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Introduction
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FIGURE 1: miR-181 is developmentally regulated, enriched in FCS-differentiated astrocytes relative to BDNF-differentiated neurons,
and can be overexpressed or depleted in cultured cells. (A) Expression levels of miR-181b-d in mouse E13 telencephalon tissue versus
mouse adult cortical tissue (181b P < 0.04, 181c P < 0.02, 181d P < 0.03, n 5 4). (B) Expression of miR-181b-d in cultured FCS-differentiated astrocytes compared to BDNF-differentiated neurons derived from neural stem cells (181b P < 0.03, 181c P < 0.01, 181d P < 0.03,
n 5 4). (C) Levels of miR-181 b and c in cultured astrocytes 24 hours after transfection with a mirR-181c mimic (181c P < 0.04, n 5 3).
(D) Levels of miR-181b and miR-181c in cultured astrocytes 48 hours after transfection with miR-181c and miR-181b microRNA hairpin
inhibitors (181b P < 0.04, 181c P < 0.00002, n 5 4). Students t test.
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To assess cytokine levels after altering miR-181 expression and treatment with LPS, astrocyte supernatant was collected 48 h after transfection and analyzed using Bio-Plex Cytokine assays according to the
provided protocol (BioRad). Cytokines assessed were IL-6, IL1-b,
fibroblast growth factor 2 (FGF2), LIF, IL-10, IL-8, HMGB1, and
FIGURE 2: Decline in miR-181 levels following exposure to LPS in the cerebral cortex of mice is TNFa-dependent and modifies cell proliferation and vulnerability to degeneration in a cell culture model of inflammation. (A) miR-181b-d expression in cerebral cortex from
LPS-treated wild-type mice versus vehicle-treated control mice, 4 h after LPS administration (181b P < 0.02, 181c P < 0.007, 181d P <
0.02, n 5 4). Students t test. (B) miR-181b-d expression in cortex from LPS treated TNFa receptor knockout mice, 4 h after IP injection
of LPS (181d P < 0.04, n 5 3). Students t test. (C) Effects of miR-181 overexpression (OE) or knockdown (KD) on the vulnerability of cultured astrocytes to cell death caused by exposure to LPS, as measured by LDH levels in the culture medium 48 h after transfection and
6 h after LPS treatment (1 lg/ml LPS: OE v KD P < 0.01, OE v CTRL P < 0.01; 250 ng/ml LPS OE v KD P < 0.05; n 5 4). 2-way ANOVA
and Bonferroni posthoc test. (D) In cultured astrocytes 48 h after miR-181 OE or miR-181 KD, followed by a 6 h LPS treatment, the relative levels of cellular metabolic activity were measured using an MTS reduction assay (250 ng/ml: OE v CTRL P < 0.001, OE v KD P <
0.001; n 5 4). 2-way ANOVA and Bonferroni posthoc test.
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TNFa. Multiplex assays were also performed for IL-5, IL-4, IL-13,
IFN-c, IL-12, IL-7, IL-2, and SAA, but all were below detection
threshold. Significance was assessed using Prism (Graphpad Software,
La Jolla, CA) using 1-way ANOVAs for each protein measured.
TNFa signaling played a role in the observed decrease in miR181 expression, we injected TNFR1/TNFR2 double knockout
(DKO) mice with LPS and examined miR-181 expression in
cortex. In contrast to WT mice, LPS did not cause a significant
reduction of miR-181b and miR-181c in DKO mice (Fig. 2B).
However, there was a significant decrease of miR-181d levels in
response to LPS in DKO mice (P < 0.04; Fig. 2B).
OE of miR-181c resulted in increased cell death as measured by LDH release, regardless of LPS concentration or control
treatment (Fig. 2C). Interestingly, a small but significant
increase in cellular proliferation as measured by MTS was
observed in astrocytes over-expressing miR-181c relative to control and knock-down at the 250 ng/ml concentration (Fig. 2D).
To ascertain the effects of reducing or overexpressing
miR-181 on cytokine and growth factor production, we performed multiplex immunoassays for factors relevant to a reactive astrocyte phenotype including LIF, FGF2, IL-6, TNF-a,
IL-1b, IL-10, IL-8, and HMGB1 (Fig. 3) Overexpression of
Results
We first investigated the developmental specificity of miR181 expression by comparing levels of miR-181b, c, and d in
cerebral cortical tissue from mice at different developmental
stages from embryonic day 13 (E13) to adult (Fig. 1A). All
three of these miR-181 family members were expressed at significantly higher levels in adult cortex relative to embryonic
telencephalon. We next examined miR-181 expression in cultured astrocytes compared to E13 neural stem cells differentiated into neurons during a 2 week culture period in the
absence of FGF2 and epidermal growth factor, and in the
presence of brain-derived neurotrophic factor (BDNF) (Fig.
1B). miR-181 family members were strongly enriched in cultured astrocytes relative to BDNF-differentiated neurons.
In anticipation of subsequent experiments, we determined
the efficacy of miR-181c OE or knockdown of miR-181b and
miR-181c on levels of these miRNAs in cultured cortical astrocytes. miR-181c was significantly elevated in astrocytes transfected with miR-181c (Fig. 1C). Levels of miR-181b and miR181c were significantly reduced in astrocytes transfected with
miRNA hairpin inhibitors against these miRNAs (Fig. 1D).
To explore the relationship between miR-181 expression
and inflammatory responses, cortex from wild-type animals
injected with either LPS or saline was examined for miR-181
expression. We found that expression levels of miR-181b, miR181c, and miR-181d were significantly decreased 4 h after LPS
injection (Fig. 2A), a time point when TNFa production was
increased in cortex (Kawamoto et al., 2013). To determine if
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FIGURE 3: Reduction of miR-181 levels alters both basal and LPSinduced cytokine production. (A) Multiplex immunoassay was used
to detect levels of LIF (CTRL v LKD P < 0.001; OE v LKD P < 0.001;
LKD v KD P < 0.001; LOE v LKD P < 0.001; LCTRL v LKD P <
0.001), IL-6 (OE v LKD P < 0.01; CTRL v LKD P < 0.01; KD v LKD
P < 0.01; LOE v LKD P < 0.05), TNF-a (CTRL v LKD P < 0.001; CTRL
v LCTRL P < 0.05; CTRL v LOE P < 0.05; OE v LKD P < 0.001; OE v
LCTRL P < 0.05; OE v LOE P < 0.05; KD v LKD P < 0.001; KD v
LCTRL P < 0.01; KD v LOE P < 0.01; LOE v LKD P < 0.01; LCTRL v
LKD P < 0.01), IL-1b (KD v LKD P < 0.01; CTRL v LKD P < 0.01; OE
v LKD P < 0.01; LOE v LKD P < 0.05; LCTRL v LKD P < 0.05), IL-10
(CTRL v LOE P < 0.001; CTRL v LCTRL P < 0.001; CTRL v LKD P <
0.05; KD v LOE P < 0.001; KD v LCTRL P < 0.001; KD v LKD P <
0.01; OE v LOE P < 0.001; OE v LKD P < 0.01; LKD v LOE P <
0.001; LKD v LCTRL P < 0.01; LCTRL v LOE P < 0.001), IL-8 (OE v
LKD P < 0.001; OE v LCTRL P < 0.001; OE v LOE P < 0.05; CTRL v
LKD P < 0.001; CTRL v LCTRL P < 0.001; CTRL v LOE P < 0.05; KD
v LKD P < 0.001; KD v LCTRL P < 0.001; KD v LOE P < 0.01; LOE v
LKD P < 0.001; LOE v LCTRL P < 0.01; LCTRL v LKD P < 0.001) and
HMGB1 (CTRL v LKD P < 0.05; OE v LKD P < 0.05; KD v LKD P <
0.05; LOE v LKD P < 0.05; LCTRL v LKD P < 0.05) in media from cultured astrocytes that had been treated with either 1 mg/ml LPS or
vehicle (n 5 3; a prefix of L indicates LPS condition). ANOVA and
Newman-Keuls posthoc test. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
FIGURE 4: Reduction of miR-181 expression alters levels of transcripts involved in cell death and inflammation responses. (A) A list of
the twenty transcripts that increased by the greatest amount, and the twenty transcripts that decreased by the greatest amount in
response to simultaneous knockdown of miR-181b and miR-181c. (B) Pathway analysis of transcriptional changes demonstrated that
alterations related to cell death, cell cycle regulation, cell differentiation, cancer and inflammatory responses, and related signaling pathways were affected. (C) Signaling pathways of interest altered by miR-181b and c knock-down included iNOS signaling. [Color figure can
be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 5: Evidence that miR-181 regulates diverse signaling pathways. (A) The mRNAs encoding SerpinB2 (P < 0.007, n 5 4), RSad2 (P
< 0.02, n 5 4), Prl2c3 (P < 0.007, n 5 4), Prl2c4 (P < 0.03, n 5 4) and CCL3 (P < 0.04, n 5 4) increased significantly after reducing miR181b and c expression. (B) Increased levels of mRNAs encoding HMGA1 (P < 0.007, n 5 4), GDNF (P < 0.004, n 5 3) and NFKBia (P <
0.008, n 5 4) after silencing miR-181b/c. (C) Lower levels of BMP4 (P < 0.05, n 5 4) and SMOC1 (P < 0.04, n 5 4) mRNAs in astrocytes
in which miR-181b/c levels were reduced. Students t test.
Discussion
We found that miR-181 expression is developmentally regulated
and that miR-181s are enriched in astrocytes compared to neurons. It was previously reported that miR-181s play roles in cell
fate specification in the hematopoietic system and developmental patterning of muscle cell fate (Chen et al., 2004; Naguibneva
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et al., 2006). Expression of miR-181 is also implicated in differentiation of stem cells by repressing Lin28 (Li et al., 2012).
Taken together with our observation that miR-181b, c, and d
are present at high levels in the mature CNS, but not early in
development prior to astrogenesis, these data support a possible
role for miR-181 in developmental specification of cell fate in
the CNS and should be an area of further investigation.
Our data suggest that in the adult brain miR-181 expression is dynamically regulated by inflammatory stimuli and
modifies the proliferation of astrocytes and their sensitivity to
death in an experimental model of neuroinflammation. Previous
studies have suggested roles for miR-181s in modifying the sensitivity of cells to death in ischemic brain injury and glioblastoma, with particular emphasis on its regulation of the Bcl-2
transcript (Chen et al., 2010; Ouyang et al., 2012a). It should
be noted that miR-181 regulates other transcripts involved in
cell survival and responses to stress including hsp-70, MCL-1
and Bim (Ouyang et al., 2012a,b). We found that an inflammatory stimulus, the TLR4 receptor ligand LPS, decreased the levels of the miR-181s in vivo, suggesting that the previously
observed alteration in miR-181 expression in ischemic stroke
models could be due to a TNF-a independent component of
the injury. Our results therefore suggest that miR-181s are
involved in a general cell stress response and cell survival-related
transcriptional profiles.
FIGURE 6: Direct interaction of miR-181 with transcriptome. (A) Top 40 most enriched transcripts in a PD of biotinylated miR-181c. (B)
Pathway analysis demonstrating that transcripts that interact with miR-181c are involved in functions of interest including cell death. (C)
Pathways of putative miR-181c targets include EIF2 signaling, mTOR signaling and mitochondrial dysfunction. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
The most striking phenotype that we observed as a consequence of miR-181 reduction was a dramatic increase in the
production of inflammatory cytokines including LIF, IL-6,
TNF-a, IL-1b, IL-8 and HMGB1. In contrast, we found that
levels of the anti-inflammatory cytokine IL-10 (de Vries, 1995;
Murray, 2005) were increased in astrocytes when miR-181 was
overexpressed. This suggests that miR-181 might play an antiinflammatory role when expressed while reduction of miR181, as observed in vivo in animals exposed to LPS, might
contribute to the production of inflammatory cytokines. We
also found that levels of FGF2, a potent mitogen for astrocytes
(Joy et al. 1997) with a potential role in regulation of astrocyte
differentiation (Irmady et al., 2011), trended towards an
increase as a result of knocking down miR-181 and exposing
astrocytes to LPS. FGF2 is also known to protect neurons
against conditions associated with inflammation including oxidative stress and excitotoxicity (Mattson et al., 1989; Mark
et al., 1997), suggesting that suppression of FGF2 production
by miR-181s could render neurons vulnerable to such adverse
conditions.
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FIGURE 7: MeCP2 and XIAP mRNAs are direct targets of miR-181. (A) NAMPT, Bcl-2 and HMGA1 mRNAs are all enriched in biotin pulldown of miR-181c (HMGA1 P < 0.08, n 5 3, Bcl-2 P < 0.02, n 5 4, NAMPT P < 0.07, n 5 4). (B) Schematic representation of Psi-CHECK2
reporter constructs generated to test putative miR-181 targets MeCP2 and XIAP mRNAs. (C) Analysis of Psi-CHECK2 MeCP2 reporter
activity 48 h after miR-181 OE (P < 0.0004, n 5 6). (D) Analysis of Psi-CHECK2 XIAP reporter activity 48 h after miR-181 OE (P < 0.001,
n 5 6). Students t test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Acknowledgment
Grant sponsor: Intramural Research Program of the National
Institute on Aging.
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