MicroRNA Expression And Its Regulatory Mechanism In Aging Mouse Macrophages

Although increasing evidence has shown that miRNA expression contributes to the regulation of aging, a multifactorial weakening and disease-prone physiological process, the role of miRNAs in age-related changes of macrophages, especially in inflammatory response remains so far unclear. Age-associated immune dysfunction, characterized by increased systemic levels of cytokines, such as TNFa and IL-6, manifests as an increased susceptibility to infections. Thus, understanding these negative regulators of the immune response has paved the way to delineating signaling pathways that cross talk and impact immune senescence.To this end, we analyzed the differential miRNA expression profile in peritoneal macrophage of natively aged mice stimulated with lipopolysaccharide (LPS), mimic an inflammatory conditions. By a LNA-based microarray assay,16 miRNAs, which were irrespective to LPS treatment in macrophages from aging mice but up-regulated or down-regulated in the young group, were considered resulting in malfunction of macrophage of aging mice. And four of them, miR-146a?miR-223?miR-28 and miR-101a were identified by qRT-PCR (quantitative real-time RT-PCR), in agreement with the results of microarray hybridization. Further comparative bioinformatics analysis, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) made function and pathway annotation for their predicted and reported target genes, revealing that 17 pathways were enriched. Moreover, the identified target genes of significant GOs and pathways were then taken to build up miRNA-gene network in immune, apoptosis, epigenetics and transcription factor.It was demonstrated by array-based hybridization and quantitative PCR that miR-146a was highly expressed and lack of response to LPS stimulation in the macrophages of senescent mice both in vitro and in vivo. Owing to miR-146a irresponsive to the stimulation of LPS and pro-inflammatory environment in the senescent mouse macrophages, we deduced that miR-146a expression was regulated by other mechanism, and abnormal miR-146a level was associated with the production of aberrant proinflammatory mediators in senescent mice. Study on the regulation of miR-146a expression showed that aberrant NF-?B binding to pre-miR-146a promoter contributed to the abnormal expression of miR-146a in the senescent mouse macrophages. DNA methylation and histone acetylization were also involved in the regulation of miR-146a expression in aged mice. Histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), significantly up-regulated miR-146a transcriptional activation by altering I?B?phosphorylation and DNA binding activity of NF-?B p65 in LPS-stimulated macrophages of senescent mice much more than those from young, suggesting that HDAC suppresses miR-146a expression in LPS-treated macrophages of aging mice and contribute to infiamm-aging.Moreover, we investigated the effects of miR-146a on the Th1/h2 cytockine expression in mouse RAW264.7 cell line and primary peritoneal macrophage. miR-146a mimics, mimics negative control (NC mimics), inhibitor miR-146a and inhibitor negative control (NC inhibitor) were transfected into RAW264.7 cells and freshly isolated peritoneal macrophage. IL-18, IL-5 and IL-10 expressions in the cells were measured by real time PCR. These data demonstrate at first time that miR-146 can regulate Thl cytockine IL-18 expression, but without effect on Th2 cytockines IL-5 and IL-10 expression.In conclusion, we analyzed the differential miRNA expression profile in peritoneal macrophage of natively aged mice, and predicted their target genes and function. These data indicate that dysregulated expression of many miRNAs including miR-146a results in age-associated dysfunction of macrophages, and NF-?B and epigenetics were involved in miR-146a expression regulation.These miRNAs might be promising biomarkers and therapeutics targets in retarding immunosenescence.