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

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be 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 was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared 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 those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted 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. miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be 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 was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared 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 those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted 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 100,000.1Hutchinson J. Fogarty A. Hubbard R. McKeever T. Global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review.Eur Respir J. 2015; 46: 795-806Crossref PubMed Scopus (543) Google Scholar 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), it appears to be increasing steadily. IPF has a poor prognosis, with a median survival of 2 to 4 years from diagnosis, making post-diagnosis survival worse than many cancers.2Ley B. Collard H.R. King T.E. Clinical course and prediction of survival in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2011; 183: 431-440Crossref PubMed Scopus (1170) Google Scholar The etiology of IPF remains unclear, but growing evidence points towards complex interactions between genetic risk factors and environmental insults on a background of age-associated predisposition as the key contributors.3Fingerlin T.E. Murphy E. Zhang W. Peljto A.L. Brown K.K. Steele M.P. et al.Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis.Nat Genet. 2013; 45: 613-620Crossref PubMed Scopus (553) Google Scholar, 4Molyneaux P.L. Cox M.J. Willis-Owen S.A.G. Mallia P. Russell K.E. Russell A.M. Murphy E. Johnston S.L. Schwartz D.A. Wells A.U. Cookson W.O.C. Maher T.M. Moffatt M.F. The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2014; 190: 906-913Crossref PubMed Scopus (377) Google Scholar, 5Alder J.K. Chen J.J.L. Lancaster L. Danoff S. Su S.C. Cogan J.D. Vulto I. Xie M. Qi X. Tuder R.M. Phillips J.A. Lansdorp P.M. Loyd J.E. Armanios M.Y. Short telomeres are a risk factor for idiopathic pulmonary fibrosis.Proc Natl Acad Sci U S A. 2008; 105: 13051-13056Crossref PubMed Scopus (589) Google Scholar Treatment options for IPF are limited and are predominately palliative. 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.6King T.E. Bradford W.Z. Castro-Bernardini S. Fagan E.A. Glaspole I. Glassberg M.K. Gorina E. Hopkins P.M. Kardatzke D. Lancaster L. Lederer D.J. Nathan S.D. Pereira C.A. Sahn S.A. Sussman R. Swigris J.J. Noble P.W. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2083-2092Crossref PubMed Scopus (2597) Google Scholar,7Richeldi L. du Bois R.M. Raghu G. Azuma A. Brown K.K. Costabel U. Cottin V. Flaherty K.R. Hansell D.M. Inoue Y. Kim D.S. Kolb M. Nicholson A.G. Noble P.W. Selman M. Taniguchi H. Brun M. Le Maulf F. Girard M. Stowasser S. Schlenker-Herceg R. Disse B. Collard H.R. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis.N Engl J Med. 2014; 370: 2071-2082Crossref PubMed Scopus (2939) Google Scholar Lung transplantation is the only option that offers hope for long-term survival, but it is only available to highly selected individuals.8Kistler K.D. Nalysnyk L. Rotella P. Esser D. Lung transplantation in idiopathic pulmonary fibrosis: a systematic review of the literature.BMC Pulm Med. 2014; 14: 139Crossref PubMed Scopus (99) Google Scholar 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.9Jones M.G. Fabre A. Schneider P. Cinetto F. Sgalla G. Mavrogordato M. Jogai S. Alzetani A. Marshall B.G. O'Reilly K.M.A. Warner J.A. Lackie P.M. Davies D.E. Hansell D.M. Nicholson A.G. Sinclair I. Brown K.K. Richeldi L. Three-dimensional characterization of fibroblast foci in idiopathic pulmonary fibrosis.JCI Insight. 2016; 1: e86375Crossref PubMed Google Scholar,10Horowitz J.C. Thannickal V.J. Epithelial-mesenchymal interactions in pulmonary fibrosis.Semin Respir Crit Care Med. 2006; 27: 600-612Crossref PubMed Scopus (100) Google Scholar The foci represent discrete sites of lung injury and repair and are 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.11Zou X.Z. Liu T. Gong Z.C. Hu C.P. Zhang Z. MicroRNAs-mediated epithelial-mesenchymal transition in fibrotic diseases.Eur J Pharmacol. 2017; 796: 190-206Crossref PubMed Scopus (53) Google Scholar The altered levels of miRNAs have been investigated in several fibrotic tissues in both animal models and human disease and have been associated with fibrosis progression.12Li H. Zhao X. Shan H. Liang H. MicroRNAs in idiopathic pulmonary fibrosis: involvement in pathogenesis and potential use in diagnosis and therapeutics.Acta Pharm Sin B. 2016; 6: 531-539Crossref PubMed Scopus (41) Google Scholar 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.13Pandit K.V. Milosevic J. MicroRNA regulatory networks in idiopathic pulmonary fibrosis1.Biochem Cell Biol. 2015; 93: 129-137Crossref PubMed Scopus (61) Google Scholar 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. This study focused on improving 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 whether the process of culturing cells on plastic altered the miRNA levels. Combining these approaches, the study uncovered novel pathways operating within FF that have not previously been described. 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 (J.Maj.. and K.J.) and later confirmed and cut out using LCM by pathology-trained technician. 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. The FF were cut from the IPF tissue sections using Zeiss Laser Capture Microdissection Microscope (Zeiss, Carl Zeiss Microscopy GmbH, Jena, 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. 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. Lung fibroblasts were isolated from donor-matched IPF lung tissue that was used for LCM and grown on plastic. Five fibroblast lines were further 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). FASTQ files obtained from a run on MiSeq were trimmed, size selected, and mapped with ChimiRa release 1.014Vitsios D.M. Enright A.J. Chimira: analysis of small RNA sequencing data and microRNA modifications.Bioinformatics. 2015; 31: 3365-3367Crossref PubMed Scopus (83) Google Scholar (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.14Vitsios D.M. Enright A.J. Chimira: analysis of small RNA sequencing data and microRNA modifications.Bioinformatics. 2015; 31: 3365-3367Crossref PubMed Scopus (83) Google Scholar Counts were analyzed using R statistics (https://www.r-project.org) and normalized using DESeq2 package for R version 3.0.115Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (38077) Google Scholar; 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 3.0.1 (https://cran.r-project.org/web/packages/gplots/gplots.pdf). 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). To determine the miRNA content of FF, histologic sections of explanted IPF lungs were obtained 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, 50 to 200 foci were identified and dissected out 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). RNA isolated from matched whole IPF lung tissue was sequenced next and a direct comparison of miRNA signatures in the LCM isolated FF from the same lung was made, 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, 20 miRNAs were 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 mitogen-activated protein kinase kinase 1 and 2 (MAP2K1/2), extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. Moreover, miRNAs present in the whole lung were related to regulation of multiple features, such as ADAM metallopeptidase with thrombospondin type 1 motif 14 (ADAMTS14) and ADAMTS15 peptidases, the transcription regulator protein atonal homolog (ATOH8), transmembrane proteins [tetraspanin (TSPAN13) and transmembrane protein 8B (TMEM8B)], insulin, chorionic gonadotropin hormone, estrogen receptor, and other features [protein tyrosine phosphatase non-receptor type 7 (PTPN7), long intergenic non-coding RNA gene FAM110C, long non-coding RNA molecule TNXA-PS1, l-gulono-gamma-lactone oxidase (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 such as TGF-β, which are classically associated with tissue fibrosis, were activated, and also identified S100A12 and tetrahydromethanopterin:alpha-l-glutamate ligase (MTPN). Serum levels of S100A12 are negatively associated with lung function in systemic scleroderma16Omatsu 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.Exp Dermatol. 2021; 30: 409-415Crossref PubMed Scopus (7) Google Scholar and IPF,17Li 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.Front Immunol. 2022; 13: 810338Crossref PubMed Scopus (5) Google Scholar whereas MTPN is reported to convert p65:p50 heterodimers to repressive p50:p50 homodimers, thus differentially regulating NF-κB target genes.18Knuefermann 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.J Biol Chem. 2002; 277: 23888-23897Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Moreover, other features, such as hepatocellular carcinoma up-regulated EZH2-associated long non-coding RNA (HEIH), kinase epoxide hydrolase B6 (EPHB6), calcifediol, synaptophysin-like protein 1 (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 CharacteristicsIdentifierSexAge, yearsFEV1, LFVC, LTLC, LTLCO, mmol CO/min/kPaKCO, mmol CO/min/kPaPack years80M541.97 (52%)2.38 (50%)4.02 (54%)3.08 (29%)0.87 (61%)27M562.37 (61%)2.9 (59%)4.84 (63%)2.9 (25%)0.7 (49%)Nil70M621.43 (51%)1.79 (51%)2.73 (45%)2.12 (26%)0.85 (63%)Nil45M442.6 (54%)2.29 (50%)3.27 (47%)3.82 (36%)1.31 (86%)Nil73M541.86 (49%)2.2 (47%)3.44 (47%)3.36 (31%)1.18 (81%)30, Stopped in 200637M621.58 (48%)2.13 (37%)4.43 (63%)2.31 (24%)0.63 (46%)Nil88M491.83 (51%)2.87 (65%)5.50 (81%)4.6 (46%)1.1 (73%)Nil90M622.07 (64%)2.49 (61%)3.54 (52%)3.59 (39%)1.10 (81%)Nil91M481.74 (50%)2.08 (49%)3.04 (46%)2.26 (23%)1.03 (69%)10Percentages of predicted values are in parentheses.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. Open table in a new tab 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; right side) and matched whole IPF lung tissue (red; 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. Significantly overexpressed miRNAs in fibroblastic foci are indicated in blue. Significantly overexpressed miRNAs in whole IPF lung are indicated in red. 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 indicates unique miRNAs in fibroblastic foci, pale blue indicates unique miRNAs in whole IPF tissue, and overlapping area indicates miRNAs found in common in both FF and whole IPF tissue. LCM, laser capture microdissection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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). Underexpressed and overexpressed miRNAs are in green and red, respectively. Prediction of activation and inhibition is in orange and blue, respectively. Molecules related to miRNAs for which no prediction of directional change could be made are indicated in white. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Percentages of predicted values are in parentheses. 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. 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. Whether it was possible to determine the miRNA expression within FF by obtaining the miRNA profile of in vitro grown fibroblasts isolated from IPF lungs, given that FF predominantly contain fibroblasts/myofibroblasts, was studied next. To this end, fibroblasts were isolated from the same IPF donor lungs used for isolation of FF by LCM, and the miRNA profiles of in vitro grown cells were compared with LCM generated FF miRNAs. Of the 25 most abundant miRNA species in the two groups, this comparison identified 11 miRNAs exclusively in FF and 11 unique miRNAs in cultured fibroblasts (Figure 4). These data were used to identify the Kyoto Encyclopedia of Genes and Genomes pathways regulated by the miRNAs (Supplemental Figure S1). Although a significant number of Kyoto Encyclopedia of Genes and Genomes pathways affected by miRNAs were found in both FF and culture-grown fibroblasts (Supplemental Figure S1A), 10 pathways regulated by miRNAs were unique to FF (Supplemental Figure S1B) and an additional 7 Kyoto Encyclopedia of Genes and Genomes pathways mapped to miRNAs were identified solely in IPF fibroblasts grown in vitro (Supplemental Figure S1C). Ingenuity Pathway Analysis of the 25 most differentially expressed miRNAs indicated putative functions in several signaling pathways unique to FF (Figure 5). In summary, caution should be taken to interpret the data related to the biology of cultured IPF fibroblasts with respect to events occurring in the FF tissue microenvironment because of the dynamic loss of cell heterogeneity and plasticity.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. Underexpressed and overexpressed miRNAs are indicated in green and red, respectively. Prediction of activation and inhibition is indicated in orange and blue, respectively. Molecules related to miRNAs for which no prediction of directional change could be made are indicated in white. Lines represent different relationships between the molecules and miRNAs, as stated in the legend.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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, whether fibroblasts cultured on plastic retain miRNA expression patterns that reflect the microenvironment and macroenvironment of the organ they were isolated from was studied next. To this end, fibroblasts from IPF lungs and/or normal lungs were isolated and cultured, and the expression profiles of miRNAs within the in vitro grown cells were compared (Figure 6A). Five miRNAs were significantly overexpressed in the normal lung fibroblasts (Supplemental Table S2 and Figure 6B) and 15 miRNAs were 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, 141 miRNAs were found to be 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 [hypoxia inducible factor 1 subunit alpha (HIF1A), hepatocyte nuclear factor 4 alpha (HNF4A), Y-box binding protein 1 (YBX1), and forkhead box O1 (FOXO1)], the translation regulator eukaryotic translation initiation factor 4E binding protein 2 (EIF4EBP2), other miRNAs (miR-1275, miR-663, miR-1908, miR-342, miR-432, and miR-154), solute carrier family 1 member 4 (SLC1A4) and SLC25A32 transporters, Gulo enzyme, cytokine (tumor necrosis factor and erythropoietin), EPHB6 kinase, insulin, roundabout guidance receptor 4 (ROBO4), and other features [taurine up-regulated 1 (TUG1); Fascin; Snhg14; visinin like 1 (VSNL1); FTX transcript, XIST regulator (FTX); and collagen type 27 alpha 1 (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 were significantly repressed compared with the fibroblasts from normal lungs treated with TGF-β1 (Supplemental Table S3 and Figure 8B). Closer inspection revealed 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, insulin like growth factor 1 (IGF1), several enzymes [methionyl-TRNA synthetase 2, mitochondrial (MARS2); thymidylate synthetase (TYMS); tripartite motif containing 71 (TRIM71); spermine oxidase (SMOX); high mobility group AT-hook 2 (Hmga2); GTP binding protein 3, mitochondrial (GTPBP3); RNA 3′-terminal phosphate cyclase (RTCA); and proline dehydrogenase 1 (PRODH)], the transcription regulator MYC proto-oncogene, BHLH transcription factor (MYC), calcifediol, and other molecules [interferon-induced protein with tetratricopeptide repeats 5 (IFIT5); RNA binding motif protein 19 (RBM19); COMM domain containing 9 (COMMD9); cytosolic iron-sulfur assembly component 2A (CIAO2A); COL5A2; and capping actin protein, gelsolin like (CAPG)] (Figure 9B). These data further confirm that the in vitro culture of fibroblasts is a potent epigenetic remodele