Idiopathic pulmonary fibrosis (IPF) is a complex disease in which a multitude of proteins and networks are disrupted. Interrogation of the transcriptome through RNA sequencing (RNA-Seq) enables the determination of genes whose differential expression is most significant in IPF, as well as the detection of alternative splicing events which are not easily observed with traditional microarray experiments. We sequenced messenger RNA from 8 IPF lung samples and 7 healthy controls on an Illumina HiSeq 2000, and found evidence for substantial differential gene expression and differential splicing. 873 genes were differentially expressed in IPF (FDR<5%), and 440 unique genes had significant differential splicing events in at least one exonic region (FDR<5%). We used qPCR to validate the differential exon usage in the second and third most significant exonic regions, in the genes COL6A3 (RNA-Seq adjusted pval = 7.18e-10) and POSTN (RNA-Seq adjusted pval = 2.06e-09), which encode the extracellular matrix proteins collagen alpha-3(VI) and periostin. The increased gene-level expression of periostin has been associated with IPF and its clinical progression, but its differential splicing has not been studied in the context of this disease. Our results suggest that alternative splicing of these and other genes may be involved in the pathogenesis of IPF. We have developed an interactive web application which allows users to explore the results of our RNA-Seq experiment, as well as those of two previously published microarray experiments, and we hope that this will serve as a resource for future investigations of gene regulation in IPF.
References
[1]
Mushiroda T, Wattanapokayakit S, Takahashi A, Nukiwa T, Kudoh S, et al. (2008) A genome-wide association study identifies an association of a common variant in TERT with susceptibility to idiopathic pulmonary fibrosis. J Med Genet 45: 654–656. doi: 10.1136/jmg.2008.057356
[2]
Seibold MA, Wise AL, Speer MC, Steele MP, Brown KK, et al. (2011) A common MUC5B promoter polymorphism and pulmonary fibrosis. N Engl J Med 364: 1503–1512. doi: 10.1056/nejmoa1013660
[3]
Chang W, Wei K, Jacobs SS, Upadhyay D, Weill D, et al. (2010) SPARC suppresses apoptosis of idiopathic pulmonary fibrosis fibroblasts through constitutive activation of beta-catenin. J Biol Chem 285: 8196–8206. doi: 10.1074/jbc.m109.025684
Fingerlin TE, Murphy E, Zhang W, Peljto AL, Brown KK, et al. (2013) Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis. Nat Genet 45: 613–620. doi: 10.1038/ng.2609
[6]
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biology 11: R106. doi: 10.1186/gb-2010-11-10-r106
[7]
Meltzer EB, Barry WT, D’Amico TA, Davis RD, Lin SS, et al. (2011) Bayesian probit regression model for the diagnosis of pulmonary fibrosis: proof-of-principle. BMC Med Genomics 4: 70. doi: 10.1186/1755-8794-4-70
[8]
Yang IV, Coldren CD, Leach SM, Seibold MA, Murphy E, et al. (2013) Expression of cilium-associated genes defines novel molecular subtypes of idiopathic pulmonary fibrosis. Thorax 68: 1114–1121. doi: 10.1136/thoraxjnl-2012-202943
[9]
Durinck S, Moreau Y, Kasprzyk A, Davis S, De Moor B, et al. (2005) BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis. Bioinformatics 21: 3439–3440. doi: 10.1093/bioinformatics/bti525
[10]
Goeman JJ, Buhlmann P (2007) Analyzing gene expression data in terms of gene sets: method-ological issues. Bioinformatics 23: 980–987. doi: 10.1093/bioinformatics/btm051
Bromberg KD, Kluger HM, Delaunay A, Abbas S, DiVito KA, et al. (2007) Increased expression of the E3 ubiquitin ligase RNF5 is associated with decreased survival in breast cancer. Cancer Res 67: 8172–8179. doi: 10.1158/0008-5472.can-07-0045
[13]
Didier C, Broday L, Bhoumik A, Israeli S, Takahashi S, et al. (2003) RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization. Mol Cell Biol 23: 5331–5345. doi: 10.1128/mcb.23.15.5331-5345.2003
[14]
Englert JM, Hanford LE, Kaminski N, Tobolewski JM, Tan RJ, et al. (2008) A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis. Am J Pathol 172: 583–591. doi: 10.2353/ajpath.2008.070569
[15]
Queisser MA, Kouri FM, Konigshoff M, Wygrecka M, Schubert U, et al. (2008) Loss of RAGE in pulmonary fibrosis: molecular relations to functional changes in pulmonary cell types. Am J Respir Cell Mol Biol 39: 337–345. doi: 10.1165/rcmb.2007-0244oc
[16]
Shirasawa M, Fujiwara N, Hirabayashi S, Ohno H, Iida J, et al. (2004) Receptor for advanced glycation end-products is a marker of type I lung alveolar cells. Genes Cells 9: 165–174. doi: 10.1111/j.1356-9597.2004.00712.x
[17]
Endris V, Wogatzky B, Leimer U, Bartsch D, Zatyka M, et al. (2002) The novel Rho-GTPase activating gene MEGAP/srGAP3 has a putative role in severe mental retardation. Proc Natl Acad Sci USA 99: 11754–11759. doi: 10.1073/pnas.162241099
[18]
Yang Y, Marcello M, Endris V, Saffrich R, Fischer R, et al. (2006) MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation. Exp Cell Res 312: 2379–2393. doi: 10.1016/j.yexcr.2006.04.001
[19]
Alcorn JF, van der Velden J, Brown AL, McElhinney B, Irvin CG, et al. (2009) c-Jun N-terminal kinase 1 is required for the development of pulmonary fibrosis. Am J Respir Cell Mol Biol 40: 422–432. doi: 10.1165/rcmb.2008-0174oc
[20]
Yoshida K, Kuwano K, Hagimoto N, Watanabe K, Matsuba T, et al. (2002) MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis. J Pathol 198: 388–396. doi: 10.1002/path.1208
[21]
Shi-Wen X, Rodriguez-Pascual F, Lamas S, Holmes A, Howat S, et al. (2006) Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pul-monary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors. Mol Cell Biol 26: 5518–5527. doi: 10.1128/mcb.00625-06
[22]
Lee MH, Padmashali R, Koria P, Andreadis ST (2011) JNK regulates binding of alpha-catenin to adherens junctions and cell-cell adhesion. FASEB J 25: 613–623. doi: 10.1096/fj.10-161380
[23]
Moeller MJ, Soofi A, Braun GS, Li X, Watzl C, et al. (2004) Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization. EMBO J 23: 3769–3779. doi: 10.1038/sj.emboj.7600380
[24]
Valletta D, Czech B, Thasler WE, Muller M, Bosserhoff AK, et al. (2012) Expression and function of the atypical cadherin FAT1 in chronic liver disease. Biochem Biophys Res Commun 426: 404–408. doi: 10.1016/j.bbrc.2012.08.104
Lian H, Ma Y, Feng J, Dong W, Yang Q, et al. (2012) Heparin-binding EGF-like growth factor induces heart interstitial fibrosis via an Akt/mTor/p70s6k pathway. PLoS ONE 7: e44946. doi: 10.1371/journal.pone.0044946
[27]
Means AL, Ray KC, Singh AB, Washington MK, Whitehead RH, et al. (2003) Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia. Gastroenterology 124: 1020–1036. doi: 10.1053/gast.2003.50150
[28]
Bonner JC (2010) Mesenchymal cell survival in airway and interstitial pulmonary fibrosis. Fibro-genesis Tissue Repair 3: 15. doi: 10.1186/1755-1536-3-15
[29]
Yagi H, Yotsumoto F, Miyamoto S (2008) Heparin-binding epidermal growth factor-like growth factor promotes transcoelomic metastasis in ovarian cancer through epithelial-mesenchymal tran-sition. Mol Cancer Ther 7: 3441–3451. doi: 10.1158/1535-7163.mct-08-0417
[30]
Poumay Y, de Rouvroit CL (2012) HB-EGF, the growth factor that accelerates keratinocyte mi-gration, but slows proliferation. J Invest Dermatol 132: 2129–2130. doi: 10.1038/jid.2012.225
[31]
Faull RJ, Stanley JM, Fraser S, Power DA, Leavesley DI (2001) HB-EGF is produced in the peritoneal cavity and enhances mesothelial cell adhesion and migration. Kidney Int 59: 614–624. doi: 10.1046/j.1523-1755.2001.059002614.x
Anders S, Reyes A, Huber W (2012) Detecting differential usage of exons from RNA-seq data. Genome Res 22: 2008–2017. doi: 10.1101/gr.133744.111
[34]
Okamoto M, Hoshino T, Kitasato Y, Sakazaki Y, Kawayama T, et al. (2011) Periostin, a matrix protein, is a novel biomarker for idiopathic interstitial pneumonias. Eur Respir J 37: 1119–1127. doi: 10.1183/09031936.00059810
[35]
Naik PK, Bozyk PD, Bentley JK, Popova AP, Birch CM, et al. (2012) Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 303: L1046–1056. doi: 10.1152/ajplung.00139.2012
[36]
Uchida M, Shiraishi H, Ohta S, Arima K, Taniguchi K, et al. (2012) Periostin, a matricellular protein, plays a role in the induction of chemokines in pulmonary fibrosis. Am J Respir Cell Mol Biol 46: 677–686. doi: 10.1165/rcmb.2011-0115oc
[37]
Yan W, Shao R (2006) Transduction of a mesenchyme-specific gene periostin into 293T cells induces cell invasive activity through epithelial-mesenchymal transformation. J Biol Chem 281: 19700–19708. doi: 10.1074/jbc.m601856200
[38]
Michaylira CZ, Wong GS, Miller CG, Gutierrez CM, Nakagawa H, et al. (2010) Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer. Cancer Res 70: 5281–5292. doi: 10.1158/0008-5472.can-10-0704
[39]
Bozyk PD, Bentley JK, Popova AP, Anyanwu AC, Linn MD, et al. (2012) Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication. PLoS ONE 7: e31336. doi: 10.1371/journal.pone.0031336
[40]
Morra L, Rechsteiner M, Casagrande S, von Teichman A, Schraml P, et al. (2012) Characterization of periostin isoform pattern in non-small cell lung cancer. Lung Cancer 76: 183–190. doi: 10.1016/j.lungcan.2011.10.013
[41]
Hoersch S, Andrade-Navarro MA (2010) Periostin shows increased evolutionary plasticity in its alternatively spliced region. BMC Evol Biol 10: 30. doi: 10.1186/1471-2148-10-30
[42]
Morra L, Moch H (2011) Periostin expression and epithelial-mesenchymal transition in cancer: a review and an update. Virchows Arch 459: 465–475. doi: 10.1007/s00428-011-1151-5
[43]
Aumailley M, Mann K, von der Mark H, Timpl R (1989) Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains. Exp Cell Res 181: 463–474. doi: 10.1016/0014-4827(89)90103-1
[44]
Arafat H, Lazar M, Salem K, Chipitsyna G, Gong Q, et al. (2011) Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer. Surgery 150: 306–315. doi: 10.1016/j.surg.2011.05.011
[45]
Gardina PJ, Clark TA, Shimada B, Staples MK, Yang Q, et al. (2006) Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics 7: 325.
[46]
Verrecchia F, Mauviel A (2002) Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol 118: 211–215. doi: 10.1046/j.1523-1747.2002.01641.x
[47]
RStudio Inc (2013) shiny: Web Application Framework for R. Available: http://CRAN.R-project.org/package = shiny. R package version 0.7.0. Accessed 2013 Jan 1.
[48]
Deng N, Sanchez CG, Lasky JA, Zhu D (2013) Detecting splicing variants in idiopathic pulmonary fibrosis from non-differentially expressed genes. PLoS ONE 8: e68352. doi: 10.1371/journal.pone.0068352
[49]
Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, et al. (2013) NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res 41: D991–995. doi: 10.1093/nar/gks1193
[50]
Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, et al. (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38: 904–909. doi: 10.1038/ng1847
[51]
Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, DePristo MA, et al (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491: 56–65.
[52]
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81: 559–575. doi: 10.1086/519795
[53]
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15–21. doi: 10.1093/bioinformatics/bts635
[54]
Anders S, Reyes A, Huber W (2011) Analyzing RNA-seq data for differential exon usage with the DEXSeq package. Available: http://watson.nci.nih.gov/bioc\_mirror/packages/2.9/bioc/html/DEXSeq.h？tml.Accessed 2013 Feb 21.
[55]
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statist Soc Ser B 57: 289–300.
[56]
Smyth GK (2005) Limma: linear models for microarray data. In: Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W, editors, Bioinformatics and Computational Biology Solutions Using R and Bioconductor, New York: Springer. 397–420.
[57]
Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3. doi: 10.2202/1544-6115.1027
[58]
Wang K, Li M, Hakonarson H (2010) Analysing biological pathways in genome-wide association studies. Nat Rev Genet 11: 843–854. doi: 10.1038/nrg2884
