Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic analysis of Urokinase plasminogen activator receptor silenced MDA-MB-231 breast cancer cells, stimulated with radiation.

ABSTRACT: Urokinase plasminogen activator receptor (PLAUR) is a GPI anchored extracellular surface receptor, playing a major role in remodeling of the extracellular matrix. It has the ability to interact with a variety of co-receptors for intracellular signaling. Over-expression of PLAUR is observed in many pathological conditions such as cancer, cardiovascular and chronic kidney diseases. We have recently demonstrated a novel role for PLAUR in DNA damage and repair processes. Here, we performed a two color microarray to understand the transcriptomic changes occuring in PLAUR silenced cells on treatment with DNA damaging radiation. Further, we would like to use this data to predict affected pathways and discover targets of therapeutic value.

ORGANISM(S): Homo sapiens

SUBMITTER: Yulia Kiyan

PROVIDER: E-MTAB-5298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. The observation of attenuated and not ablated checkpoint responses in AT cells supports a hypothesis that the ATM- and rad3-related checkpoint kinase ATR also responds to IR-induced DNA damage and complements ATM signaling. Experiment Overall Design: Logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâs Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScanÂ® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.

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Transcriptomic analysis of Urokinase plasminogen activator receptor silenced MDA-MB-231 breast cancer cells, stimulated with radiation.

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