Unknown,Transcriptomics,Genomics,Proteomics

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An extracellular matrix-driven positive feedback loop regulates translation in Idiopathic Pulmonary Fibrosis

ABSTRACT: Analysis of gene expression of lung fibroblasts seeded onto decellularized extracellular matrix (ECM). Experiment had 2x2 design where fibroblasts from idiopathic pulmonary fibrosis (IPF) or control patients were seeded onto decelluarized lung tissue from IPF or control patients allowing for determination of gene expression differences that were driven by IPF ECM and which differences were driven by the IPF fibroblast. Lung fibroblasts from 5 patients with idiopathic pulmonary fibrosis and 5 control patients were cultured on decellularized ECM from IPF or control lung. Total RNA and polyribosome RNA were isolated after the cells were cultured on the decellularized ECM for 18 hours. When possible, a control cell line and a diseased cell line were cultured (and processed) simultaneously to minimize the effect of experimental variance induced by running the experiment at different times.Samples with the same batch number (provied in the sample 'characteristics' field) were cultured and processed at the same time.

ORGANISM(S): Homo sapiens

SUBMITTER: Matthew Parker

PROVIDER: E-GEOD-45686 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RNA sequencing has been performed to investigate the transcriptomic profile of fibroblasts derived from apical and basal regions of idiopathic pulmonary fibrosis (IPF) and control (CTR) lungs. Lung fibroblasts were cultured onto T25 flasks until full confluence was reached. Fibroblasts were then harvested by trypsinization using 0.05% trypsin-EDTA and washed in Phosphate Buffered Saline (PBS). RNA was extracted using the RNeasy Mini kit protocol. Samples were then sent to the Australian Genome Research Facility (AGRF) where 1μg of RNA was submitted for next-generation sequencing. Once raw data returned, bioinformatics analysis was conducted. Results revealed little difference at the transcriptomic level between apical lung fibroblasts isolated from IPF and CTR donors. In contrast, there was a significant difference in gene expression between IPF and CTR lung basal fibroblasts with 90 differentially expressed genes (DEGs) identified in IPF basal fibroblasts. Gene ontology analysis of these 90 DEGs suggested that the most important functions were associated with receptor ligand activity, cell adhesion molecule binding, and integrin binding. Furthermore, fibroblasts isolated from the basal and apical sites of control lung were not significantly different. Interestingly, the lung basal fibroblasts from IPF patients were significantly different in their transcriptomic profile to that of apical fibroblasts from the same patients. 303 DEGs were identified in IPF basal fibroblasts compared with IPF apical fibroblasts. According to the GO analysis on these 303 DEGs, the most important function of the identified dysregulated genes was associated with the composition and function of the extracellular (ECM). Further pathway analysis identified 4 signaling pathways, namely arrhythmogenic right ventricular cardiomyopathy, calcium signaling pathway, dilated cardiomyopathy, and hematopoietic cell lineage. An interesting finding was that these IPF basal fibroblasts were clustered into two groups (Group 1 and Group 2). Analysis on these two groups found 3594 DEGs in Group 1 compared to Group 2. Go analysis suggested most of the identified dysregulated genes was involved in the composition and function of the ECM. Pathway analysis identified 28 dysregulated signaling pathways, including PI3K-Akt signaling pathway, TGF-β signaling pathway and Wnt signaling pathway. The results of the transcriptome analysis intimate that the IPF is more predominant in the basal lungs and the fibroblast-derived from the basal region of the fibrotic lungs may serve as a central role in IPF. Moreover, the dramatic difference between two groups of IPF basal fibroblasts might be linked to the differential response to the same drug occurred in IPF patients.

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An extracellular matrix-driven positive feedback loop regulates translation in Idiopathic Pulmonary Fibrosis

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