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MiRNA expression profile of pre-transfected HEY cells

ABSTRACT: MicroRNAs (miRNAs) are short (~22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. We wanted to quantify the levels of endogenous miRNAs in pre-transfected HEY ovarian cancer cells to identify the highly expressed miRNAs. It was previously established that transfection of small RNAs can globally perturb gene expression, and one mechanism responsible for such alterations may involve de-repression of targets of endogenous miRNAs because of the saturation of the RISC complexes by the exogenously administered small RNA (Khan et al., 2009. Nat Biotechnol 27, 549-555). By measuring the levels of miRNAs in the pre-transfected HEY cells, we wanted to determine the miRNAs most highly expressed in HEY cells, and therefore, most likely to experience the effects of such competition.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE29126 | GEO | 2012/04/09

SECONDARY ACCESSION(S): PRJNA140393

REPOSITORIES: GEO

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Project description:The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. To understand the effect of exosomal cargo released from locally stimulated cells on distal cell expression, we collected exosomes from local ovarian adenocarcinoma (HEY) cells that were either unstimulated (control-exosomes), stimulated with pIC (pIC-exosomes), or lipopolysaccharide (LPS-exosomes) for 48 hours. The three groups of exosomes were added to naÃ¯ve (distal) cells and the gene expression profiles were compared between local TLR stimulation (for 6 hours) and distal stimulation mediated by exosomes at the 48-hour time point The goal of the study was to delineate the differential effector function of TLR-exosomes based on the innate immune activation state (control-, LPS-, pIC-stimulation) of the cell of origin in vitro. We used microarrays to understand the changes in gene expression in 2 samples of local hey cells either unstimulated(control) or stimulated with poly(I:C) or LPS each. We also profiled 2 samples of distal naÃ¯ve cells that were exposed to either control, LPS or poly(I:C) exosomes from local cells. Here we show that exosomes from TLR stimulated cells (TLR-exosomes) can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. This work definitively establishes the differential effector function for TLR-exosomes in communicating the activation state of the cell of origin. HEY cells were either unstimulated (Local control) or stimulated with LPS or poly (I:C) for 6 hours and the RNA from these cells was profiled using a microarray. Exosomes were collected from local cells that were unstimulated or stimulated for 48 hours and added to distal (naive) HEY cells that were exposed to control exosomes, LPS exosomes or pIC exosomes and then the RNA was collected for analysis by microarray. mRNA expression was captured on Affymetrix U133 Plus 2 chips. mRNA microarray data were analyzed using the Expression Console software (Affymetrix) and Bioconductor tools written in the R statistical programming language. Pre-processing of raw signal intensities and normalization was performed using GCRMA (R). Linear modelling of the transformed data was determined by using Limma in R with the Benjamini and Hochberg correction. Differentially expressed probesets were identified using a threshold 5% FDR correction and a fold change â¥ 1.4 was applied

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MiRNA expression profile of pre-transfected HEY cells

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