Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Silencing of the WFS1 gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage.

ABSTRACT: The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72 and 96 hours after transfection with three different WFS1 siRNAs. In order to verify silencing we performed quantitative RT-PCR and western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and western blot confirmed clear silencing of WFS1 gene expression after 48 hours. Eleven genes had an FDR value less than 10% and most of them were genes related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlation between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest the role of WSF1 gene in cell survival and its involvement in the degenerative diseases. 54 samples; whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72 and 96 hours after transfection with three different WFS1 siRNAs.

ORGANISM(S): Homo sapiens

SUBMITTER: Sulev Koks

PROVIDER: E-GEOD-40585 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:NOX1 gene is member of the NADPH-dependent superoxide producing enzyme family. It generates reactive oxygen species (ROS) in regulated manner in response to growth factors, cytokines and calcium signal. Low levels of ROS play role in many processes including cell proliferation, inflammatory response and mitogenesis. NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequence carrying U6 promoter/shRNA cassettes were cloned into GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. Clone 6A and 6C showed 70-80% decrease in NOX1 expression. The cells were expanded and analyzed further. We measured decreased ROS production, cell proliferation and G1/S block of the cell cycle. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. Significant decrease was measured in the volume of forming tumors. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer. Experiment Overall Design: We have found that NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequences carrying the U6 promoter/shRNA cassettes were cloned into a GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. A significant decrease was measured in the volume of tumors formed. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer.

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Silencing of the WFS1 gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage.

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