MiRNA expression in aging skin
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Steffen Sass
PROVIDER: E-GEOD-55299 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In this study, we have analyzed DNA methylation changes upon aging of human dermal fibroblasts by using the HumanMethylation27 BeadChip assessing 27,578 unique CpG sites. Cells were isolated from skin samples donated by young (6-23 years) and elderly (60-73 years) patients undergoing surgical interventions. Strikingly, global DNA-methylation profiles of fibroblasts from the same anatomical site clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. Skin samples from younger or elderly donors were treated with dispase (Roche Diagnostics, Mannheim, Germany) for 12 hours to separate the dermis from the epidermis. The dermis was digested with 0,2% collagenase and 1,5% BSA in collagenase buffer (100mM HEPES, 120mM NaCl, 50mM KCl, 1mM CaCl2, 5mM Glucose) for 45 minutes. Dermal remnants were removed by filtering the digest through a 100µm nylon strainer (Falcon, Becton Dickinson [BD], San Jose, USA). The cells were subsequently washed and expanded in standard medium consisting of DMEM (PAA; 1g/L glucose) supplemented with glutamine (PAA), penicillin/sptrepamycin (PAA) and 10% fetal calf serum (Biochrom, Berlin, Germany) in a humidified atmosphere at 5% CO2. Cells were always replated when grown to 80% confluency. For methylation profiles upon aging we have isolated DNA from the samples of passage 3 of younger and elderly donors. For methylation profiles upon long-term culture we have isolated DNA from the samples of early passage (P3) and late passage (P21)
2012-02-14 | E-GEOD-22595 | biostudies-arrayexpress
Project description:Gene expression approach to differences in stress response between human skin fibroblasts derived from chronologically young (~20 years) and old (~90 years) subjects. Expression of CDKN2A was most different between fibroblasts from young and old subjects and Human skin fibroblasts were isolated from skin biopsies of 6 young subjects and 6 old subjects and were treated for 3 hours and 3 days with 0.6 uM rotenone. Also there were control samples, not treated but grown for 3 days, making a total of 36 samples. No experimental replicates were included. The expression of CDKN2A was quantified in the same RNA samples by real-time PCR.
2011-04-01 | E-GEOD-28300 | biostudies-arrayexpress
Project description:Gene expression approach to differences in stress response between human skin fibroblasts derived from chronologically young (~20 years) and old (~90 years) subjects. Expression of CDKN2A was most different between fibroblasts from young and old subjects and
2011-04-01 | GSE28300 | GEO
Project description:BACKGROUND: Vasectomized men who wish for restoration of fertility commonly face problems to conceive despite high surgical success rates of vasovasostomy, a surgery routinely used to retrieve duct patency. In human, vasectomy has consequences on the epididymal transcriptome and is responsible for non-reversible changes that affect the biochemical parameters and functions of ejaculated sperm in vasovasotomized men. Since microRNAs (miRNAs) are key regulators of post-transcriptional gene expression, we explored the consequences of vasectomy on miRNA pattern in the human epididymis and determined the repercussion and reversibility of these changes in seminal microvesicles (SMVs) from semen of vasovasostomized men. METHOD: miRNA signatures from a unique set of human epididymal segments (caput, corpus, cauda) provided by controls and vasectomized donors as well as miRNA content of SMVs isolated from semen of normal, vasectomized and vasovasostomized donors were determined by using high-throughput microarray technology. Epididymal miRNA signature was correlated with gene expression profiles by employing an integrated genomic approach. RESULTS: While the segmented pattern of miRNA expression was maintained along the human epididymis following vasectomy, the array of miRNAs from each anatomical region significantly differed between normal and vasectomized donors. Greatest changes were observed in cauda epididymidis and were negatively correlated with transcription factors. Vasectomy also impacted expression of miRNAs found in SMVs from normal donors, including miRNAs of epididymal origin contained in epididymosomes. Among seminal miRNAs sequelae, 52 were reversible according to miRNA expression level following duct patency retrieval. In contrast, 66 of miRNAs found in SMVs were not retrieved in SMVs after vasovasostomy. CONCLUSION: We showed that vasectomy has a substantial impact on the miRNA signature of the epididymis and SMVs, and that vasovasostomy does not restore all molecular players found in normal fertile individuals. According to the critical role played by miRNAs in the control of male fertility, we believe that miRNA sequelae occurring upstream and downstream of the vasectomy site may be in part responsible for the reduced fertility outcome reported after surgically successful vasectomy reversal. Human epididymides from 3 control (26, 47 and 50 years old. GEO accession number GSE35522) and 3 vasectomized donors (45, 47 and 48 years old), were collected and dissected into 3 anatomical regions (caput, corpus and cauda). Seminal microvesicles (SMVs) were isolated from the semen of normal (pool of 3 donnors), vasectomized (pool of 3 donnors) and vasovasostomized donors (pool of 5 donnors). A cross-comparison between the miRNA signature found in different segments and seminal vesicles was performed using GeneChip miRNA Array 2.0 (Affimetrix, Santa Clara, CA).
2013-02-25 | E-GEOD-43676 | biostudies-arrayexpress
Project description:Fibroblasts were isolated from healthy skin, donors were young, middlege and old. We used Affymetrix miRNA microarrays to detail the global expression of miRNAs.
2014-02-25 | GSE55299 | GEO
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2015-09-02 | E-GEOD-69052 | biostudies-arrayexpress
Project description:To screen for potential miRNA that may contribute to the etiopathogenesis of EDS-HT/JHS miRNA expression profiling was performed using the Affymetrix GeneChip® miRNA 3.0 Array and comparing the miRNA expression changes of skin fibroblasts of five EDS-HT/JHS patients with those of six healthy individuals Comparison of miRNA expression profiles between five EDS-HT/JHS human fibroblasts and six controls
2016-08-05 | E-GEOD-77756 | biostudies-arrayexpress
Project description:Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo-and-extra-terminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2). Co-crystal structures revealed binding modes of RVX-208 and its synthetic precursor and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation HepG2 Cells were treated with eitther DMSO or 0.5uM JQ1 or 5uM RVX-208. Three samples per condition, total of nine samples. Inhbitor treatment was carried out for 4h before RNA was extracted. HepG2 cells (ATCC: HB-8065) were maintained in M-NM-1-MEM (Cat.#BE12-169F; BioWhittaker) supplemented with 10 % heat-inactivated foetal calf serum (PAA #A15-152), non-essential amino acids (Cat. #M7145; Sigma), glutamine (Cat.#M11-004; PAA), and vitamins (Cat.#M6895; Sigma). Cells were grown at 37 M-BM-0C in a humidified cabinet at 5 % CO2 (Heraeus Function Line). For experiments, cells were seeded the day prior to treatment at 2x105/ml. Treatments were performed for 4 h so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved. At harvest, cells were washed once with PBS (Cat.#H15-002; PAA), and lysed in situ using RLT buffer supplemented with 10 M-NM-<l/ml M-NM-2-mercaptoethanol (Cat.#M7522; Sigma). Total RNA was extracted and prepared using RNeasy columns (Cat.#74106 plus; Qiagen) including a Qia shredding step (Cat.#79656; Qiagen) and an on-column DNAse digestion (Cat.#EN0521; Fermentas), according to the manufacturerM-bM-^@M-^Ys instructions. The resulting RNA was quantified and quality controlled using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher). RNA integrity was assessed on a BioAnalyzer (model G2938C; Agilent Laboratories, USA) and all samples had a RNA Integrity Number (RIN) M-bM-^IM-% 9. Labelled sense ssDNA for hybridization was generated from 200 ng starting RNA with the Ambion WT expression kit (Cat.#4411973; Ambion) and the Affymetrix GeneChip WT Terminal Labelling and Controls Kit (Cat.#901525; Affymetrix) according to the manufacturerM-bM-^@M-^Ys instructions. The distribution of fragmented sense ssDNA lengths was measured on the BioAnalyser. The fragmented ssDNA was labelled and hybridized for 17 hours at 45 M-BM-0C on the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000 and the affymetrix Command Console (v.3.2.4; Affymetrix) was used to generate CEL files.
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