Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles due to common seed sequence of members of miR-302/372/373/520 family miRNAs and dsEcad640 in human PC-3 cells

ABSTRACT: To examine the genome-wide gene regulation due to the common seed sequence of the members of miR-302/3272/3273/520 family miRNAs and dsEcad640, we performed microarray profiling of gene expression by the transfection of chemically synthesized dsEcad640, miR-302a,miR-372, miR-373, miR-520c,and miR-520f duplexes into PC-3 cells. Chemically synthesized dsEcad640, miR-302a, miR-372, miR-373, miR-520c,and miR-520f duplexes were transfected into PC-3 cells. At 24 hours after transfection,the total RNA was extracted using the RNeasy Mini Kit (Qiagen). As a control, mock transfected cells treated with transfection reagent without siRNA and miRNA were used.Transfection was carried out four times independently, and the RNA quality was examined using Bioanalyzer (Agilent). Mixed RNA samples (150 ng total) of four experiments were used for the microarray analysis. After cDNA was synthesized with the Agilent One Color Spike Mix Kit, cRNA was prepared using the Quick Amp Labeling Kit (Agilent), labeled with Cy-3, and purified with the RNeasy Mini Kit (Qiagen).The cRNA was hybridized against the Agilent Whole Human Genome Microarray (4 × 44 K multipack format) at 65°C for 17 hours, then washed with Gene Expression Wash Buffer 1 and Wash Buffer 2 (Agilent) in 0.005% Triton X-102. The microarray slide was scanned using the DNA Microarray Scanner (Agilent) and quantified using Feature Extraction Software (Agilent).

ORGANISM(S): Homo sapiens

SUBMITTER: Kumiko Ui-Tei

PROVIDER: E-GEOD-32999 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) are an evolutionarily conserved large class of small non-coding RNAs that mediate post-transcriptional silencing of genes and influence a broad spectrum of biological processes ranging from embryonic development to organismal death. Our previous study identified the miR-29 family, three paralogous species of miR-29a/b/c, as the most predominantly expressed small RNA in aged mouse brain compared to neonate one. The mouse brain miR-29 is highly astrocytic. Its expression is quiescent during early brain development, and thereafter steadily increases to a saturating level around the end of the reproductive phase. To explore the functional relevance of miR-29 expression to the neural physiology of mouse brain from the mechanistic perspective of mammalian species-specific lifespan, we here designed a gain-of-function approach through forced expression of miR-29 in astrocyte from mouse fetus and surveyed the resulting alteration in the transcriptional and translational levels. DNA microarray analyses retrieved a total of 5,589 genes showing a temporal significant expression changes in the miR-29-transfected fetal astrocytes, and classified them into two gene groups positively or negatively regulated by miR-29. Mass spectrometry (MS)-based quantification of translational products of miR-29-responsive genes identified 18 species of miR-29 target candidates. We performed functional enrichment analyses using bioinformatics resources to elucidate the biological roles of the gene sets thus identified, and obtained their expression trend that favor the processes for facilitating cell differentiation while supporting normal cell proliferation /survival, which was different from the functional signatures of miR-29 at adult stages, implying the bifunctional property of miR-29 depending on the developmental context. Our present results strongly suggest that miR-29 serves as the central coordinator to shift the global gene expression in the developing mouse brain toward matured phenotype, and through which ensure the programmed transition to the post-developmental stage that is inherently set for the mouse lifespan. DNA microarray experiment: Sample labeling, hybridization and washing were performed following the standard protocol detailed in the Agilent One-Color Microarray-Based Gene Expression Analysis ver.5.7. Briefly, a One-color Spike-Mix was diluted 1000-fold and a 5-μl aliquot of the diluted mix were added to every 0.5 μg of total RNA samples (three each of extraction replicates of miR-29-treated sample and control sample named elsewhere) prior to labeling reaction. The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP. The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2. The processed microarrays were scanned using an Agilent DNA Microarray Scanner. Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.

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Gene expression profiles due to common seed sequence of members of miR-302/372/373/520 family miRNAs and dsEcad640 in human PC-3 cells

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