TNFalpha and IL1beta stimulate differential gene expression in endometrial stromal cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE40007 | GEO | 2014/08/01
SECONDARY ACCESSION(S): PRJNA172340
REPOSITORIES: GEO
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Project description:Purpose: This study was undertaken to evaluate the effect of peritoneal fluid from subjects with differing stages of endometriosis on gene expression in endometrial stromal cells. Methods: Peritoneal fluid from subjects with minimal, moderate, and severe stages of endometriosis or without endometriosis (controls) was collected, filtered and separated from peritoneal cells. Telomerase – immortalized human endometrial stromal cells (T-HESC) were treated with the peritoneal fluid samples for 48 hours. RNA was isolated from treated cells and processed for DNA microarray. Results: 162 genes were found to be differentially expressed in T-HESC cells treated with peritoneal fluid from subjects with differing stages of endometriosis. Three of the differentially expressed genes were chosen for confirmatory studies using quantitative RT-PCR; these were interleukin 8, corin and matrix metalloproteinase 3. The primary gene ontologies identified by microarray highlight functions expected to be involved in the establishment of endometriosis, such as proteases, signal transduction, defense and immune system, cell adhesion, cell cycle and cell death, cytoskeleton, transcriptional regulation and translation, extracellular matrix, and enzymes and metabolism. Conclusions: This study demonstrated that the severity of endometriosis affected the factors present in peritoneal fluid which, in turn, affected gene expression in endometrial stromal cells.
2019-07-01 | GSE101176 | GEO
Project description:Cytokines are implicated in the development of inflammatory diseases such as endometriosis. This project was designed to test the hypothesis that specific cytokines that are secreted by macrophages, such as tumor necrosis factor ? (TNF?) and interleukin 1 beta (IL1?), cause gene expression changes in endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with TNF? (5ng/ml) ± IL1? (1ng/ml). DNA microarray and real time RT-PCR were used to study the gene expression changes in T-HESC cells. Two hundred and nineteen genes featuring in various gene ontologies were found to be differentially expressed in T-HESC cells treated with TNF? ± ILI?. The gene ontologies included functions expected to be associated with the development of endometriosis such as peptidases, cell adhesion, cell death/apoptosis, cell cycle, growth factors, cytoskeletal organization, defense/immune system, signal transduction, and transcriptional regulation. The differential expression of 4 genes (interleukin 8 (IL8), interleukin 6 (IL6), IL1? and matrix metalloproteinase (MMP3) was confirmed by real time RT-PCR. All four genes were up-regulated in response to TNF? ± ILI? in T-HESC cells. The effect of TNF? ± ILI? on migration and invasion of T-HESC cells, as measured with Boyden chambers, was not affected by treatment with these cytokines. Goal of the experiment: To examine differential gene expression in telomerase-immortalized human endometrial stromal cells (T-HESC) in response to treatment with tumor necrosis factor alpha (TNF?) ± interleukin 1? (IL1?). Keywords: cytokines, endometrial stromal cells, endometriosis Experimental factors: cytokine treatment Experimental design: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were treated with vehicle or with the cytokines, TNF? (5ng/ml) ± IL1? (1ng/ml). Three different passages of cells were collected for each treatment group to provide biological replicates. Quality control steps: The cRNA that was synthesized from each cell culture sample was used for hybridization to a single CodeLink (Applied Microarrays, Tempe, AZ) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labeling: The origin of each biological sample: The samples were cultured cells of the telomerase-immortalized human endometrial stromal cell (T-HESC) line obtained from ATCC (CRL-4003). Manipulations of biological samples and protocols used: The telomerase immortalized - human endometrial stromal cell line (T-HESC, ATCC #CRL-4003, Manassas, VA) was used for all experiments. The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO) with 10% fetal bovine serum albumin (FBS, Hyclone, South Logan, UT), insulin, selenious acid, transferrin, bovine serum albumin and linoleic acid (ITS+ Premix, BD Biosciences, Bedford, MA), puromycin (Cellgro, Manassas, VA) and penicillin/ streptomycin (Hyclone, South Logan, UT). After the cells reached 80% confluence they were starved for 24 hours in T-HESC starvation medium (DMEM+ ITS+ puromycin+ penicillin/streptomycin) before treatment. The cells were treated with vehicle or with TNF? (5ng/ml) alone, IL1? (1ng/ml) alone, or with the two cytokines combined at the same concentrations for 48 hours. Total RNA was then isolated from the cell cultures for use in the DNA microarray experiments. Each treatment was carried out with 3 different passages of cells. Technical protocols: The cultured endometrial cells were rinsed with PBS and TRI reagent (MRC) was added. The cells were scraped into tubes and centrifuged in the presence of bromochloropropane and sodium acetate. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity was evaluated with the RNA 6000 NanoLabChip in an Agilent Bioanalyzer. Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total T-HESC cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on columns. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on columns. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94°C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37°C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46°C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT each and once in 0.05% Tween 20 for 20 sec. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2014-08-01 | E-GEOD-40007 | biostudies-arrayexpress
Project description:We used microarray to determine the miRNA whose expression was changed at 6 hours after inflammatory cytokines (TNFα, IL1α, IL1β, or IL6) treatment. Two-condition experiment; Caco2 control vs Caco2 treated with TNFα, IL1α, IL1β,or IL6 for 6 hours
2012-04-21 | E-GEOD-37288 | biostudies-arrayexpress
Project description:We performed microarray profiling of microRNAs of dysfunctional human primary aortic SMCs induced by different cytokines (PDGF-BB, TGFβ1, TNFα, IL1β).
2021-11-03 | GSE185784 | GEO
Project description:We used microarray to determine the miRNA whose expression was changed at 6 hours after inflammatory cytokines (TNFα, IL1α, IL1β, or IL6) treatment.
2012-04-22 | GSE37288 | GEO
Project description:It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in endometriosis patients. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from healthy individuals or with endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified novel differences in key pathways between normal and endometriosis, including defective bone morphogenetic protein (BMP) signaling (ID1, ID2, ID3, BMP6), altered iron transport (SLC40A1, TFRC), endometrial stem cell markers (CD44, SUSD2), and retinoid signaling pathways (RORB, ALDH1L1, RARA). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 in decidualized stromal cells from endometriosis patients relative to normal individuals, with an overrepresentation of gene ontologies related to signaling by transforming growth factor β (TGFβ), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation of patient-derived assembloids from endometriosis patients restored decidualization. In summary, transcriptomic and genomic profiling of patient-derived endometrial cells and assembloids identified that restoring BMP/SMAD1/5/4 signaling is crucial for engaging a robust decidualization program in women with endometriosis.
2024-02-17 | GSE243157 | GEO
Project description:It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in endometriosis patients. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from healthy individuals or with endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified novel differences in key pathways between normal and endometriosis, including defective bone morphogenetic protein (BMP) signaling (ID1, ID2, ID3, BMP6), altered iron transport (SLC40A1, TFRC), endometrial stem cell markers (CD44, SUSD2), and retinoid signaling pathways (RORB, ALDH1L1, RARA). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 in decidualized stromal cells from endometriosis patients relative to normal individuals, with an overrepresentation of gene ontologies related to signaling by transforming growth factor β (TGFβ), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation of patient-derived assembloids from endometriosis patients restored decidualization. In summary, transcriptomic and genomic profiling of patient-derived endometrial cells and assembloids identified that restoring BMP/SMAD1/5/4 signaling is crucial for engaging a robust decidualization program in women with endometriosis.
2024-02-17 | GSE243156 | GEO
Project description:It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in endometriosis patients. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from healthy individuals or with endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified novel differences in key pathways between normal and endometriosis, including defective bone morphogenetic protein (BMP) signaling (ID1, ID2, ID3, BMP6), altered iron transport (SLC40A1, TFRC), endometrial stem cell markers (CD44, SUSD2), and retinoid signaling pathways (RORB, ALDH1L1, RARA). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 in decidualized stromal cells from endometriosis patients relative to normal individuals, with an overrepresentation of gene ontologies related to signaling by transforming growth factor β (TGFβ), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation of patient-derived assembloids from endometriosis patients restored decidualization. In summary, transcriptomic and genomic profiling of patient-derived endometrial cells and assembloids identified that restoring BMP/SMAD1/5/4 signaling is crucial for engaging a robust decidualization program in women with endometriosis.
2024-02-17 | GSE243039 | GEO
Project description:Studies indicate that high-grade serous ovarian carcinoma (HGSOC), the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE). Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events. Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1), tumor necrosis factor (TNF), and cortisol. Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect. The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated. To examine the response of FTE cells and the ability of glucocorticoids to oppose this response, an immortalized human FTE cell line, OE-E6/E7, were treated with IL1β, dexamethasone (DEX), IL1β and DEX, or vehicle and genome-wide gene expression profiling was performed. IL1β altered the expression of 47 genes of which 17 were reversed by DEX. DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1β treatment altered the expression of 784 genes. Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, TACC2 and PI3. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1β that was blocked by DEX. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that dysregulation of glucocorticoid signaling could contribute to increased risk for HGSOC. An immortalized human oviductal cell line, OE-E6/E7 was treated with 10 nM dexamethasone and/or 50 ng/ml IL1b. Cells were harvested 18 hours after treatment and rna was extracted and used for genome-wide gene expression.
2015-01-27 | E-GEOD-54608 | biostudies-arrayexpress
Project description:Endometriosis is a common, chronic inflammatory condition that frequently results in pain and infertility and is estimated to affect 6–10% of women of reproductive age. However, the mechanisms underlying the induction of inflammation in endometriosis remain unclear. Here, we identified that ectopic endometrial T-cell-derived active interleukin (IL)-16 acts as an inflammation-driving cytokine in endometriosis. Furthermore, we found that in ectopic endometrial T cells, the activation of IL-16 requires iron overload-induced caspase-3 activity and that the release of active IL-16 is reliant on GSDME-mediated pyroptosis. These observations suggest that active IL-16 may represent a promising target in the treatment of endometriosis.
2024-04-10 | GSE229735 | GEO