Comparative analysis of gene expression in Fra-1+/+ and Fra-1-/- mice lung
ABSTRACT: We hypothesized that gene expression in lungs of Fra-1+/+ and Fra-1-/- mice are divergent thus contributing fibrosis. More specifically, Fra-1-/- mice are increased susceptible to fibrosis. In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between Fra-1+/+ and Fra-1-/- mice at early time point. This study utilizes microarray analysis to test these hypotheses. 5 days PBS and bleomycin treated lung samples from both Fra-1+/+ and Fra-1-/- mice were used. RNA was isolated and used for global gene expression profiling (Affymetrix MoGene 1.0ST v1 Array). The raw probe signal intensities were quantile normalized over all samples, summarized with the robust multi-array average (RMA) algorithm and log2 transformed with a median polish, using the Affymetrix Power Tools. We considered a transcript cluster (gene-level) to be reliably expressed in a sample if the Affymetrix implemented DABG (detection above ground) p-value was less than 0.05. We used local-pooled-error (LPE) estimates and robust statistical tests for evaluating significance of each gene's differential expression in a comparison.
Project description:The aim of this experiment is to identify novel gene candidates being regulated in Fra-2 overexpressing mice. Fra-2 over-expressing mice (Fra-2 TG) develop vascular remodeling and pulmonary fibrosis. Fra-2, as a trascription factor, is responsible for downstream target genes expression. Comparison of the mRNA expressed from WT and Fra-2 TG animals will help to identify Fra-2 target genes. The microarray analysis will be performed in mRNA isolated from lung homogenate. The lungs of the mice were extensively perfused. The right part was snap-froen and transfered to (-80°C). The total RNA was isolated and quantified. Comparison of transgenes vs. wild-types. Hybridization of samples from 4 wild-type and 4 transgene animals = 8 single-color hybridizations
Project description:Background and Aim: Fra-1 (Fos-related antigen-1) is a member of the AP1 (activator protein-1) family of transcription factors. We have recently shown that Fra-1 is necessary for breast cancer cells to metastasize in vivo, and that breast cancer outcome can be predicted by a classifier comprising genes that are expressed in a Fra-1-dependent fashion. Here, we show that Fra-1 plays an important role also in colon cancer progression. Methods: We compared proliferation rates of parental and Fra-1-depleted colon cancer cells in vitro under 2D, 3D, and attachment-free conditions and in vivo upon subcutaneous and intravenous injections into mice. We also compared RNA expression profiles of colon cancer cells with and without Fra-1 expression. Results: Fra-1 depletion impair colony outgrowth of human colon cancer cells in soft agar and in suspension, whereas it does not affect proliferation on 2D culture plates. Consistent with this, upon subcutaneous injection into mice, tumors formed by Fra-1-depleted colon cancer cells are only three times smaller than those produced by control cells. In contrast, when injected intravenously, Fra-1 depletion causes 200-fold reduction in tumor burden. Consistent with the more aggressive characteristics of Fra-1-proficient tumors, the prognosis of colon cancer patients can be predicted by a Fra-1 classifier generated by comparing RNA profiles of parental and Fra-1-depleted colon cancer cells. Conclusions: Our results demonstrate that Fra-1 is an important determinant of the metastatic potential of human colon cancer cells, and suggest that a Fra-1 classifier can be used as a prognostic predictor in colon cancer patients. Overall design: HT29 cell line, two shRNAs against Fra-1, one empty vector control, three biological replicates
Project description:In order to analyze the global changes in gene expression resulting from induction of NetA-Fra signaling, we carried out a microarray experiment comparing Drosophila third instar wing imaginal discs in which Net+Fra had been overexpressed to age matched wild type wing imaginal discs. RNA extracted from both +NetA-Fra overexpression and wildtype third instar imaginal discs were hybridized to the Affymetrix GeneChip Drosophila Genome 2.0 . Heat shock induced GFP-marked clones ectopically expressing NetA+Fra in larvae were generated. Controls for this study included age matched wildtype third instar wing imaginal discs bearing GFP clones which were prepared in the same manner. Total RNA was extracted from dissected +NetA-Fra vs. control third instar wing imaginal discs and hybridized to the Affymetrix GeneChip Drosophila Genome 2.0.
Project description:Purpose: Compare the transciptome between lung myofibroblasts that contituatively express Fra-2 to control lung myofibroblasts to determine genes that may contribute to regulation of secondary alveolar septation Methods: mRNa sequences from lung myofibroblasts isolated from mutant mice that contituatively express Fra-2 in α-SMA expressing cells (sma-Fra2) were compared to control mice. n=4 in each group Results: 43,432 transcripts wre analyzed and 361 genes were differentially expressed in the smaFra2 mice compared to wild type control. Functional analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. Changes in genes from lung myofibroblasts in smaFra2 mice: genes involved with extracellular matrix and cell adhesion were upregulated; Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 (Enpp2) and LPA receptor1 (LPA1) were downregulated. Coclusion: The secondary alveolar septation defect in smaFra2 mice could be explaned by Fra-2 mediated changes in extracellular matrix and cell adhesion in developing lung myofibroblasts. Overall design: Genome-wide sequencing of lung myofiblasts that constituatively express Fra-2
Project description:Hepatic mRNA expression was compared between control and Fra-1hep mice, which were fed chow diet Overall design: Liver RNA pooled from 5 chow-diet fed control mice (control pool) was Cy3-labeled and liver RNA from 6 individual Fra-1hep mice was Cy5-labeled. On each microarray, Cy3‐labeled control pool and one Cy5-labeled mutant sample were hybridized at 65°C for 17 hours.
Project description:In order to analyze the global changes in gene expression resulting from loss of Fra signaling, we performed a microarray experiment comparing Drosophila embryos containing a loss of function fra mutation to age matched wildtype embryos. RNA extracted from fra mutant vs. wildtype embryos were hybridized to the Affymetrix GeneChip Drosophila Genome 2.0 . We selected Drosophila embryos at stage 13, which marks the onset of Fra expression in the embryonic CNS and the early stages of commissural axon guidance in the ventral nerve cord. RNA was extracted from both fra and wildtype embryos. Hybridization experiments were performed on Affymetrix Drosophila Genome 2.0 microarrays.
Project description:In order to analyze the global changes in gene expression resulting from loss of Fra signaling, we performed a microarray experiment comparing Drosophila embryos containing a loss of function fra mutation to age matched wildtype embryos. RNA extracted from fra mutant vs. wildtype embryos were hybridized to the Affymetrix GeneChip Drosophila Genome 2.0 . Overall design: We selected Drosophila embryos at stage 13, which marks the onset of Fra expression in the embryonic CNS and the early stages of commissural axon guidance in the ventral nerve cord. RNA was extracted from both fra and wildtype embryos. Hybridization experiments were performed on Affymetrix Drosophila Genome 2.0 microarrays.
Project description:Hepatic mRNA expression was compared between control and Fra-1hep mice, which were fed High fat diet (45%kcal fat) Overall design: Liver RNA pooled from 5 HFD fed control mice (control pool) was Cy3-labeled and liver RNA from 4 individual Fra-1hep mice was Cy5-labeled. On each microarray, Cy3‐labeled control pool and one Cy5-labeled mutant sample were hybridized at 65°C for 17 hours.
Project description:The transcription factor complex AP-1 (Activator protein 1) is composed of Jun (c-Jun, JunB, JunD) and Fos proteins (c-Fos, FosB, Fra-1, Fra-2) which control a variety of stress responses, including cell proliferation, apoptosis, inflammation, wound healing, and cancer. Individual Fos proteins have been thoroughly studied in gain- and loss-of-function mouse models, which revealed important functions in bone cell proliferation and differentiation. We have recently demonstrated that loss of Fra-2 causes perinatal lethality and severe osteopenia due to several cellular defects, including a chondrocyte differentiation defect and a control of osteoclast survival and size. Moreover, we have reported a profibrogenic function of Fra-2 in transgenic mice, in which ectopic expression of Fra-2 in various organs resulted in generalized fibrosis with predominant manifestations in the lung. Fra-2 knock-out newborns have increased numbers and size of osteoclasts in vivo. The pulmonary phenotype observed in Fra-2Tg mice is characterized by vascular remodeling and obliteration of pulmonary arteries, which coincides with expression of osteopontin, an AP-1 target gene involved in vascular remodeling and fibrogenesis. These alterations are followed by inflammation; release of profibrogenic factors, such as IL-4, insulin-like growth factor 1, and CXCL5. The expression profiling study was performed to analyse changes in transcript levels in lung over a period of time. Total RNA of four mutant male animals at each time point (age: 6, 10 and 14 weeks) were hybridised versus a pool of total RNA of four wild type mice of the corresponding age. For each mutant animal, two technical chip hybridisations were performed, including a dye-swap experiment (in total, 8 hybridisation of each time point = 2 technical replicates x 4 biological replicates).