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In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

ABSTRACT: MicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response.Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Temporal changes in the expression of cytokines and key molecules associated with cytokine signaling were assessed at 7, 14, and 21 days after dMCAO, using mouse cytokine gene and protein arrays and Western blot analyses. Electron and immunofluorescence confocal microscopy techniques were used to evaluate the ultrastructural changes, as well as altered expression of specific phenotypic markers, at different time points after dMCAO.In the inhibitor-injected mice (inhibitor group), there was a significant decrease in CCL12 and CXCL3 cytokine expression at 7 days and significantly increased levels of major cytokines IL-10, IL-4, IL-6, MIP-1?, IL-5, and IL-17 at 14 days after dMCAO. These temporal changes correlated with altered expression of miR-155 target proteins SOCS-1, SHIP-1, and C/EBP-? and phosphorylation levels of cytokine signaling regulator STAT-3. Electron microscopy showed decreased number of phagocytically active peri-vascular microglia/macrophages in the inhibitor samples. Immunofluorescence and Western blot of these samples demonstrated that expression of leukocyte/ macrophage marker CD45 and phagocytosis marker CD68 was reduced at 7 days, and in contrast, significantly increased at 14 days after dMCAO, as compared to controls.Based on our findings, we propose that in vivo miR-155 inhibition following mouse stroke significantly alters the time course of the expression of major cytokines and inflammation-associated molecules, which could influence inflammation process and tissue repair after experimental cerebral ischemia.

SUBMITTER: Pena-Philippides JC

PROVIDER: S-EPMC5103429 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

Pena-Philippides Juan Carlos JC Caballero-Garrido Ernesto E Lordkipanidze Tamar T Roitbak Tamara T

Journal of neuroinflammation 20161109 1

<h4>Background</h4>MicroRNA miR-155 is implicated in modulation of the inflammatory processes in various pathological conditions. In our previous studies, we demonstrated that in vivo inhibition of miR-155 promotes functional recovery after mouse experimental stroke. In the present study, we explored if this beneficial effect is associated with miR-155 inhibition-induced alterations in post-stroke inflammatory response.<h4>Methods</h4>Intravenous injections of a specific miR-155 inhibitor were i ...[more]

PMID: 27829437

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Project description:Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.

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In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

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In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response.

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