Gene expression profiles of lymphatic endothelial cells after knockdown of Prox1 and/or NR2F2
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE12846 | GEO | 2008/09/20
SECONDARY ACCESSION(S): PRJNA111071
REPOSITORIES: GEO
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Project description:The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses and promotes cancer metastasis. MicroRNAs (miRNAs) have recently emerged as key and potent regulators of the genome that control virtually all aspects of cell and organism biology. Surprisingly, the physiological importance and functional activities of miRNAs in the lymphatic vascular system have not been explored. To address this, we first defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs). Comparative analysis of these profiles identified 4 BVEC-signature and 2 LEC-signature miRNAs. Further expression analysis by quantitative real-time PCR analysis and by in situ hybridization (ISH) studies confirmed these vascular lineage-specific expression patterns in vivo. Functional characterization of the BVEC-signature miRNA, miR-31, identified a novel BVEC-specific post-transcriptional regulatory mechanism that inhibits lymphatic-specific transcription programs in vitro and lymphatic vascular development during Xenopus embryogenesis. These effects are, in part, mediated via direct post-transcriptional repression of PROX1, a master regulator of lymphatic lineage-specific differentiation. Together, these findings indicate that miR-31, and miRNAs in general, are potent regulators of vascular lineage-specific differentiation and development. 500,000 LECs were transfected with 2 µM Pre-miR-31 or Pre-miR-Neg molecules in biological duplicate and total RNA isolated using the mirVana isolation kit 48 hours post-transfection. The transcriptome profiles of these cells were defined using the Applied Biosystems Human Genome Survey Microarray v2.0 as described. Briefly, dioxigenin-UTP-labeled cRNA was generated from 1.5 µg of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems). 20 µg cRNA were fragmented and hybridized to the microarrays using the Applied Biosystems Chemiluminescence Detection Kit. Signal detection, image acquisition and initial analyses were performed using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer. Raw data were normalized using Quantile normalization available from R/Bioconductor. Present calls were defined based on average signal-to-noise ratios (S/N ratio) >3 and quality (error) values <5,00025. Feature signal intensities were converted to log2 values. miR-31-repressed genes were identified based on present calls in both Pre-miR-Neg arrays, log2(Pre31/PreNeg) �-0.59 and p-values <0.05, while miR-31-induced genes were present in both Pre-miR-31 arrays, had log2(Pre31/PreNeg) �0.59 and p-values <0.05. P-values were calculated using empirical Bayes statistics for differential expression.
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