Comment[ArrayExpressAccession] E-GEOD-39122 MAGE-TAB Version 1.1 Public Release Date 2013-07-20 Investigation Title Gene expression-based, inflammatory response prediction by bronchial epithelial cell line treated with signal peptide of eosinophil cationic protein Comment[Submitted Name] Gene expression-based, inflammatory response prediction by bronchial epithelial cell line treated with signal peptide of eosinophil cationic protein Experiment Description Background Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-a (TGF-a) and epidermal growth factor receptor (EGFR). Results In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then generated a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes. This revealed that STAT1[/2], an important transcriptional factor that regulates cytokine expression and release, served as a hub to connect the pathways of cytokine stimulation (TGF-a and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-a and EGFR with the functional linkage network indicated that STAT1 served as a hub that connects two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Therefore, we hypothesize that ECPsp may regulate the migration of macrophages in vivo. Conclusion The increased expression and release of various cytokines triggered by ECPsp may attract macrophages to bronchia to purge damaged cells. Our approach, involving experimental and computational systems biology, predicts pathways and potential biological functions for further characterization of this novel function of ECPsp under inflammatory conditions. The control group of this study is Beas-2B cells treated with pEGFP-C1, and the experiment group is the same cell line treated with pEGFPN1-ECPsp. Each group was conducted by two biological repeats and two technical repeats were done in DNA microarray analyses. On the microarray chip, 29,187 probes correspond to the annotated genes in the RefSeq v38 and Ensembl v56 databases. Furthermore, 1,088 control probes are also included for monitoring the sample quality and the hybridization process. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Chang Chang Liu Tsai Fan Wang Hsieh Chang Pai Huang Lan Person First Name Hao-Teng Hao-Teng Yu-Shu Pei-Wen Tan-chi Yong Chia-Hung Margaret Tun-Wen Chien-Fu Chung-Yu Person Mid Initials D Person Email htchang@mail.cmu.edu.tw Person Affiliation China Medical University Person Address China Medical University, No.91, Hsueh-shih Rd., Taichung Ciy, Taiwan Person Roles submitter Protocol Name P-GSE39122-1 P-GSE39122-3 P-GSE39122-4 P-GSE39122-2 P-GSE39122-5 Protocol Description The raw data were re-scaled to account for the differences in individual hybridization intensities. We employed a rank-invariant scaling method to select genes (Tseng et al., 2001), which were then used for fitting of a non-linear normalization curve. Rosetta Resolver System (Rosetta Biosoftware) was used to normalize the data. ID_REF = VALUE = Normalized signal intensity Five mg of total RNA was reverse-transcribed using oligo-dT primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen) in the presence of 100 mCi of [33P]dCTP (Amersham Bioscience, Piscataway, NJ) according to the instructions for the Gene The GeneFilters were prehybridized for 2 h at 51C with 0.5 mg/ml of poly-dA (Invitrogen) and 0.5 mg/ml Cot-1 DNA (Invitrogen) in 10 ml of AlkPhos DIRECT hybridization buffer (Amersham Bioscience) and then hybridized for 17 h at 51C with the denatured radi On average, 50 7 um-thick cryostat sections were prepared for the extraction of total RNA from tumor cell-rich areas, which were identified by a surgical pathologist (YY) using every 10th section stained by May-Giemsa. RNA was extracted using Trizol. The arrays were then exposed for 2 hours to an Imaging Plate and scanned at 25-mm resolution with a BAS5000 phosphoimager (Fuji Photo Film), images of the hybridized arrays were processed with L Process (Fuji Photo Film) and signal intensities quantified Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name GENOTYPE Experimental Factor Type genotype Comment[SecondaryAccession] GSE39122 Comment[GEOReleaseDate] 2013-07-20 Comment[ArrayExpressSubmissionDate] 2012-07-05 Comment[GEOLastUpdateDate] 2013-07-21 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-39122.sdrf.txt