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.

Project description:Neonates are highly susceptible to repeat infection and respond poorly to vaccination; these attributes derive from intrinsic differences between neonatal and adult naïve CD8+ T cells. In contrast to adult cells, naïve neonatal cells exhibit a greatly limited ability to differentiate into memory cells, a fundamental property of their adult counterparts. Here, we describe the role of the miR-29 microRNA in naïve T cells, focusing on age-related differences in miR-29 expression and the consequences of these differences in adult and neonatal cells, from mice and humans. In adults, high expression of miR-29 licenses naïve cells towards eventual memory cell fates; whereas neonatal naïve cells, which lack high expression of miR-29, are predisposed towards effector cell fates in response to an infection. Multiple lines of evidence support this model, including analysis of a mouse model deficient in miR-29, which we examine with adoptive transfer experiments to define the functional consequences of reduction of miR-29, together with genomic assays to define the regulatory impact of miR-29. Adult miR 29 deficient naïve CD8+ T cells cell are primed for activation and therefore secrete elevated levels of cytolytic molecules, and express transcription factors at levels associated with effector cell differentiation; moreover, these cells exhibit an altered CD8+ T cell memory repertoire, akin to that of neonatal CD8+ T cells. Importantly, we use a method that exploits extracellular vesicles as a delivery vehicle with which to modulate levels of miR-29 in neonatal and adult naïve T cells, examining both human and mouse cells. For example, increasing miR 29 expression in mouse naïve neonatal CD8+ T cells significantly improved the memory response during infection, concomitant with alterations to the chromatin landscape characteristic of cells primed for memory differentiation. Delivery of miR-29 antagomirs to human adult naïve CD8+ T cells was sufficient to induce the adult cells to adopt phenotypes and gene expression signatures normally found in cells present in newborns. This study establishes miR-29 as a key conserved regulator in naïve CD8+ T cells, and by adjusting levels of miR-29, has the potential to underlie therapeutic strategies for controlling the balance of effector versus memory fates in human T cells.

Dataset Information

Global profiling of gene expression in mouse astrocyte in response to the potential longevity determinant miR-29

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