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Genome-wide DNA methylation analysis of different cell types

ABSTRACT: We performed whole-genome methylation analysis using 450K Illumina BeadArrays on different human cell types. In total 24 experiments were performed. Dermal fibroblasts, three different epidermal melanocytes (dark, medium and light pigmentation), epidermal keratinocytes, mammary fibroblasts, mammary epithelial cells, mammary endothelial cells and mesenchymal stem cells were analyzed in technical duplicates. Unmethylated DNA were analyzed in technical duplicates. Two different normal breast tissue samples were analyzed. Finally peripheral blood leukocytes and an enzymatically methylated sample were analyzed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE74877 | GEO | 2016/02/29

SECONDARY ACCESSION(S): PRJNA301715

REPOSITORIES: GEO

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Project description:Analysis of the transcriptional profiles of mRNA and microRNA in Rasless fibroblasts. 4-Hydroxy-tamoxifen (4-OHT) treatment triggers removal of K-Ras expression in [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/ert] mouse fibroblasts (named K-Raslox) generating M-bM-^@M-^\RaslessM-bM-^@M-^] MEFs which are unable to proliferate, but recover proliferative ability after ectopic expression of constitutively active downstream kinases such as BRAF and MEK1. Using Affymetrix microarrays, we compared the transcriptional profiles of Rasless fibroblasts to those of K-Raslox (MEFs lacking only H-Ras and N-Ras). We also compared the transcriptional profile of Rasless cells with those of BRAF and MEK1-transfected Rasless cells which recovered proliferative ability. Re-entry of the Rasless cells into cell cycle progression through ectopic expression of activated BRAF or MEK1 resulted in full reversion of most transcriptional alterations identified in cells lacking the three canonical Ras proteins. Pre-confluent cell cultures corresponding to the different Ras genotypes were harvested for extraction of total RNA and miRNA samples, and subsequent hybridization on Affymetrix microarrays. The different sets of experimental mRNA samples analyzed here included (i) 8 samples K-Raslox (proliferating cells expressing only K-Ras, considered as Control): 4 biological replicates, technical duplicates each; (ii) 7 samples Rasless: the same cells after treatment with 4-OHT for 12 days (to become non-proliferating fibroblasts), including 4 biological replicates and technical duplicates from 3 of them; (iii) 3 samples BRAF-rescued MEFs (Rasless cells harboring activated BRAF constructs, with regained proliferative ability): 3 biological replicates; (iv) 3 samples MEK1-rescued MEFs (Rasless cells harboring activated MEK1 constructs, with regained proliferative ability): 3 biological replicates; and their respective controls harbouring the vempty vectors (v) 2 samples Hygromycin-resistant (control for BRAF-transfected Rasless cells) including 2 biological replicates, and (vi) 4 samples Puromycin-resistant (control for MEK1-transfected Rasless cells) including 2 biological replicates, technical duplicates each. The gene expression study contains a total of 27 samples. Regarding the miRNA expression study, the experimental samples analyzed included (i) 4 samples samples K-Raslox (used as Control): 2 biological replicates, technical duplicates each; (ii) 8 samples Rasless: the same cells after treatment with 4-OHT for 6 days (2 biological replicates, technical duplicates each) and 12 days (2 biological replicates, technical duplicates each); (iii) 4 samples BRAF-rescued, including 2 biological replicates, technical duplicates each; (iv) 4 samples MEK1-rescued,including 2 biological replicates, technical duplicates each, and (v) 4 samples Puromycin-resistant empty vector, containing 2 biological replicates, technical duplicates each. The miRNA expression study contains a total of 24 samples.

Project description:BACKGROUND: Ductal carcinoma in situ (DCIS) is the earliest stage of breast cancer. During DCIS, tumor cells remain inside the mammary duct, growing under a microenvironment characterized by hypoxia, nutrient starvation, and waste product accumulation; this harsh microenvironment promotes genomic instability and eventually cell invasion. However, there is a lack of biomarkers to predict what patients will transition to a more invasive tumor or how DCIS cells manage to survive in this harsh microenvironment. </br> METHODS: In this work, we have developed a microfluidic model that recapitulates the DCIS microenvironment. In the microdevice, a DCIS model cell line was grown inside a luminal mammary duct model, embedded in a 3D hydrogel with mammary fibroblasts. Cell behavior was monitored by confocal microscopy and optical metabolic imaging. Additionally, metabolite profile was studied by NMR whereas gene expression was analyzed by RT-qPCR. </br> FINDINGS: DCIS cell metabolism led to hypoxia and nutrient starvation; revealing an altered metabolism focused on glycolysis and other hypoxia-associated pathways. In response to this starvation and hypoxia, DCIS cells modified the expression of multiple genes, and a gradient of different metabolic phenotypes was observed across the mammary duct model. These genetic changes observed in the model were in good agreement with patient genomic profiles; identifying multiple compounds targeting the affected pathways. In this context, the hypoxia-activated prodrug tirapazamine selectively destroyed hypoxic DCIS cells. </br> INTERPRETATION: The results showed the capacity of the microfluidic model to mimic the DCIS structure, identifying multiple cellular adaptations to endure the hypoxia and nutrient starvation generated within the mammary duct. These findings may suggest new potential therapeutic directions to treat DCIS. In summary, given the lack of in vitro models to study DCIS, this microfluidic device holds great potential to find new DCIS predictors and therapies and translate them to the clinic.

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Genome-wide DNA methylation analysis of different cell types

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