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miRNA Expression in Fibroblastic Foci within Idiopathic Pulmonary Fibrosis Lungs Reveals Novel Disease-Relevant Pathways

Open AccessPublished:January 20, 2023DOI:https://doi.org/10.1016/j.ajpath.2022.12.015
      miRNAs are a class of noncoding RNAs of approximately 22 nucleotides long that play an important role in regulating gene expression at a post-transcriptional level. Aberrant levels of miRNAs have been associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or in vitro monocultures in which fibrosis has been artificially induced. In this study, we used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, then isolate miRNAs and compare their expression levels with those found in whole IPF lung tissue and/or in vitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue; of these molecules, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impact on several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotypically and functionally discrete tissue microenvironment.
      Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease characterized by a restrictive ventilatory defect and impaired gas transfer due to deposition of fibrotic tissue in the lung interstitium. The incidence of IPF has been reported as ranging from 2.8 to 18 cases per 100,000,
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      but it appears to be steadily increasing, with 6000 new patients/year presenting and a disease prevalence of approximately 32,000 in the United Kingdom (British Lung Foundation UK IPF Statistics, https://www.blf.org.uk/support-for-you/idiopathic-pulmonary-fibrosis-ipf/statistics, last accessed February 2, 2022). IPF has a poor prognosis, with a median survival from diagnosis of only 2 to 4 years, making post-diagnosis survival worse than many cancers.
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      The etiology of IPF remains unclear, but evidence is growing that complex interactions between genetic risk factors and environmental insults on a background of age-associated predisposition are most likely to be the key contributors.
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      Short telomeres are a risk factor for idiopathic pulmonary fibrosis.
      Treatment options for IPF are limited and are predominately palliative, yet two distinct pharmaceutical agents, pirfenidone and nintedanib, are licensed as novel IPF treatments. However, at best, these agents decrease the rate of decline in patient lung function and reduce the risk of acute deteriorations of lung function rather than halt or reverse the fibrogenic process.
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      A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis.
      ,
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      Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis.
      Lung transplantation is the only option that offers hope for long-term survival, but it is only available to highly selected individuals.
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      Lung transplantation in idiopathic pulmonary fibrosis: a systematic review of the literature.
      The pathology of IPF is characterized by disruption of normal lung architecture due to deposition of excessive collagen and extracellular matrix in the alveolar walls and development of aggregates of proliferating fibroblasts and myofibroblasts, which are recognized as fibroblastic foci (FF) on histologic evaluation of diseased tissue.
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      • Sinclair I.
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      Three-dimensional characterization of fibroblast foci in idiopathic pulmonary fibrosis.
      ,
      • Horowitz J.C.
      • Thannickal V.J.
      Epithelial-mesenchymal interactions in pulmonary fibrosis.
      The foci represent discrete sites of lung injury and repair and are thought to be of pivotal importance to the progression of IPF. An improved understanding of the mechanisms that lead the fibroblast/myofibroblast population within the FF in the lung to proliferate and produce excessive extracellular matrix is critical to identifying potential pathways to target new therapies that might halt or even reverse the disease process.
      miRNAs are short noncoding RNAs that regulate gene expression in a post-transcriptional manner through binding to the 3′-untranslated region of their target mRNAs. This interferes with protein production by destabilizing the mRNA and causing translational fine-tuning. As a result, miRNA expression levels can influence several cellular processes, including differentiation, proliferation, activation, and apoptosis.
      • Zou X.Z.
      • Liu T.
      • Gong Z.C.
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      • Zhang Z.
      MicroRNAs-mediated epithelial-mesenchymal transition in fibrotic diseases.
      The altered quantities of miRNAs have been investigated in several fibrotic tissues in both animal models and human disease and have been associated with fibrosis progression.
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      • Shan H.
      • Liang H.
      MicroRNAs in idiopathic pulmonary fibrosis: involvement in pathogenesis and potential use in diagnosis and therapeutics.
      However, many of these studies have used whole organ tissues, comparing the diseased organ with the normal healthy control. Alternatively, studies were performed on single-cell types, predominantly epithelium or fibroblasts, cultured from diseased or normal tissue or from animal organs in which fibrosis has been artificially induced.
      • Pandit K.V.
      • Milosevic J.
      MicroRNA regulatory networks in idiopathic pulmonary fibrosis1.
      All of these approaches carry inherent problems associated with the heterogeneity of cell types and their ratios in the diseased versus healthy organ context or issues emanating from isolated cells cultured on stiff tissue culture plastic.
      In this study, we wanted to improve our understanding of the role of miRNAs in the pathophysiology of IPF by investigating the expression patterns of miRNAs within FF themselves, the hallmark lesions of IPF that are rich in fibroblasts and myofibroblasts, which are cell types known to drive fibrogenesis. To do this, multiple foci from IPF lung tissue samples were collected using laser capture microdissection (LCM), and the miRNA profile was quantified by next-generation sequencing. The levels of miRNAs in FF were compared with those found in total IPF tissue or in fibroblast cultures isolated from matched IPF or normal control lung tissue to investigate if the process of culturing cells on plastic alters the miRNA levels. Combining these approaches, we have uncovered novel pathways operating within FF that have not previously been described.

      Materials and Methods

      IPF Lung Tissue Preparation for Laser Microdissection

      Formalin-fixed, paraffin-embedded blocks of IPF lung tissue from nine patients were sectioned and stained with Mayer’s hematoxylin. The slides were used for LCM isolation of FF on the same day. FF were detected and selected by a pathologist and later confirmed and cut out using LCM by pathology-trained technician.

      Study Approval

      Human Subjects

      Use of human tissue was approved by Newcastle and North Tyneside Local Research Ethics number 11/NE/0291. All samples were collected and used subject to patient's written consent. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). IPF tissue was obtained from patients undergoing lung transplantation at the Institute of Transplantation, Newcastle Upon Tyne Hospitals NHS Foundation Trust.

      Laser Microdissection of IPF Lung Tissue

      The FF were cut from the IPF tissue sections using Zeiss Laser Capture Microdissection Microscope (Zeiss, Carl Zeiss Microscopy GmbH, Germany). The area of interest was cut out using an automated laser pressure catapulting method. Microdissected areas were collected in an AdhesiveCap (Zeiss, Carl Zeiss Microscopy GmbH), and RNA was isolated using RNeasy FFPE kit (Qiagen, Hilden, Germany), as per manufacturer's instructions. The surface area of 9,000,000 ± 1,000,000 μm2 was found to generate 50 to 200 ng of RNA, which comprised at least 70 to 90 pooled individual foci. The foci were pooled from the same lung only, and never between patients. Total RNAs were extracted using FFPE RNeasy extraction kit (Qiagen), according to the manufacturer's protocol.

      Small RNA Library Preparation and Sequencing

      For each patient, NEBNext small RNA libraries for next-generation sequencing (New England Bio Labs Inc., Ipswich, MA) were prepared from total RNA. QIAquick PCR purification kit (Qiagen) and a 6% polyacrylamide gel were used to perform the library quality control and size selection of 21-nucleotide RNA fragments. Qubit dsDNA HS Assay kit and a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA) were used to measure the abundance of the libraries, and the size of the fragments contained in the libraries was measured with a DNA high-sensitivity chip and an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA). The libraries were sequenced on Illumina (San Diego, CA) MiSeq, according to the manufacturer's protocols at 50-bp read length.

      Isolation and Cell Culture of Fibroblasts from IPF Lung or Control Normal Human Lung and RNA Isolation

      Lung fibroblasts were isolated from donor-matched IPF lung tissue that was used for LCM and grown on plastic. A further five fibroblast lines were grown by the outgrowth method from normal human lungs and were used as controls. The cells were grown to 90% confluence, then serum starved for 24 hours in media containing 0.4% fetal calf serum. After the 24-hour period, the cells were incubated for 48 hours with complete media only or media supplemented with 3 ng/mL transforming growth factor (TGF)-β1. The cells were harvested at 48 hours, and total RNA was extracted using the RNeasy mini kit (Qiagen).

      Small RNA Data Processing

      FASTQ files obtained from a run on MiSeq were trimmed, size selected, and mapped with ChimiRa release 1.0
      • Vitsios D.M.
      • Enright A.J.
      Chimira: analysis of small RNA sequencing data and microRNA modifications.
      (http://wwwdev.ebi.ac.uk/enright-dev/chimiRa/index.php, last accessed March 24, 2020). ChimiRa was used to trim the sequences from sequencing adapters, using as adapter sequence AGATCGGAAGAGC, then map them against human miRNA hairpin sequences from miRBase, and extract count-based miRNA expression data.
      • Vitsios D.M.
      • Enright A.J.
      Chimira: analysis of small RNA sequencing data and microRNA modifications.
      Counts were analyzed using R statistics (https://www.r-project.org) and normalized using DESeq2 package for R version 3.01
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ; heat maps and volcano plots (fold change > 1 and P = 0.01) were plotted for all samples and conditions using gplots package for R version 3.01 (https://cran.r-project.org/web/packages/gplots/gplots.pdf).

      Availability of Data and Material

      Raw and processed miRNA sequencing data can be found at Gene Expression Omnibus using the accession number GSE220107 (https://www.ncbi.nlm.nih.gov/geo).

      Results

      To determine the miRNA content of FF, the authors obtained histologic sections of explanted IPF lungs from nine patients (Table 1). Sections were stained with hematoxylin and eosin, and foci were identified using light microscopy (Figure 1A). Despite some interpatient variability in the numbers of FF present within different explanted IPF lungs, the authors were able to identify and dissect out 50 to 200 foci from each lung using LCM (Figure 1B). RNA isolated from foci was sequenced, generating on average 3 to 4 million reads for each donor lung. The sequences were mapped onto the human genome, and the number of individual miRNAs sequenced was quantified. The mean of counts for each miRNA shows the most abundant miRNA species expressed in the FF (Figure 1C). The authors next sequenced RNA isolated from matched whole IPF lung tissue and made a direct comparison of miRNA signatures in the LCM isolated FF from the same lung, generating a heat map with 43 significantly different miRNAs (Figure 2A and Supplemental Table S1). This direct comparison highlighted 25 miRNAs that were significantly overexpressed in the whole IPF lung tissue compared with the foci (Figure 2B), and 18 miRNAs that are significantly overexpressed in FF compared with the whole IPF lung tissue (Figure 2B) based on a fold change of two or greater and P < 0.01. Although there were significant differences in the levels of their expression, the authors found 20 miRNAs to be present in both samples, with 13 miRNAs unique to IPF lung tissue and 15 miRNAs unique to fibroblastic foci (Figure 2C). To put these miRNAs into a biological context, Ingenuity Pathway Analysis (Qiagen) was performed. Network analysis revealed numerous miRNAs related to IPF and inflammation, as evidenced by their relationship with proinflammatory cytokines, TGF-β, THEMIS, and kinases, such as MAP2K1/2, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. Moreover, miRNAs present in the whole lung appeared to be related to regulation of multiple features, such ADAMTS14 and ADAMTS15 peptidases, the transcription regulator ATOH8, transmembrane proteins (TSPAN13 and TMEM8B), insulin, CG hormone, estrogen receptor, and other features (PTPN7, FAM110C, Tnxa-ps1, Gulo, and Snhg14) (Figure 3A). However, network analyses based on miRNAs found in LCM isolated fibroblastic foci revealed several molecules not found in the analyses based on whole IPF tissue (Figure 3B). This analysis predicted that pathways classically associated with tissue fibrosis were activated, such as TGF-β, but also identified S100A12 and MTPN. Serum levels of S100A12 were found to be negatively associated with lung function in systemic scleroderma
      • Omatsu J.
      • Saigusa R.
      • Miyagawa T.
      • Fukui Y.
      • Toyama S.
      • Awaji K.
      • Ikawa T.
      • Norimatsu Y.
      • Yoshizaki A.
      • Sato S.
      • Asano Y.
      Serum S100A12 levels: possible association with skin sclerosis and interstitial lung disease in systemic sclerosis.
      and IPF,
      • Li Y.
      • He Y.
      • Chen S.
      • Wang Q.
      • Yang Y.
      • Shen D.
      • Ma J.
      • Wen Z.
      • Ning S.
      • Chen H.
      S100A12 as biomarker of disease severity and prognosis in patients with idiopathic pulmonary fibrosis.
      whereas MTPN was reported to convert p65:p50 heterodimers to repressive p50:p50 homodimers, thus differentially regulating NF-κB target genes.
      • Knuefermann P.
      • Chen P.
      • Misra A.
      • Shi S.P.
      • Abdellatif M.
      • Sivasubramanian N.
      Myotrophin/V-1, a protein up-regulated in the failing human heart and in postnatal cerebellum, converts NFκB p50-p65 heterodimers to p50-p50 and p65-p65 homodimers.
      Moreover, other features, such the long noncoding RNA HEIH, kinase EPHB6, calcifediol, SYPL1 transporter, and several miRNAs (miR-126, miR-143, miR-221, miR-26, and miR-423), were also predicted to be associated to miRNAs from LCM isolated FF but were not found in network analysis based on whole lung (Figure 3B).
      Table 1Explanted IPF Lungs: Patient Characteristics
      IdentifierSexAge, yearsFEV1 (%)FVC (%)TLC (%)TLCO (%)KCO (%)Pack years
      80M541.97 (52)2.38 (50)4.02 (54)0.87 (61)3.08
      27M562.37 (61)2.9 (59)4.84 (63)2.9 (25)0.7 (49)Nil
      70M621.43 (51)1.79 (51)2.73 (45)2.12 (26)0.85 (63)Nil
      45M442.6 (54)2.29 (50)3.27 (47)3.82 (36)1.31 (86)Nil
      73M541.86 (49)2.2 (47)3.44 (47)3.36 (31)1.18 (81)30, Stopped in 2006
      37M621.58 (48)2.13 (37)4.43 (63)2.31 (24)0.63 (46)Nil
      88M491.83 (51)2.87 (65)5.50 (81)4.6 (46)1.1 (73)Nil
      90M622.07 (64)2.49 (61)3.54 (52)3.59 (39)1.10 (81)Nil
      91M481.74 (50)2.08 (49)3.04 (46)2.26 (23)1.03 (69)10
      M, male; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; IPF, idiopathic pulmonary fibrosis; KCO, carbon monoxide transfer coefficient; TLC, total lung capacity; TLCO, carbon monoxide transfer factor.
      Figure thumbnail gr1
      Figure 1Fibroblastic foci identification from histology sections of idiopathic pulmonary fibrosis (IPF) explanted lungs. A: Representative images of hematoxylin and eosin–stained IPF explanted lungs showing fibroblastic foci outlined in light blue dotted line. B: Representative images of the identification and laser cut microdissected fibroblastic foci in hematoxylin IPF lung sections. Top panels: Fibroblastic foci identification in hematoxylin IPF lung sections before laser capture microdissection (LCM). Bottom panels: Matched IPF lung sections after LCM of the fibroblastic foci. C: Top 25 most expressed miRNAs in IPF fibroblastic foci. Scale bars: 100 μm (A); 150 μm (B).
      Figure thumbnail gr2
      Figure 2Differential expression of miRNAs in human idiopathic pulmonary fibrosis (IPF) lungs and fibroblastic foci (FF) isolated from the same lungs (heat map). A: Heat map of the 43 significant differently expressed miRNAs in the comparison between fibroblastic foci isolated from IPF lungs (blue label; right side) and matched whole IPF lung tissue (red label; left side); P < 0.01 and fold change >2. Color scale from pale green to dark blue being lower to higher expression, respectively. B: Volcano plot of the significantly differently expressed miRNAs. In blue, significant overexpressed miRNAs in fibroblastic foci. In red, significantly overexpressed miRNAs in whole IPF lung; P < 0.01 and fold change >2. C: Venn diagram of the miRNAs with >10 counts in at least three samples per group. Pale orange unique miRNAs in fibroblastic foci, pale blue unique miRNAs in whole IPF tissue, and overlapping area miRNAs found in common in both FF and whole IPF tissue. LCM, laser capture microdissection.
      Figure thumbnail gr3
      Figure 3Differential expression of miRNAs in human idiopathic pulmonary fibrosis (IPF) lungs and fibroblastic foci isolated from the same lungs [Ingenuity Pathway Analysis (IPA) plots]. IPA-generated biological network of differentially expressed miRNAs found in whole IPF lung tissue (A) and fibroblastic foci (B). In green and red, underexpressed and overexpressed miRNAs, respectively. In orange and blue, prediction of activation and inhibition, respectively. In white are molecules related to miRNAs for which no prediction of directional change could be made. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.
      Taken together, these data show that the use of heterogeneous mixture of cells (ie, whole tissue) can mask the identity and expression patterns of miRNAs emanating from a particular, highly disease-relevant subpopulation of cells within the organ.
      The authors next asked if it is possible to study the miRNA expression within FF by determining the miRNA profile of in vitro grown fibroblasts isolated from IPF lungs, given that FF predominantly contain fibroblasts/myofibroblasts. To this end, fibroblasts were isolated from the same IPF donor lungs that were used for isolation of FF by LCM, and the miRNA profiles of in vitro grown cells were then compared with LCM generated FF miRNAs. This comparison identified 11 miRNAs that were found exclusively in FF and 11 unique miRNAs found only in cultured fibroblasts of the 25 most abundant miRNA species in both groups (Figure 4). Using these data, the authors determined the Kyoto Encyclopedia of Genes and Genomes pathways regulated by the miRNAs (Supplemental Figure S1). Although there was a significant number of Kyoto Encyclopedia of Genes and Genomes pathways that were affected by miRNAs found in both FF and culture-grown fibroblasts (Supplemental Figure S1A), there were 10 pathways regulated by miRNAs unique to FF (Supplemental Figure S1B) and a further 7 Kyoto Encyclopedia of Genes and Genomes pathways that mapped to miRNAs solely identified in IPF fibroblasts grown in vitro (Supplemental Figure S1C). Furthermore, the authors performed Ingenuity Pathway Analysis of the 25 most differentially expressed miRNAs, which indicate putative functions in several signaling pathways unique only to FF (Figure 5). In summary, the data interpretation of the biology of cultured IPF fibroblasts must be cautious with respect to events occurring in the FF tissue microenvironment because of loss of cell heterogeneity and plasticity that are dynamic in the FF.
      Figure thumbnail gr4
      Figure 4Most expressed miRNAs in fibroblastic foci and cultured primary fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) lungs (counts). A: Top 25 most abundant miRNAs in IPF fibroblastic foci. In blue, miRNAs uniquely found in fibroblastic foci. In gray, common miRNAs found in fibroblastic foci and primary fibroblasts isolated from IPF lungs. B: Top 25 most abundant miRNAs in cultured primary fibroblasts isolated from IPF lungs. In red, miRNAs uniquely found in cultured fibroblasts isolated from IPF lungs.
      Figure thumbnail gr5
      Figure 5Most expressed miRNAs in fibroblastic foci and cultured primary fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) lungs [Ingenuity Pathway Analysis (IPA) plots]. A and B: IPA-generated biological network of miRNAs with highest expression in fibroblastic foci and cultured primary IPF fibroblasts. In green and red, underexpressed and overexpressed miRNAs, respectively. In orange and blue, prediction of activation and inhibition, respectively. In white are molecules related to miRNAs for which no prediction of directional change could be made. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.
      Despite gene regulation alterations mediated by in vitro culture, isolated lung fibroblasts are a valuable resource in research. To further understand the limitations of in vitro culturing, the authors sought to assess if fibroblasts cultured on plastic retain miRNA expression patterns that reflect the microenvironment and macroenvironment of the organ they were isolated from. To this end, the authors isolated and cultured fibroblasts from IPF lungs and/or normal lungs and then compared the expression profiles of miRNAs within the in vitro grown cells (Figure 6A). The comparison shows 5 miRNAs that were significantly overexpressed in the normal lung fibroblasts (Supplemental Table S2 and Figure 6B) and 15 miRNAs that are significantly overexpressed in IPF fibroblasts (Supplemental Table S2 and Figure 6B). These data also show a small difference in the fold change and P value between the IPF and normal lung fibroblasts, suggesting that the process of in vitro culturing may remodel the epigenome such that original tissue of origin signatures is at least diminished, if not completely lost. Moreover, when considering miRNAs with >10 counts in at least three samples per group, the authors found that 141 miRNAs were overlapping, whereas 24 miRNAs were present only in normal cells and 24 were unique to IPF cells (Figure 7A). Ingenuity Pathway Analysis revealed that significant differentially expressed miRNAs in fibroblasts from IPF lungs were found to be related to the transcription regulators (HIF1A, HNF4A, YBX1, and FOXO1), the translation regulator EIF4EBP2, other miRNAs (miR-1275, miR-663, miR-1908, miR-342, miR-432, and miR-154), SLC1A4 and SLC25A32 transporters, Gulo enzyme, cytokine (tumor necrosis factor and EPO), EPHB6 kinase, insulin, the receptor ROBO4, and other features (TUG1, Fascin, Snhg14, VSNL1, FTX, and COL27A1) (Figure 7B). To further explore the biological behavior of IPF and normal lung fibroblasts, the cells were treated with TGF-β1 to ascertain the response to profibrogenic stimulus; miRNAs were then isolated and sequenced (Figure 8A). The comparison identified 13 miRNAs that were significantly overexpressed in the IPF fibroblasts treated with TGF-β1 (Supplemental Table S3 and Figure 8B) and 3 miRNAs that are significantly repressed compared with the fibroblasts from normal lungs treated with TGF-β1 (Supplemental Table S3 and Figure 8B). Closer inspection reveals that 3 of 13 significantly overexpressed miRNAs in the IPF lung fibroblasts are due to treatment with TGF-β1 (Figure 8B), with the other 10 miRNAs present as baseline difference between the IPF and normal lung fibroblasts (Figure 8B). Likewise, the remaining three overexpressed miRNAs in normal lung fibroblasts were present at baseline (Figure 8B). Of the 239 TGF-β1–dependent differentially regulated miRNAs in cultured lung fibroblasts, 195 were common to normal and IPF fibroblasts, whereas 21 were unique to normal fibroblasts and 23 were unique to the IPF cultured fibroblasts (Figure 9A). Ingenuity Pathway Analysis showed these significant miRNAs to be associated with several features, some of which were also found related to miRNAs differently expressed in fibroblasts from IPF lungs, such the transporter SLC25A32, TUG1, EPHB6 kinase, miRNAs (miR-342 and miR-1908), and the transcription regulators FOXO1 and HNF4A. In addition, the relationship with different molecules was also revealed, including miR-320, the peptidase ADAMTS2, IGF1 growth factor, several enzymes (MARS2, TYMS, TRIM71, SMOX, Hmga2, GTPBP3, RTCA, and PRODH), the transcription regulator MYC, calcifediol, and other molecules (IFIT5, RBM19, COMMD9, CIAO2A, COL5A2, and CAPG) (Figure 9B). These data further confirm that the in vitro culture of fibroblasts is a potent epigenetic remodeler such that even the treatment with TGF-β1, a potent inducer of fibrogenic signaling, has little effect. These data show that culturing on plastic may alter miRNA expression levels in primary fibroblasts such that organ of origin epigenetic signatures may be diminished or erased.
      Figure thumbnail gr6
      Figure 6Differential expression of miRNAs in fibroblasts isolated from normal and idiopathic pulmonary fibrosis (IPF) lungs (heat map). A: Heat map of the 20 significant differentially expressed miRNAs in the comparison between IPF lung (red; left side) and normal lung primary cultured fibroblasts (green; right side); P < 0.01 and fold change >2. B: Volcano plot of the significant differentially expressed miRNAs comparing IPF lung and normal lung primary cultured fibroblasts. In blue, five significantly overexpressed miRNAs in normal lung fibroblasts. In red, 15 significantly overexpressed miRNAs in IPF fibroblasts.
      Figure thumbnail gr7
      Figure 7Differential expression of miRNAs in fibroblasts isolated from normal and idiopathic pulmonary fibrosis (IPF) lungs [Ingenuity Pathway Analysis (IPA) plot]. A: Venn diagram of the miRNAs with >10 counts in at least three samples per group. Pale orange indicates unique miRNAs in IPF fibroblasts; pale blue, unique miRNAs in fibroblasts from normal lungs, with overlapping area miRNAs found in common in both fibroblasts isolated from normal and IPF lungs. B: IPA-generated biological network of differentially expressed miRNAs in fibroblasts isolated from normal and IPF lungs. In green and red, underexpressed and overexpressed miRNAs, respectively. In orange and blue, prediction of activation and inhibition, respectively. In white are molecules related to miRNAs for which no prediction of directional change could be made. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.
      Figure thumbnail gr8
      Figure 8Differential expression of miRNAs in fibroblasts isolated from normal and idiopathic pulmonary fibrosis (IPF) lungs treated with transforming growth factor (TGF)-β1 (heat map). A: Heat map of the 16 significant differentially expressed miRNAs in the comparison between IPF lung (blue; left side) and normal lung primary cultured fibroblasts (purple; right side), both treated with TGF-β1; P < 0.01 and fold change >2. B: Volcano plot of the significant differentially expressed miRNAs comparing IPF lung and normal lung primary cultured fibroblasts treated with TGF-β1. In blue, three significantly overexpressed miRNAs in normal lung fibroblasts treated with TGF-β1. In red, 13 significantly overexpressed miRNAs in IPF fibroblasts treated with TGF-β1. In green text, three miRNAs that change as a result of treatment with TGF-β1.
      Figure thumbnail gr9
      Figure 9Differential expression of miRNAs in fibroblasts isolated from normal and idiopathic pulmonary fibrosis (IPF) lungs treated with transforming growth factor (TGF)-β1 (heat map). A: Venn diagram of the miRNAs with >10 counts in at least three samples per group. Pale orange indicates unique miRNAs in IPF fibroblasts treated with TGF-β1; pale blue, unique miRNAs in TGF-β–treated fibroblasts from normal lungs, with overlapping area miRNAs found in common in both. B: Ingenuity Pathway Analysis–generated biological network of differentially expressed miRNAs in fibroblasts isolated from normal and IPF lungs, both treated with TGF-β1. In green and red, underexpressed and overexpressed miRNAs, respectively. In orange and blue, prediction of activation and inhibition, respectively. In white are molecules related to miRNAs for which no prediction of directional change could be made. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.

      Discussion

      miRNAs are short noncoding RNA molecules that function in post-transcriptional regulation of gene expression. Along with other epigenetic mechanisms, miRNAs regulate gene expression in physiological and pathophysiological conditions. In this study, we address the role of miRNAs in the pathophysiology of IPF by investigating their expression patterns within fibroblastic foci, the hallmark IPF lesions highly rich in extracellular matrix–producing cell types, such as fibroblasts and myofibroblasts. Extracellular matrix–generating activities of these cells are of cardinal importance in development of IPF; therefore, understanding the underlying epigenetic regulation may illuminate novel ways of manipulating disease progression.
      To this end, we isolated fibroblastic foci from IPF lungs using laser microdissection. This approach generated a pure population of cells that were fixed in situ, thus preserving all of the miRNA epigenetic signatures determined by microenvironmental influences, including those linked to the disease process itself. From these signatures, we have been able to gain insight into novel signaling pathways and genes affected by miRNAs identified. More important, our data show that it is not possible to gain equivalent understanding from either the isolated fibroblasts or whole IPF lung tissue. As such, these findings represent significant and useful incremental gain in our knowledge of the subject.
      Previous studies collectively identified several miRNAs commonly found in IPF tissue or IPF fibroblasts, with these being the members of let-7 family (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7I, and miR-98), miR-21, miR-9, miR-26a, miR-29a, miR-30 family, miR-145, miR-155, miR-200a-c, miR-375, miR-326, miR-153, and miR-1343. We confirm the presence of most of these miRNAs in our data but also uncover some additional species present in IPF lungs, fibroblastic foci, and IPF cultured fibroblasts.
      It is well established that epigenetic determinants, including miRNAs, are exquisitely cell type specific. Previously published studies used either whole IPF lung tissue or isolated and cultured lung fibroblasts to determine miRNA expression. Fibroblastic foci within the IPF lung almost exclusively contain fibroblasts/myofibroblasts, which are the cell types known to drive IPF progression. We, therefore, asked if heterogeneous mixture of lung cells (as found in a whole lung) can be used to learn about processes that regulate progression of IPF, given that those processes are thought to be driven by events emanating from the FF?
      We were therefore interested to ask several outstanding important questions:
      • i)
        Can novel and valuable insight be gained by determining epigenetic signatures that are unique to FF in IPF lungs?
      • ii)
        FF almost exclusively contain fibroblasts/myofibroblasts; therefore, is it possible to understand the miRNA expression within FF by isolating fibroblasts, growing them in vitro, and assessing their miRNA content?
      • iii)
        Do fibroblasts cultured on plastic retain miRNA expression patterns that reflect the microenvironment and macroenvironment of the organ they were isolated from, or does culturing lung fibroblasts on plastic cause major alterations in miRNA expression?
      Interestingly, only five common miRNAs were found to be significantly different in FF and IPF fibroblasts: miR-145-5p, miR-4443, miR-4488, miR-4516, and miR-5100. From these miRNAs, miR-145-5p and miR-5100 have previously been described in IPF.
      • Li H.
      • Zhao X.
      • Shan H.
      • Liang H.
      MicroRNAs in idiopathic pulmonary fibrosis: involvement in pathogenesis and potential use in diagnosis and therapeutics.
      ,
      • Perera U.E.
      • Derseh H.B.
      • Dewage S.N.V.
      • Stent A.
      • Wijayarathna R.
      • Snibson K.J.
      Evaluation of microRNA expression in a sheep model for lung fibrosis.
      • Yang S.
      • Cui H.
      • Xie N.
      • Icyuz M.
      • Banerjee S.
      • Antony V.B.
      • Abraham E.
      • Thannickal V.J.
      • Liu G.
      MiR-145 regulates myofibroblast differentiation and lung fibrosis.
      • Li C.Y.
      • Wang Y.H.
      • Lin Z.Y.
      • Yang L.W.
      • Gao S.L.
      • Liu T.
      • Zou B.A.
      • Pan Z.C.
      • Song Z.Q.
      • Liu G.
      Mir-5100 targets TOB2 to drive epithelial-mesenchymal transition associated with activating smad2/3 in lung epithelial cells.
      However, miR-4443, miR-4488, and miR-4516 have not to date been shown related to IPF.
      Previous studies identified miR-4443 as a key regulator of tumorigenesis and metastasis in a variety of tumors.
      • Ebrahimi S.O.
      • Reiisi S.
      Downregulation of miR-4443 and miR-5195-3p in ovarian cancer tissue contributes to metastasis and tumorigenesis.
      • Meerson A.
      • Yehuda H.
      Leptin and insulin up-regulate miR-4443 to suppress NCOA1 and TRAF4, and decrease the invasiveness of human colon cancer cells.
      • Li M.
      • Zhang X.
      • Ding X.
      • Zheng Y.
      • Du H.
      • Li H.
      • Ji H.
      • Wang Z.
      • Jiao P.
      • Song X.
      • Zhong Y.
      • Wu H.M.
      Long noncoding RNA LINC00460 promotes cell progression by sponging miR-4443 in head and neck squamous cell carcinoma.
      • Gao Y.
      • Xu Y.
      • Wang J.
      • Yang X.
      • Wen L.
      • Feng J.
      Lncrna mnx1-as1 promotes glioblastoma progression through inhibition of mir-4443.
      In particular, miR-4443 up-regulation in lung has been related to chemotherapy resistance in patients with non–small-cell lung cancer.
      • Song Z.
      • Jia G.
      • Ma P.
      • Cang S.
      Exosomal miR-4443 promotes cisplatin resistance in non-small cell lung carcinoma by regulating FSP1 m6A modification-mediated ferroptosis.
      ,
      • Zhang W.
      • Qiao B.
      • Fan J.
      Overexpression of miR-4443 promotes the resistance of non-small cell lung cancer cells to epirubicin by targeting INPP4A and regulating the activation of JAK2/STAT3 pathway.
      The results of Zhang et al
      • Zhang W.
      • Qiao B.
      • Fan J.
      Overexpression of miR-4443 promotes the resistance of non-small cell lung cancer cells to epirubicin by targeting INPP4A and regulating the activation of JAK2/STAT3 pathway.
      suggested that the overexpression of miR-4443 promoted the resistance to epirubicin-based chemotherapy of non–small-cell lung cancer cells via the activation of the JAK2/STAT3 pathway. Moreover, Chowdhari and Saini
      • Chowdhari S.
      • Saini N.
      hsa-miR-4516 Mediated downregulation of STAT3/CDK6/UBE2N plays a role in PUVA induced apoptosis in keratinocytes.
      identified miR-4516 as negative regulator of STAT3 in cultured human keratinocytes. Interestingly, the JAK2/STAT3 pathway has been associated with fibrosis, including pulmonary fibrosis.
      • She Y.X.
      • Yu Q.Y.
      • Tang X.X.
      Role of interleukins in the pathogenesis of pulmonary fibrosis.
      • Montero P.
      • Milara J.
      • Roger I.
      • Cortijo J.
      Role of jak/stat in interstitial lung diseases; molecular and cellular mechanisms.
      • Shi K.
      • Jiang J.
      • Ma T.
      • Xie J.
      • Duan L.
      • Chen R.
      • Song P.
      • Yu Z.
      • Liu C.
      • Zhu Q.
      • Zheng J.
      Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-β, Smad3 and JAK-STAT pathway.
      Our results show miR-4443 and miR-4516 to be up-regulated in both FF and IPF fibroblasts.
      Up-regulation of miR-4488 was observed in the exosome of patients with dermatomyositis complicated with interstitial lung diseases before treatment, when compared with patients with dermatomyositis without prior interstitial lung disease complications. Bioinformatic analysis of the putative miR-4488 targets suggested that miR-4488 may contribute to systemic inflammation in patients with dermatomyositis complicated with interstitial lung diseases.
      • Zhong D.
      • Wu C.
      • Xu D.
      • Bai J.
      • Wang Q.
      • Zeng X.
      Plasma-derived exosomal hsa-miR-4488 and hsa-miR-1228-5p: novel biomarkers for dermatomyositis-associated interstitial lung disease with anti-melanoma differentiation-associated protein 5 antibody-positive subset.
      However, miR-4488 implication in IPF has not been described.
      Interestingly, 38 of 43 miRNAs were found to be expressed only in FF, but not in IPF, fibroblasts, either with or without TGF-β1 stimulation. From these 38 miRNAs, 25 have been already described in IPF (Table 2
      • Li H.
      • Zhao X.
      • Shan H.
      • Liang H.
      MicroRNAs in idiopathic pulmonary fibrosis: involvement in pathogenesis and potential use in diagnosis and therapeutics.
      ,
      • Perera U.E.
      • Derseh H.B.
      • Dewage S.N.V.
      • Stent A.
      • Wijayarathna R.
      • Snibson K.J.
      Evaluation of microRNA expression in a sheep model for lung fibrosis.
      • Yang S.
      • Cui H.
      • Xie N.
      • Icyuz M.
      • Banerjee S.
      • Antony V.B.
      • Abraham E.
      • Thannickal V.J.
      • Liu G.
      MiR-145 regulates myofibroblast differentiation and lung fibrosis.
      • Li C.Y.
      • Wang Y.H.
      • Lin Z.Y.
      • Yang L.W.
      • Gao S.L.
      • Liu T.
      • Zou B.A.
      • Pan Z.C.
      • Song Z.Q.
      • Liu G.
      Mir-5100 targets TOB2 to drive epithelial-mesenchymal transition associated with activating smad2/3 in lung epithelial cells.
      ,
      • Kaur G.
      • Maremanda K.P.
      • Campos M.
      • Chand H.S.
      • Li F.
      • Hirani N.
      • Haseeb M.A.
      • Li D.
      • Rahman I.
      Distinct exosomal miRNA profiles from BALF and lung tissue of COPD and IPF patients.
      • McDonough J.E.
      • Ahangari F.
      • Li Q.
      • Jain S.
      • Verleden S.E.
      • Maya J.H.
      • Vukmirovic M.
      • DeIuliis G.
      • Tzouvelekis A.
      • Tanabe N.
      • Chu F.
      • Yan X.
      • Verschakelen J.
      • Homer R.J.
      • Manatakis Dv
      • Zhang J.
      • Ding J.
      • Maes K.
      • de Sadeleer L.
      • Vos R.
      • Neyrinck A.
      • Benos Pv
      • Joseph Z.B.
      • Tantin D.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      • Oak S.R.
      • Murray L.
      • Herath A.
      • Sleeman M.
      • Anderson I.
      • Joshi A.D.
      • Coelho A.L.
      • Flaherty K.R.
      • Toews G.B.
      • Knight D.
      • Martinez F.J.
      • Hogaboam C.M.
      A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis.
      • Ogawa T.
      • Enomoto M.
      • Fujii H.
      • Sekiya Y.
      • Yoshizato K.
      • Ikeda K.
      • Kawada N.
      MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis.
      • Wang X.
      • Wang J.
      • Huang G.
      • Li Y.
      • Guo S.
      miR-320a-3P alleviates the epithelial-mesenchymal transition of A549 cells by activation of STAT3/SMAD3 signaling in a pulmonary fibrosis model.
      • Wang Y.C.
      • Liu J.S.
      • Tang H.K.
      • Nie J.
      • Zhu J.X.
      • Wen L.L.
      • Guo Q.L.
      MiR?221 targets HMGA2 to inhibit bleomycin induced pulmonary fibrosis by regulating TGF1/Smad3-induced EMT.
      • Long X.R.
      • Zhang Y.J.
      • Zhang M.Y.
      • Chen K.
      • Zheng X.F.S.
      • Wang H.Y.
      Identification of an 88-microRNA signature in whole blood for diagnosis of hepatocellular carcinoma and other chronic liver diseases.
      • Dirol H.
      • Toylu A.
      • Ogus A.C.
      • Cilli A.
      • Ozbudak O.
      • Clark O.A.
      • Ozdemir T.
      Alterations in plasma miR-21, miR-590, miR-192 and miR-215 in idiopathic pulmonary fibrosis and their clinical importance.
      • Wang Z.
      • Zhao Y.
      • Zhao H.
      • Zhou J.
      • Feng D.
      • Tang F.
      • Li Y.
      • Lv L.
      • Chen Z.
      • Ma X.
      • Tian X.
      • Yao J.
      Inhibition of p66Shc oxidative signaling via CA-induced upregulation of miR-203a-3p alleviates liver fibrosis progression.
      • Li P.
      • Li J.
      • Chen T.
      • Wang H.
      • Chu H.
      • Chang J.
      • Zang W.
      • Wang Y.
      • Ma Y.
      • Du Y.
      • Zhao G.
      • Zhang G.
      Expression analysis of serum microRNAs in idiopathic pulmonary fibrosis.
      • Xie T.
      • Liang J.
      • Guo R.
      • Liu N.
      • Noble P.W.
      • Jiang D.
      Comprehensive microRNA analysis in bleomycin-induced pulmonary fibrosis identifies multiple sites of molecular regulation.
      • Cui H.
      • Banerjee S.
      • Xie N.
      • Ge J.
      • Liu R.M.
      • Matalon S.
      • Thannickal V.J.
      • Liu G.
      MicroRNA-27a-3p is a negative regulator of lung fibrosis by targeting myofibroblast differentiation.
      • Disayabutr S.
      • Kim E.K.
      • Cha S.I.
      • Green G.
      • Naikawadi R.P.
      • Jones K.D.
      • Golden J.A.
      • Schroeder A.
      • Matthay M.A.
      • Kukreja J.
      • Erle D.J.
      • Collard H.R.
      • Wolters P.J.
      MIR-34 MIRNAs regulate cellular senescence in type II alveolar epithelial cells of patients with idiopathic pulmonary fibrosis.
      • Zhang S.
      • Liu H.
      • Liu Y.
      • Zhang J.
      • Li H.
      • Liu W.
      • Cao G.
      • Xv P.
      • Zhang J.
      • Lv C.
      • Song X.
      miR-30a As potential therapeutics by targeting tet1 through regulation of Drp-1 promoter hydroxymethylation in idiopathic pulmonary fibrosis.
      • Liu B.
      • Jiang T.
      • Hu X.
      • Liu Z.
      • Zhao L.
      • Liu H.
      • Liu Z.
      • Ma L.
      Downregulation of microRNA-30a in bronchoalveolar lavage fluid from idiopathic pulmonary fibrosis patients.
      • Thottakara T.
      • Lund N.
      • Krämer E.
      • Kirchhof P.
      • Carrier L.
      • Patten M.
      A novel miRNA screen identifies miRNA-4454 as a candidate biomarker for ventricular fibrosis in patients with hypertrophic cardiomyopathy.
      • Liang H.
      • Xu C.
      • Pan Z.
      • Zhang Y.
      • Xu Z.
      • Chen Y.
      • Li T.
      • Li X.
      • Liu Y.
      • Huangfu L.
      • Lu Y.
      • Zhang Z.
      • Yang B.
      • Gitau S.
      • Lu Y.
      • Shan H.
      • Du Z.
      The antifibrotic effects and mechanisms of microRNA-26a action in idiopathic pulmonary fibrosis.
      • Yang Z.
      • Peng Y.
      • Yang S.
      MicroRNA-146a regulates the transformation from liver fibrosis to cirrhosis in patients with hepatitis B via interleukin-6.
      • Ebrahimpour A.
      • Shrestha S.
      • Bonnen M.D.
      • Tony Eissa N.
      • Raghu G.
      • Ghebre Y.T.
      Nicotine modulates growth factors and microRNA to promote inflammatory and fibrotic processess.
      • Huang C.
      • Xiao X.
      • Yang Y.
      • Mishra A.
      • Liang Y.
      • Zeng X.
      • Yang X.
      • Xu D.
      • Blackburn M.R.
      • Henke C.A.
      • Liu L.
      MicroRNA-101 attenuates pulmonary fibrosis by inhibiting fibroblast proliferation and activation.
      • Yang S.
      • Banerjee S.
      • de Freitas A.
      • Sanders Y.Y.
      • Ding Q.
      • Matalon S.
      • Thannickal V.J.
      • Abraham E.
      • Liu G.
      Participation of miR-200 in pulmonary fibrosis.
      • Moimas S.
      • Salton F.
      • Kosmider B.
      • Ring N.
      • Volpe M.C.
      • Bahmed K.
      • Braga L.
      • Rehman M.
      • Vodret S.
      • Graziani M.L.
      • Wolfson M.R.
      • Marchetti N.
      • Rogers T.J.
      • Giacca M.
      • Criner G.J.
      • Zacchigna S.
      • Confalonieri M.
      MiR-200 family members reduce senescence and restore idiopathic pulmonary fibrosis type II alveolar epithelial cell transdifferentiation.
      • Sheng S.
      • Zou M.
      • Yang Y.
      • Guan M.
      • Ren S.
      • Wang X.
      • Wang L.
      • Xue Y.
      miR-23a-3p regulates the inflammatory response and fibrosis in diabetic kidney disease by targeting early growth response 1.
      • Berschneider B.
      • Ellwanger D.C.
      • Baarsma H.A.
      • Thiel C.
      • Shimbori C.
      • White E.S.
      • Kolb M.
      • Neth P.
      • Königshoff M.
      MiR-92a regulates TGF-β1-induced WISP1 expression in pulmonary fibrosis.
      • Pandit K.V.
      • Corcoran D.
      • Yousef H.
      • Yarlagadda M.
      • Tzouvelekis A.
      • Gibson K.F.
      • Konishi K.
      • Yousem S.A.
      • Singh M.
      • Handley D.
      • Richards T.
      • Selman M.
      • Watkins S.C.
      • Pardo A.
      • Ben-Yehudah A.
      • Bouros D.
      • Eickelberg O.
      • Ray P.
      • Benos Pv
      • Kaminski N.
      Inhibition and role of let-7d in idiopathic pulmonary fibrosis.
      • Lino Cardenas C.L.
      • Henaoui I.S.
      • Courcot E.
      • Roderburg C.
      • Cauffiez C.
      • Aubert S.
      • Copin M.C.
      • Wallaert B.
      • Glowacki F.
      • Dewaeles E.
      • Milosevic J.
      • Maurizio J.
      • Tedrow J.
      • Marcet B.
      • Lo-Guidice J.M.
      • Kaminski N.
      • Barbry P.
      • Luedde T.
      • Perrais M.
      • Mari B.
      • Pottier N.
      miR-199a-5p is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.
      ); however, to the extent of our knowledge, 13 have never been described as related to IPF. Among these, several miRNAs have been described in fibrotic disease in other tissues. For instance, miR-370-3p, miR-222-3p, miR-146a-5p, and miR-203a-3p, among others (Table 2), have been described in liver fibrosis; and miR-4454 and miR-23a-3p have been linked to cardiac and renal fibrosis (Table 2). Of note, miR-4448 and miR-4284 have no obvious links to IPF or fibrosis, and as such given their expression in FF, it will therefore be instructive to examine these miRNAs for potential novel functions in IPF. Altogether, these findings suggest that the use of whole organ or in vitro grown fibroblast likely masks microenvironmental epigenetic changes occurring within the FF.
      Table 2Fibroblastic Foci from IPF Lungs Compared with Whole IPF Lung Tissue
      VariablemiRNAsFibrosisReference
      Up-regulated in FFhsa-miR-122-5pIPF
      • Kaur G.
      • Maremanda K.P.
      • Campos M.
      • Chand H.S.
      • Li F.
      • Hirani N.
      • Haseeb M.A.
      • Li D.
      • Rahman I.
      Distinct exosomal miRNA profiles from BALF and lung tissue of COPD and IPF patients.
      hsa-miR-127-3pIPF
      • McDonough J.E.
      • Ahangari F.
      • Li Q.
      • Jain S.
      • Verleden S.E.
      • Maya J.H.
      • Vukmirovic M.
      • DeIuliis G.
      • Tzouvelekis A.
      • Tanabe N.
      • Chu F.
      • Yan X.
      • Verschakelen J.
      • Homer R.J.
      • Manatakis Dv
      • Zhang J.
      • Ding J.
      • Maes K.
      • de Sadeleer L.
      • Vos R.
      • Neyrinck A.
      • Benos Pv
      • Joseph Z.B.
      • Tantin D.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      hsa-miR-370-3pHSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      hsa-miR-423-5pIPF
      • McDonough J.E.
      • Ahangari F.
      • Li Q.
      • Jain S.
      • Verleden S.E.
      • Maya J.H.
      • Vukmirovic M.
      • DeIuliis G.
      • Tzouvelekis A.
      • Tanabe N.
      • Chu F.
      • Yan X.
      • Verschakelen J.
      • Homer R.J.
      • Manatakis Dv
      • Zhang J.
      • Ding J.
      • Maes K.
      • de Sadeleer L.
      • Vos R.
      • Neyrinck A.
      • Benos Pv
      • Joseph Z.B.
      • Tantin D.
      • Hogg J.C.
      • Vanaudenaerde B.M.
      • Wuyts W.A.
      • Kaminski N.
      Transcriptional regulatory model of fibrosis progression in the human lung.
      ,
      • Oak S.R.
      • Murray L.
      • Herath A.
      • Sleeman M.
      • Anderson I.
      • Joshi A.D.
      • Coelho A.L.
      • Flaherty K.R.
      • Toews G.B.
      • Knight D.
      • Martinez F.J.
      • Hogaboam C.M.
      A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis.
      hsa-miR-222-3pHSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      ,
      • Ogawa T.
      • Enomoto M.
      • Fujii H.
      • Sekiya Y.
      • Yoshizato K.
      • Ikeda K.
      • Kawada N.
      MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis.
      hsa-miR-4488
      hsa-miR-320aIPF
      • Wang X.
      • Wang J.
      • Huang G.
      • Li Y.
      • Guo S.
      miR-320a-3P alleviates the epithelial-mesenchymal transition of A549 cells by activation of STAT3/SMAD3 signaling in a pulmonary fibrosis model.
      hsa-miR-7641HSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      hsa-miR-1246HSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      hsa-let-7e-5pHSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      hsa-miR-221-3pIPF
      • Wang Y.C.
      • Liu J.S.
      • Tang H.K.
      • Nie J.
      • Zhu J.X.
      • Wen L.L.
      • Guo Q.L.
      MiR?221 targets HMGA2 to inhibit bleomycin induced pulmonary fibrosis by regulating TGF1/Smad3-induced EMT.
      hsa-miR-4508HSC and cirrhosis
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      ,
      • Long X.R.
      • Zhang Y.J.
      • Zhang M.Y.
      • Chen K.
      • Zheng X.F.S.
      • Wang H.Y.
      Identification of an 88-microRNA signature in whole blood for diagnosis of hepatocellular carcinoma and other chronic liver diseases.
      hsa-miR-4516
      hsa-miR-4497HSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
      • Mann D.A.
      • Mann J.
      RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.
      hsa-miR-192-5pIPF
      • Dirol H.
      • Toylu A.
      • Ogus A.C.
      • Cilli A.
      • Ozbudak O.
      • Clark O.A.
      • Ozdemir T.
      Alterations in plasma miR-21, miR-590, miR-192 and miR-215 in idiopathic pulmonary fibrosis and their clinical importance.
      hsa-miR-4443
      hsa-miR-203a-3pHSC
      • Sabater L.
      • Locatelli L.
      • Oakley F.
      • Hardy T.
      • French J.
      • Robinson S.M.
      • Sen G.
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      Transcriptional regulatory model of fibrosis progression in the human lung.
      hsa-miR-145-5pIPF
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      MicroRNAs in idiopathic pulmonary fibrosis: involvement in pathogenesis and potential use in diagnosis and therapeutics.
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      List includes highlighted miRNAs that are significant in FF, primary fibroblasts, and/or primary fibroblasts + transforming growth factor-β1.
      FF, fibroblastic foci; IPF, idiopathic pulmonary fibrosis.
      In summary, by analyzing miRNA expression specifically in FF, we were able to identify up to 38 miRNAs that were exclusively expressed in FF, which could potentially contribute to IPF development and progression. Future studies into biology of IPF should utilize platforms that better reflect the distinct biology of the FF tissue niche, such as precision cut IPF lung slices that may be a more suitable preclinical model that closely recapitulates in vivo environment of diseased lung.

      Author Contributions

      J.B.G. performed most of the laboratory-based work, and L.S. performed the bioinformatics analyses presented in the manuscript; G.A. and A.C. performed additional laboratory-based work; I.H. and D.R. provided guidance and performed a proportion of the bioinformatics analyses; D.A.M., A.J.F., A.B., J.Maj, K.J., F.O., and L.A.B. provided advice, human tissue samples, and/or contributed to the experimental design and writing; and J.Man conceived the studies, designed the experiments, and wrote the manuscript. All authors read and commented on the final manuscript.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Enrichment Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the top 25 most expressed miRNAs in fibrotic foci and cultured fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) lung. A: KEGG pathways associated with 14 common miRNAs of the top 25 most expressed found in fibrotic foci (blue) and in cultured fibroblasts isolated from IPF lungs (red). The x axis shows the –log10 enrichment P value, whereas the y axis shows the different KEGG pathways. B: KEGG pathways associated with the unique 11 miRNAs of the top 25 found in fibrotic foci. The x axis shows the –log10 enrichment P value, whereas the y axis shows the different KEGG pathways. C: KEGG pathways associated with unique 11 miRNAs found in isolated fibroblasts of the top 25. The x axis shows the –log10 enrichment P value, whereas the y axis shows the different KEGG pathways. ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase.

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