Identification of common microRNA between COPD and non-small cell lung cancer through pathway enrichment analysis

BMC Genomic Data - Tập 22 Số 1 - Trang 1-14 - 2021
Fathinavid, Amirhossein1, Ghobadi, Mohadeseh Zarei2, Najafi, Ali3, Masoudi-Nejad, Ali2
1Laboratory of Systems Biology and Bioinformatics (LBB), Department of Bioinformatics, Kish International Campus, Kish Island, Iran
2Laboratory of Systems Biology and Bioinformatics (LBB), Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
3Molecular Biology Research Center, System Biology and Poisoning Institute, Tehran, Iran

Tóm tắt

Different factors have been introduced which influence the pathogenesis of chronic obstructive pulmonary disease (COPD) and non-small cell lung cancer (NSCLC). COPD as an independent factor is involved in the development of lung cancer. Moreover, there are certain resemblances between NSCLC and COPD, such as growth factors, activation of intracellular pathways, as well as epigenetic factors. One of the best approaches to understand the possible shared pathogenesis routes between COPD and NSCLC is to study the biological pathways that are activated. MicroRNAs (miRNAs) are critical biomolecules that implicate the regulation of several biological and cellular processes. As such, the main goal of this study was to use a systems biology approach to discover common dysregulated miRNAs between COPD and NSCLC, one that targets most genes within common enriched pathways. To reconstruct the miRNA-pathways for each disease, we used the microarray miRNA expression data. Then, we employed “miRNA set enrichment analysis” (MiRSEA) to identify the most significant joint miRNAs between COPD and NSCLC based on the enrichment scores. Overall, our study revealed the involvement of the targets of miRNAs (such as has-miR-15b, hsa-miR-106a, has-miR-17, has-miR-103, and has-miR-107) in the most important common biological pathways. According to the promising results of the pathway analysis, the identified miRNAs can be utilized as the new potential signatures for therapy through understanding the molecular mechanisms of both diseases.

Tài liệu tham khảo

citation_journal_title=Am J Respir Crit Care Med; citation_title=Global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease 2017 report; citation_author=CF Vogelmeier, GJ Criner, FJ Martinez, A Anzueto, PJ Barnes, J Bourbeau, BR Celli, R Chen, M Decramer, LM Fabbri, P Frith, DMG Halpin, MV López Varela, M Nishimura, N Roche, R Rodriguez-Roisin, DD Sin, D Singh, R Stockley, J Vestbo, JA Wedzicha, A Agustí; citation_volume=195; citation_issue=5; citation_publication_date=2017; citation_pages=557-582; citation_doi=10.1164/rccm.201701-0218PP; citation_id=CR1 citation_journal_title=Respir Med X; citation_title=Prevalence of lung cancer in chronic obstructive pulmonary disease: a systematic review; citation_author=SJ Butler, L Ellerton, RS Goldstein, D Brooks; citation_volume=1; citation_publication_date=2019; citation_pages=100003; citation_doi=10.1016/j.yrmex.2019.100003; citation_id=CR2 citation_journal_title=Lung Cancer; citation_title=The relationship between COPD and lung cancer; citation_author=AL Durham, IM Adcock; citation_volume=90; citation_issue=2; citation_publication_date=2015; citation_pages=121-127; citation_doi=10.1016/j.lungcan.2015.08.017; citation_id=CR3 citation_journal_title=Clin Chest Med; citation_title=Lung Cancer 2020; citation_author=BC Bade, CS Cruz; citation_volume=41; citation_issue=1; citation_publication_date=2020; citation_pages=1-24; citation_doi=10.1016/j.ccm.2019.10.001; citation_id=CR4 citation_journal_title=Thorax; citation_title=Chronic obstructive pulmonary disease and lung cancer incidence in never smokers: a cohort study; citation_author=HY Park, D Kang, SH Shin, K-H Yoo, CK Rhee, GY Suh, H Kim, YM Shim, E Guallar, J Cho, OJ Kwon; citation_volume=75; citation_issue=6; citation_publication_date=2020; citation_pages=506-509; citation_doi=10.1136/thoraxjnl-2019-213732; citation_id=CR5 Van Roosbroeck K, Calin GA. Cancer hallmarks and MicroRNAs: the therapeutic connection. Adv Cancer Res. 2017;(135):119-49. https://doi.org/10.1016/bs.acr.2017.06.002 . citation_journal_title=Nicotine Tob Res; citation_title=Cigarette smoking reduction and health risks: a systematic review and Meta-analysis; citation_author=JT Chang, GM Anic, BL Rostron, M Tanwar, CM Chang; citation_volume=23; citation_issue=4; citation_publication_date=2020; citation_pages=635-642; citation_doi=10.1093/ntr/ntaa156; citation_id=CR7 citation_journal_title=J Thorac Dis; citation_title=Chronic obstructive pulmonary disease (COPD) and lung cancer: common pathways for pathogenesis; citation_author=BA Parris, HE O’Farrell, KM Fong, IA Yang; citation_volume=11; citation_publication_date=2019; citation_pages=S2155-S2172; citation_doi=10.21037/jtd.2019.10.54; citation_id=CR8 citation_journal_title=Lancet; citation_title=Management of non-small-cell lung cancer: recent developments; citation_author=M Reck, DF Heigener, T Mok, JC Soria, KF Rabe; citation_volume=382; citation_issue=9893; citation_publication_date=2013; citation_pages=709-719; citation_doi=10.1016/S0140-6736(13)61502-0; citation_id=CR9 citation_journal_title=Curr Opin Pharmacol; citation_title=Current concepts on the role of inflammation in COPD and lung cancer; citation_author=H Yao, I Rahman; citation_volume=9; citation_issue=4; citation_publication_date=2009; citation_pages=375-383; citation_doi=10.1016/j.coph.2009.06.009; citation_id=CR10 citation_journal_title=Lung Cancer; citation_title=Identification of novel epigenetic abnormalities as sputum biomarkers for lung cancer risk among smokers and COPD patients; citation_author=M Tessema, DD Tassew, CM Yingling, K Do, MA Picchi, G Wu, H Petersen, S Randell, Y Lin, SA Belinsky, Y Tesfaigzi; citation_volume=146; citation_publication_date=2020; citation_pages=189-196; citation_doi=10.1016/j.lungcan.2020.05.017; citation_id=CR11 Bartel DP. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell. 2004;116(2):281-97. https://doi.org/10.1016/S0092-8674(04)00045-5 . citation_journal_title=Int J Mol Sci; citation_title=Regulatory mechanism of MicroRNA expression in Cancer; citation_author=Z Ali Syeda, SSS Langden, C Munkhzul, M Lee, SJ Song; citation_volume=21; citation_issue=5; citation_publication_date=2020; citation_pages=1723; citation_doi=10.3390/ijms21051723; citation_id=CR13 citation_journal_title=J Clin Immunol; citation_title=Association study of common genetic variants in pre-microRNAs in patients with ulcerative colitis; citation_author=M Okubo, T Tahara, T Shibata, H Yamashita, M Nakamura, D Yoshioka, J Yonemura, Y Kamiya, T Ishizuka, Y Nakagawa, M Nagasaka, M Iwata, H Yamada, I Hirata, T Arisawa; citation_volume=31; citation_issue=1; citation_publication_date=2011; citation_pages=69-73; citation_doi=10.1007/s10875-010-9461-y; citation_id=CR14 citation_journal_title=Thorac Cancer; citation_title=MicroRNA-based biomarkers for diagnosis of non-small cell lung cancer (NSCLC); citation_author=J Liao, J Shen, Q Leng, M Qin, M Zhan, F Jiang; citation_volume=11; citation_issue=3; citation_publication_date=2020; citation_pages=762-768; citation_doi=10.1111/1759-7714.13337; citation_id=CR15 citation_journal_title=J Integr Bioinform; citation_title=Visualization and analysis of MicroRNAs within KEGG pathways using VANESA; citation_author=H Hamzeiy, R Suluyayla, C Brinkrolf, SJ Janowski, R Hofestaedt, J Allmer; citation_volume=14; citation_publication_date=2017; citation_pages=9; citation_doi=10.1515/jib-2016-0004; citation_id=CR16 citation_journal_title=Expert Opin Ther Targets; citation_title=Common pathogenic mechanisms and pathways in the development of COPD and lung cancer; citation_author=IA Yang, V Relan, CM Wright, MR Davidson, KB Sriram, SM Savarimuthu Francis, BE Clarke, EE Duhig, RV Bowman, KM Fong; citation_volume=15; citation_issue=4; citation_publication_date=2011; citation_pages=439-456; citation_doi=10.1517/14728222.2011.555400; citation_id=CR17 citation_journal_title=Pharmacol Ther; citation_title=Pathobiological mechanisms underlying metabolic syndrome (MetS) in chronic obstructive pulmonary disease (COPD): clinical significance and therapeutic strategies; citation_author=SMH Chan, S Selemidis, S Bozinovski, R Vlahos; citation_volume=198; citation_publication_date=2019; citation_pages=160-188; citation_doi=10.1016/j.pharmthera.2019.02.013; citation_id=CR18 citation_journal_title=Carcinogenesis; citation_title=Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto-oncogenes; citation_author=Y Ibuki, T Toyooka, X Zhao, I Yoshida; citation_volume=35; citation_issue=6; citation_publication_date=2014; citation_pages=1228-1237; citation_doi=10.1093/carcin/bgt492; citation_id=CR19 Yun CL, Zierath JR. AMP-activated protein kinase signaling in metabolic regulation. J Clin Invest. 2006;100:328–41. https://doi.org/10.1161/01 . citation_journal_title=Brief Bioinform; citation_title=miR+pathway: the integration and visualization of miRNA and KEGG pathways; citation_author=C Pian, G Zhang, L Gao, X Fan, F Li; citation_volume=21; citation_issue=2; citation_publication_date=2020; citation_pages=699-708; citation_doi=10.1093/bib/bby128; citation_id=CR21 citation_journal_title=J Integr Bioinform; citation_title=VANESA - a software application for the visualization and analysis of networks in system biology applications; citation_author=C Brinkrolf, SJ Janowski, B Kormeier, M Lewinski, K Hippe, D Borck, R Hofestädt; citation_volume=11; citation_issue=2; citation_publication_date=2014; citation_pages=239; citation_doi=10.2390/biecoll-jib-2014-239; citation_id=CR22 Akbas F, Coskunpinar E, Aynaci E, Müsteri Oltulu Y, Yildiz P. Analysis of Serum Micro-RNAs as potential biomarker in chronic obstructive pulmonary disease. Exp Lung Res. 2012;38(6):286-94. https://doi.org/10.3109/01902148.2012.689088 . citation_journal_title=J Thorac Oncol; citation_title=Chronic obstructive pulmonary disease and risk of lung cancer: the importance of smoking and timing of diagnosis; citation_author=HA Powell, B Iyen-Omofoman, DR Baldwin, RB Hubbard, LJ Tata; citation_volume=8; citation_issue=1; citation_publication_date=2013; citation_pages=6-11; citation_doi=10.1097/JTO.0b013e318274a7dc; citation_id=CR24 citation_journal_title=Respirology; citation_title=Impact of COPD and emphysema on survival of patients with lung cancer: a meta-analysis of observational studies; citation_author=Y-H Gao, W-J Guan, Q Liu, H-Q Wang, Y-N Zhu, R-C Chen, GJ Zhang; citation_volume=21; citation_issue=2; citation_publication_date=2016; citation_pages=269-279; citation_doi=10.1111/resp.12661; citation_id=CR25 citation_journal_title=Curr Opin Pulm Med; citation_title=Lung cancer in chronic obstructive pulmonary disease patients, it is not just the cigarette smoke; citation_author=P Sanchez-Salcedo, JJ Zulueta; citation_volume=22; citation_issue=4; citation_publication_date=2016; citation_pages=344-349; citation_doi=10.1097/MCP.0000000000000283; citation_id=CR26 citation_journal_title=J Thor Dis; citation_title=Relationships between chronic obstructive pulmonary disease and lung cancer: biological insights; citation_author=E Barreiro, V Bustamante, V Curull, J Gea, JL López-Campos, X Muñoz; citation_volume=8; citation_issue=10; citation_publication_date=2016; citation_pages=E1122-E1135; citation_doi=10.21037/jtd.2016.09.54; citation_id=CR27 citation_journal_title=Chronic Obstr Pulm Dis; citation_title=MicroRNAs as therapeutic targets in lung disease: prospects and challenges; citation_author=T Sato, H Baskoro, SI Rennard, K Seyama, K Takahashi; citation_volume=3; citation_issue=1; citation_publication_date=2016; citation_pages=382-388; citation_doi=10.15326/jcopdf.3.1.2015.0160; citation_id=CR28 citation_title=The Roles of MicroRNA in Lung Cancer; citation_publication_date=2019; citation_id=CR29; citation_author=K Wu; citation_author=Y Tsai; citation_author=C Lien; citation_author=P Kuo citation_journal_title=Adv Dermatol Allergol; citation_title=Preliminary studies: differences in microRNA expression in asthma and chronic obstructive pulmonary disease; citation_author=M Pietrusińska, A Pająk, P Górski, P Kuna, J Szemraj, A Goździńska-Nielepkowicz, T Pietras; citation_volume=4; citation_issue=4; citation_publication_date=2016; citation_pages=276-280; citation_doi=10.5114/ada.2016.61603; citation_id=CR30 citation_journal_title=Int J COPD; citation_title=Peripheral leukocyte microRNAs as novel biomarkers for COPD; citation_author=R Wang, J Xu, H Liu, Z Zhao; citation_volume=Volume 12; citation_publication_date=2017; citation_pages=1101-1112; citation_doi=10.2147/COPD.S130416; citation_id=CR31 citation_journal_title=Int J Mol Med; citation_title=Clinical relevance of plasma miR-106b levels in patients with chronic obstructive pulmonary disease; citation_author=S Soeda, JH Ohyashiki, K Ohtsuki, T Umezu, Y Setoguchi, K Ohyashiki; citation_volume=31; citation_issue=3; citation_publication_date=2013; citation_pages=533-539; citation_doi=10.3892/ijmm.2013.1251; citation_id=CR32 citation_journal_title=Thorax; citation_title=Gene expression networks in COPD: microRNA and mRNA regulation; citation_author=ME Ezzie, M Crawford, J-H Cho, R Orellana, S Zhang, R Gelinas, K Batte, L Yu, G Nuovo, D Galas, P Diaz, K Wang, SP Nana-Sinkam; citation_volume=67; citation_issue=2; citation_publication_date=2012; citation_pages=122-131; citation_doi=10.1136/thoraxjnl-2011-200089; citation_id=CR33 Xie X, Liu HT, Mei J, Ding FB, Xiao HB, Hu FQ, et al. miR-106a promotes growth and metastasis of non-small cell lung cancer by targeting PTEN. Int J Clin Exp Pathol. 2015;8(4):3827-34. https://doi.org/10.3892/ol.2018.9697 . Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell. 2006;9(3):189-98. https://doi.org/10.1016/j.ccr.2006.01.025 . citation_journal_title=J Clin Med; citation_title=microRNAs are key regulators in Chronic Lung Disease: Exploring the vital link between disease progression and Lung Cancer; citation_author=MS Eapen, KD McAlinden, S Myers, W Lu, SS Sohal; citation_volume=8; citation_issue=11; citation_publication_date=2019; citation_pages=1986; citation_doi=10.3390/jcm8111986; citation_id=CR36 citation_journal_title=Gen Physiol Biophys; citation_title=Investigation of key miRNAs and potential mechanisms in non-small cell lung cancer development from chronic obstructive pulmonary disease; citation_author=T Denkçeken, E Pala; citation_volume=39; citation_issue=01; citation_publication_date=2020; citation_pages=69-77; citation_doi=10.4149/gpb_2019042; citation_id=CR37 citation_journal_title=Eur Respir J; citation_title=Pathogenesis of lung cancer signalling pathways: roadmap for therapies; citation_author=E Brambilla, A Gazdar; citation_volume=33; citation_issue=6; citation_publication_date=2009; citation_pages=1485-1497; citation_doi=10.1183/09031936.00014009; citation_id=CR38 Suzuki M, Wada H, Yoshino M, Tian L, Shigematsu H, Suzuki H, et al. Molecular characterization of chronic obstructive pulmonary disease-related non-small cell lung cancer through aberrant methylation and alterations of EGFR signaling. Ann Surg Oncol. 2010;17(3):878-88. https://doi.org/10.1245/s10434-009-0739-3 . citation_journal_title=Int J Oncol; citation_title=MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer; citation_author=H Xia, Y Li, X Lv; citation_volume=49; citation_issue=4; citation_publication_date=2016; citation_pages=1325-1333; citation_doi=10.3892/ijo.2016.3628; citation_id=CR40 citation_journal_title=Oncotarget; citation_title=MicroRNA-107-5p suppresses non-small cell lung cancer by directly targeting oncogene epidermal growth factor receptor; citation_author=P Wang, X Liu, Y Shao, H Wang, C Liang, B Han; citation_volume=8; citation_publication_date=2017; citation_pages=57012-57023; citation_doi=10.18632/oncotarget.18505; citation_id=CR41 citation_journal_title=PLoS One; citation_title=MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines; citation_author=Y Takahashi, ARR Forrest, E Maeno, T Hashimoto, CO Daub, J Yasuda; citation_volume=4; citation_issue=8; citation_publication_date=2009; citation_pages=e6677; citation_doi=10.1371/journal.pone.0006677; citation_id=CR42 citation_journal_title=Cancer Res; citation_title=FOXO3a is a major target of inactivation by PI3K/AKT signaling in aggressive neuroblastoma; citation_author=EE Santo, P Stroeken, PV Sluis, J Koster, R Versteeg, EM Westerhout; citation_volume=73; citation_issue=7; citation_publication_date=2013; citation_pages=2189-2198; citation_doi=10.1158/0008-5472.CAN-12-3767; citation_id=CR43 citation_journal_title=Oncol Res; citation_title=MiR-103 functions as a tumor suppressor by directly targeting programmed cell death 10 in NSCLC; citation_author=D Yang, JJ Wang, JS Li, QY Xu; citation_volume=26; citation_issue=4; citation_publication_date=2018; citation_pages=519-528; citation_doi=10.3727/096504017X15000757094686; citation_id=CR44 citation_journal_title=Lung Cancer; citation_title=Co-occurrence of targetable mutations in non-small cell lung cancer (NSCLC) patients harboring MAP2K1 mutations; citation_author=M Scheffler, A Holzem, A Kron, L Nogova, MA Ihle, C Levetzow, J Fassunke, C Wömpner, E Bitter, S Koleczko, DSY Abdulla, S Michels, R Fischer, R Riedel, JP Weber, T Westphal, U Gerigk, J Kern, B Kaminsky, W Randerath, KO Kambartel, S Merkelbach-Bruse, R Büttner, J Wolf; citation_volume=144; citation_publication_date=2020; citation_pages=40-48; citation_doi=10.1016/j.lungcan.2020.04.020; citation_id=CR45 Li W, Cui K, Prochownik EV, Li Y. The deubiquitinase USP21 stabilizes MEK2 to promote tumor growth. Cell Death Dis. 2018;9(5):482-94. https://doi.org/10.1038/s41419-018-0523-z . citation_journal_title=Mol Cancer; citation_title=MicroRNA-505 functions as a tumor suppressor in endometrial cancer by targeting TGF-aα; citation_author=S Chen, KX Sun, BL Liu, ZH Zong, Y Zhao; citation_volume=15; citation_issue=1; citation_publication_date=2016; citation_pages=11; citation_doi=10.1186/s12943-016-0496-4; citation_id=CR47 Chen L, Kong G, Zhang C, Dong H, Yang C, Song G, et al. MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma. Oncotarget. 2016;7(15):20041–53. https://doi.org/10.18632/oncotarget.7884 . citation_journal_title=Mol Cancer; citation_title=miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner; citation_author=N Bandi, E Vassella; citation_volume=10; citation_publication_date=2011; citation_pages=55; citation_doi=10.1186/1476-4598-10-55; citation_id=CR49 citation_journal_title=Tumor Biol; citation_title=MicroRNA-15a inhibits the growth and invasiveness of malignant melanoma and directly targets on CDCA4 gene; citation_author=C Alderman, A Sehlaoui, Z Xiao, Y Yang; citation_volume=37; citation_issue=10; citation_publication_date=2016; citation_pages=13941-13950; citation_doi=10.1007/s13277-016-5271-z; citation_id=CR50 Li B, Xie Z, Li B. miR-152 functions as a tumor suppressor in colorectal cancer by targeting PIK3R3. Tumor Biol. 2016;37(8):10075-84. https://doi.org/10.1007/s13277-016-4888-2 . Yang T, Thakur A, Chen T, Yang L, Lei G, Liang Y, et al. MicroRNA-15a induces cell apoptosis and inhibits metastasis by targeting BCL2L2 in non-small cell lung cancer. Tumor Biol. 2015;36(6):4357-65. https://doi.org/10.1007/s13277-015-3075-1 . citation_journal_title=Proc Natl Acad Sci U S A; citation_title=P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis; citation_author=M Yamakuchi, CD Lotterman, C Bao, RH Hruban, B Karim, JT Mendell, D Huso, CJ Lowenstein; citation_volume=107; citation_issue=14; citation_publication_date=2010; citation_pages=6334-6339; citation_doi=10.1073/pnas.0911082107; citation_id=CR53 citation_journal_title=Development; citation_title=Cdks, cyclins and CKIs: roles beyond cell cycle regulation; citation_author=S Lim, P Kaldis; citation_volume=140; citation_issue=15; citation_publication_date=2013; citation_pages=3079-3093; citation_doi=10.1242/dev.091744; citation_id=CR54 Gordon E, Ravicz J, Liu S, Chawla S, Hall F. Cell cycle checkpoint control: the cyclin G1/Mdm2/p53 axis emerges as a strategic target for broad-spectrum cancer gene therapy - a review of molecular mechanisms for oncologists. Mol Clin Oncol. 2018. https://doi.org/10.3892/mco.2018.1657 . citation_journal_title=Sci Rep; citation_title=Keratin 19 regulates cell cycle pathway and sensitivity of breast cancer cells to CDK inhibitors; citation_author=P Sharma, S Alsharif, K Bursch, S Parvathaneni, DG Anastasakis, J Chahine, A Fallatah, K Nicolas, S Sharma, M Hafner, B Kallakury, BM Chung; citation_volume=9; citation_issue=1; citation_publication_date=2019; citation_pages=14650; citation_doi=10.1038/s41598-019-51195-9; citation_id=CR56 Sean D, Meltzer PS. GEOquery: a bridge between the gene expression omnibus (GEO) and BioConductor. Bioinformatics. 2007;23(14):1846–7. https://doi.org/10.1093/bioinformatics/btm254 . Robinson MD, McCarthy DJ, Smyth GK. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009;26(1):139–40. https://doi.org/10.1093/bioinformatics/btp616 . Han J, Liu S, Zhang Y, Xu Y, Jiang Y, Zhang C, et al. MiRSEA: Discovering the pathways regulated by dysfunctional MicroRNAs. Oncotarget. 2016;7(34):55012-25. https://doi.org/10.18632/oncotarget.10839 . citation_journal_title=PLoS Biol; citation_title=WikiPathways: pathway editing for the people; citation_author=AR Pico, T Kelder, MP Iersel, K Hanspers, BR Conklin, C Evelo; citation_volume=6; citation_issue=7; citation_publication_date=2008; citation_pages=e184; citation_doi=10.1371/journal.pbio.0060184; citation_id=CR60 citation_journal_title=PLoS Comput Biol; citation_title=PathVisio 3: an extendable pathway analysis toolbox; citation_author=M Kutmon, MP Iersel, A Bohler, T Kelder, N Nunes, AR Pico, CT Evelo; citation_volume=11; citation_issue=2; citation_publication_date=2015; citation_pages=e1004085; citation_doi=10.1371/journal.pcbi.1004085; citation_id=CR61