrosis and cancer, such as the metabolism process and specific markers, is different.
Other lncRNAs in epithelial cells are lncRNAs-uc.77 and 2700086A05Rik, which directly target zinc finger E-box-binding homeobox 2 and homeobox protein Hox-A3 to promote paraquatinduced pulmonary fibrosis by enhancing epithelial-mesenchymal transition (EMT) with increased α-smooth muscle actin (SMA) and vimentin and reduced E-cadherin. 36 Overall, because the current strategy for treating fibrosis is mainly focused on fibroblasts, these lncRNAs, as initial mediators in fibrotic pathogenesis, provide promising new targets in epithelial cells for the early treatment of fibrosis.

| lncRNA in fibroblasts
The fundamental pathogenic hallmarks of pulmonary fibrosis are uncontrolled proliferation and the high migration of activated fibroblasts. In pulmonary fibrosis, Song et al. 37  There are many reasons for this, including the detection method (RNA-sequencing or microarray), sample selection (cell type, rat, mice, patient blood or tissue) and the development of detection technology, and so on. These lncRNAs are roughly classified as sense-exon-overlap, sense-intron-overlap, antisense-exon-overlap, antisense-intron-overlap, bidirectional and intergenic according to their positions in genes. Among these lncRNAs, 15 lncRNAs show 90% sequence similarity to exons of protein-coding genes spread across different chromosomes. 37 The working mechanism of lncITPF is further elucidated in fibroblasts from IPF patients and human embryonic lung fibroblasts. 39 lncITPF is transcribed from the tenth intron to the eleventh exon of its host gene integrin b-like 1 (ITGBL1) and upregulated in the nucleus, which indicates that lncITPF regulates ITGBL1 transcription. ITGBL1 contains 10 repeats of epidermal growth factor-like domain and encodes a β integrin-related protein TIED; its high expression facilitates metastasis and invasion and correlates with poor survival in cancer patients. 40 In pulmonary fibrosis, high ITGBL1 expression increases the fibrotic markers, such as a-SMA, vimentin and collagen, and promotes myofibroblast proliferation and migration, thus resulting in fibrogenesis. The pro-fibrotic role of lncITPF depends on ITGBL1 by enhancing H3 and H4 histone acetylation of the ITGBL1 promoter, in which lncITPF forms an RNA-protein complex with its direct binding protein Certain studies have confirmed an incidence of lung cancer of 4.8%-48% and, without pulmonary fibrosis, of 2.0%-6.4% 43 ; thus, IPF has been considered a precancerous condition or another form of cancer. 44,45 p53 is an important tumor gene because approximately 50% mutated p53 gene appear in tumors. 46 Recently, lncRNA cyclin-dependent kinase inhibitor-2B-antisense RNA 1 (CDKN2B-AS1) in IPF patients was reported to predict lung cancer by regulating the p53-signaling pathway. lncCDKN2B-AS1 is reduced in the peripheral blood of IPF patients, and its neighboring gene CDKN2A is decreased simultaneously. 47   Silicosis is a progressive pulmonary fibrosis and is initiated via the phagocytosis of silica particles by alveolar macrophages. An alteration profile of lncRNAs has been investigated in lung tissues of silicainduced rats. 55 Upregulated lncRNA LOC103691771 in the silicotic rat lung was further confirmed to activate macrophages and promote fibroblast differentiation via regulation of the TGFβ1-Smad2/3 signaling pathway. 56 Thus, investigations of lncRNA in macrophages will modulate the alveolar microenvironment, which will ameliorate pulmonary fibrosis. EndMT and the upregulated lncRNA Gm16410 was further investigated and it was found that lncRNA Gm16410 mediates PM2.5-induced EndMT by regulating the TGF-β1/Smad3/p-Smad3 pathway, which highlights the potential of lncRNAs to promote pulmonary fibrosis under environmental pollution. 57 The above-mentioned lncRNAs, located in various cells that directly target protein-coding genes to regulate pulmonary fibrosis, are listed in Table 1.

| LNCRNA DIRECTLY TARGETS NON-CODING GENES TO REGULATE PULMONARY FIBROSIS
High-throughput technologies reveal that less than 2% of the human genome encodes protein, with the majority of the genome comprising ncRNAs that are not translated into proteins. 58 Among these ncRNAs, regulatory ncRNAs including miRNA, lncRNA and circRNA can function as vital regulators of gene expression.  65 this regulatory mode has been reported in many diseases. 66 Huang et al. 67  The hypoxia-induced H19/YB-1 cascade modulates cardiac remodeling after infarction and may be a prognostic marker for cancer. 74,75 Several studies have shown that H19 is highly upregulated in pulmonary fibrosis in vivo and in vitro. Additional detailed information regarding the above-mentioned lncRNAs that regulate pulmonary fibrosis through miRNAs is summarized in Table 2.

| lncRNA targets circRNA
CircRNA is a closed-loop structure generated by pre-mRNA back  102 Astragaloside IV treatment increases lncsirt1 AS and lncsirt1 F I G U R E 1 Molecular mechanisms of lncRNAs and lncRNA-based clinical applications in pulmonary fibrosis. Aging, environmental exposure or genetic predisposition to alveolar epithelial cell results in the release of pro-fibrotic factors, such as growth factors, cytokines, chemokines and matrix metalloproteinases, which promote abnormal and persistent fibroblast activation and remodeling, macrophage M1/M2 polarization classification, and endothelial-mesenchymal transition. Meanwhile, many abnormal organelles, including telomere attrition, endoplasmic reticulum stress and mitochondrial dysfunction, are also involved in fibrogenesis. lncRNAs participate in these abnormal biological processes in the nucleus or cytoplasm via diverse mechanisms and thus regulate numerous signaling pathways in different cell types to control pulmonary fibrogenesis.
(1) The images present the various methodologies for lncRNA as the diagnostic marker.
(2) lncRNA-based therapeutic strategies are proposed depending on their different regulatory patterns AS and is validated to enhance the stability of sirt1 and increase sirt1 expression, thereby preventing IPF. 103 Certainly, lncRNA-based clinical application still has some challenges. In some cases, several lncRNAs cover the intronic and exonic sequences of their host genes, which suggests the possibility of lncRNA-based pharmacological approaches producing off-target effects. 105 In addition, a few lncRNAs actually encode small proteins. 17,106 Individual lncRNA can also act as both a protein-coding gene and an ncRNA. 107 Nevertheless, the protein-coding potential of lncRNA in pulmonary fibrosis is not still documented. Despite potential issues, the spatial-, temporal-and tissue-specific expression patterns of lncRNAs suggest that this is a promising area for new targets in pulmonary fibrosis. Potential lncRNA-based clinical applications are listed in Figure 1.

CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
The datasets used and analysed during the current study are available from the corresponding author upon reasonable request.