Glucose depletion activates mmu-miR-466h-5p expression through oxidative stress and inhibition of histone deacetylation.
ABSTRACT MicroRNAs (miRNAs) are involved in the regulation of multiple cellular processes. Changes of miRNA expression have been linked to the development of various diseases including cancer, but the molecular events leading to these changes at different physiological conditions are not well characterized. Here we examined the intracellular events responsible for the miR-466h-5p activation in mouse cells exposed to glucose deprivation. MiR-466h-5p is a member of the miR-297-669 cluster located in intron 10 of Sfmbt2 gene on mouse chromosome 2 and has a pro-apoptotic role. We showed that the time-dependant activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation caused by glucose deprivation-induced oxidative stress. This oxidative stress causes the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity, reduced protein levels of HDAC2 and increased acetylation in miR-466h-5p promoter region, which led to the activation of this miRNA. Based on this study and previous work, we suggest a possible role of miR-466h-5p (and miR 297-669 cluster) in the cells during toxic metabolites accumulation. Improved characterization of the molecular events that lead to the activation of miR-466h-5p may provide a better understanding of the relation between cellular environment and miRNA activation.
- SourceAvailable from: Stephen Kwok-Wing Tsui[Show abstract] [Hide abstract]
ABSTRACT: This study aimed at elucidating how Coxsackie B virus (CVB) perturbs the host's microRNA (miRNA) regulatory pathways that lead to antiviral events. The results of miRNA profiling in rat pancreatic cells infection models revealed that rat rno-miR-466d was up-regulated in CVB infection. Furthermore, in silico studies showed that Coxsackie virus and Adenovirus Receptor (CAR), a cellular receptor, was one of the rno-miR-466d targets involved in viral entry. Subsequent experiments also proved that both the rno-miR-466d and the human hsa-miR-466, which are orthologs of the miR-467 gene family, could effectively down-regulate the levels of rat and human CAR protein expression, respectively. Copyright © 2014. Published by Elsevier B.V.FEBS Letters 12/2014; · 3.34 Impact Factor
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ABSTRACT: Both exposure to ionizing radiation and obesity have been associated with various pathologies including cancer. There is a crucial need in better understanding the interactions between ionizing radiation effects (especially at low doses) and other risk factors, such as obesity. In order to evaluate radiation responses in obese animals, C3H and C57BL/6J mice fed a control normal fat or a high fat (HF) diet were exposed to fractionated doses of X-rays (0.75 Gy ×4). Bone marrow micronucleus assays did not suggest a modulation of radiation-induced genotoxicity by HF diet. Using MSP, we observed that the promoters of p16 and Dapk genes were methylated in the livers of C57BL/6J mice fed a HF diet (irradiated and non-irradiated); Mgmt promoter was methylated in irradiated and/or HF diet-fed mice. In addition, methylation PCR arrays identified Ep300 and Socs1 (whose promoters exhibited higher methylation levels in non-irradiated HF diet-fed mice) as potential targets for further studies. We then compared microRNA regulations after radiation exposure in the livers of C57BL/6J mice fed a normal or an HF diet, using microRNA arrays. Interestingly, radiation-triggered microRNA regulations observed in normal mice were not observed in obese mice. miR-466e was upregulated in non-irradiated obese mice. In vitro free fatty acid (palmitic acid, oleic acid) administration sensitized AML12 mouse liver cells to ionizing radiation, but the inhibition of miR-466e counteracted this radio-sensitization, suggesting that the modulation of radiation responses by diet-induced obesity might involve miR-466e expression. All together, our results suggested the existence of dietary effects on radiation responses (especially epigenetic regulations) in mice, possibly in relationship with obesity-induced chronic oxidative stress.PLoS ONE 08/2014; 9(8):e106277. · 3.53 Impact Factor
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ABSTRACT: Background Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1.ResultsAmong the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control.Conclusions Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.Journal of biomedical science. 01/2015; 22(1):3.
Glucose depletion activates mmu-miR-466h-5p
expression through oxidative stress and inhibition
of histone deacetylation
Aliaksandr Druz1,2, Michael Betenbaugh2and Joseph Shiloach1,*
1Biotechnology Core Laboratory National Institute of Diabetes and Digestive and Kidney Diseases, National
Institute of Health Bldg 14A Bethesda, MD 20892 and2Department of Chemical and Biomolecular Engineering,
Johns Hopkins University, Baltimore MD 21218, USA
Received March 10, 2012; Revised April 27, 2012; Accepted April 28, 2012
MicroRNAs (miRNAs) are involved in the regulation
of multiple cellular processes. Changes of miRNA
expression have been linked to the development of
various diseases including cancer, but the molecu-
lar events leading to these changes at different
physiological conditions are not well characterized.
Here we examined the intracellular events respon-
sible for the miR-466h-5p activation in mouse cells
exposed to glucose deprivation. MiR-466h-5p is a
member of the miR-297-669 cluster located in
intron 10 of Sfmbt2 gene on mouse chromosome 2
and has a pro-apoptotic role. We showed that the
miR-669c and the Sfmbt2 gene followed the inhib-
ition of histone deacetylation caused by glucose
deprivation-induced oxidative stress. This oxidative
stress causes the accumulation of reactive oxygen
species (ROS) and depletion of reduced glutathione
(GSH) that together inhibited histone deacetylases
(HDACs) activity, reduced protein levels of HDAC2
promoter region, which led to the activation of this
miRNA. Based on this study and previous work, we
suggest a possible role of miR-466h-5p (and miR
297-669 cluster) in the cells during toxic metabolites
accumulation. Improved characterization of the mo-
miR-466h-5p may provide a better understanding
of the relation between cellular environment and
MicroRNAs (miRNAs), small non-coding single-stranded
RNAs (18–25 nucleotides) have been shown to regulate
gene expression in various cellular processes and functions
such as cell development, differentiation, metabolism, pro-
liferation and apoptosis (1–3). Alterations in miRNA ex-
pression profiles have been linked to cancer development
and progression (1,4). Changes in miRNA expression
profiles have also been explored as biomarkers for
various diseases (5–7).
Although the mechanisms of miRNA-guided regulation
of mRNAs expression have received considerable atten-
tion (4,8), the molecular events leading to miRNAs acti-
vation are not well known. Several studies demonstrated
that miRNA expression can be regulated by transcription
factors such as c-Myc, Hif-1alpha, p53 and NF-kB (8–10).
Some miRNAs were shown to be regulated by the inhib-
ition of DNA methylation and histone deacetylation
(11–13), but the molecular events leading to these events
under different physiological conditions are not clear.
In our previous study, we showed that the mouse miR
297-669 cluster was activated in nutrient depleted condi-
tions (2). This cluster is located in intron 10 of the mouse
Sfmbt2 gene on Chromosome 2 and is composed of more
than 40 miRNAs. All detected members of this cluster had
low expression levels in CHO cells when grown in fresh
media but were up-regulated in response to nutrients de-
pletion. One member of this cluster, mmu-miR-466h-5p,
was shown to have a pro-apoptotic role through targeting
of several anti-apoptotic genes which led to Caspase-3/7
activation and loss of cell viability. In another study,
several other members of this cluster were up-regulated
when mice liver were exposed to high acetaminophen con-
centrations and therefore, these miRNAs were suggested
as potential biomarkers for drug-induced liver injury (7).
Another member of the miR 297-669 cluster, mmu-
miR-669c, was shown to be associated with regulation of
glutathione metabolism in the liver of aging mice (14).
In the current study, we investigated the possibility
that the low expression of miR-466h-5p in cells grown in
fresh media may be the result of transcriptional silencing
in the promoter region of this miRNA, and that its
*To whom correspondence should be addressed. Tel: +1 301 496 9719; Fax: +1 301 451 5911; Email: firstname.lastname@example.org
Published online 25 May 2012Nucleic Acids Research, 2012, Vol. 40, No. 15 7291–7302
Published by Oxford University Press 2012.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
correlated to metabolic stress and changes in transcrip-
tional regulation. Stress from intracellular toxicity is
known to facilitate the accumulation of various reactive
species, including reactive oxygen species (ROS), espe-
cially when the concentration of intracellular reduced
glutathione (GSH) is low (7,15) as GSH is known to neu-
tralize ROS and other toxic metabolites. We therefore,
hypothesized that nutrient depletion leads to the accumu-
lation of ROS and to GSH depletion that causes oxidative
stress and loss of cellular detoxification capacity, trigger-
ing miR-466h-5p activation.
Glucose deprivation is a commonly investigated meta-
bolic stress that induces signal transduction and gene ex-
pression (16,17). Indeed, glucose deprivation was shown
to cause cytotoxicity and oxidative stress in human cancer
cells (17–19). The lack of intracellular glucose leads to
production of intracellular ROS by uncoupling glucose
metabolism from the oxidative transport chain activity.
Mammalian cells were shown to respond to glucose
diminish ROS generation (i.e. H2O2). But prolonged
which is necessary to keep GSH in a reduced form (17).
The accumulation of ROS can mediate the signal
transduction cascade, the activation of stress kinases
and substrates phosphorylation
studies have shown the reduced activity of histone
deacetylases (HDACs) during oxidative stress (20–22).
Histone deacetylase 2 was shown to be phosphorylated,
ubiquitinated and degraded in response to oxidative stress
induced by cigarette smoke (21). Reduction of HDACs
activity leads to increased acetylation of histones which
results in DNA uncoiling that allows the binding of tran-
scriptionalfactors and increased
In this study, we demonstrate that glucose deprivation
leads to accumulation of ROS in a time-dependent
manner that decreases HDACs activity and particularly
reduces levels of HDAC2. The inhibition of HDACs
activityresults in the
miR-466h-5p promoter region and up-regulation of this
miRNA. This study presents a novel approach linking
the regulation of miRNA expression to the molecular
events at the known physiological condition.
in nutrient-depletedconditions (2)is
MATERIALS AND METHODS
Mouse cell lines (B/CMBA.Ov) were purchased from
ATCC, Manassas, VA, USA (Cat. No. CRL-6331_FL)
and grown in DMEM media supplemented with 5%
non-dialyzed fetal bovine serum (Life Technologies,
Gaithersburg, MD, USA). Cells were grown in 37?C,
5% CO2humidified incubator. Prior to any treatment,
cells were counted (Cedex Roche, Indianapolis, IN,
USA) and 8?105cells were seeded in 100-mm tissue
culture dishes and grown for 3 days in DMEM supple-
mented with 5% dialyzed serum (Life Technologies,
Gaithersburg, MD, USA).
Cells treatment with chromatin-modifying drugs
Cells in 100mm tissue culture dishes were treated with
1mM or 3mM 5-aza-20-deoxycytidine (Sigma-Aldrich,
St. Louis,MO, USA)and/or
4-phenylbutyric acid (Sigma-Aldrich, St. Louis, MO,
USA) (12). The drugs were dissolved in 70% ethanol at
500–1000 times their above indicated final concentration
in the media. Cells were washed three times with PBS and
7ml of fresh DMEM+5% dialyzed serum media contain-
ing epigenetic drugs alone or in combinations were added
to the plates. Two plates were used as controls: one con-
tained the same amount of drug-free and solvent-free fresh
media, and the other contained the maximum relative
amount of solvent (without drugs). All measurements
were performed 24h after drug treatments.
Glucose deprivation treatment
Cells grown in 100mm dishes in DMEM+5% dialyzed
serum media were rinsed 3 times with PBS. A quantity of
7ml of glucose-free DMEM (Life Technologies, Gaith-
ersburg, MD, USA) supplemented with 5% dialyzed
serum were added to the plates and the samples were
taken at specified time points. For control growth condi-
tions, fresh glucose-containing DMEM+5% dialyzed
serum was added instead of glucose-free media, and
samples were taken after 24h.
Hydrogen peroxide treatments
Cells grown in 100mm dishes in DMEM+5% dialyzed
serum media were washed 3 times with PBS. Hydrogen
peroxide (H2O2) solutions at concentrations of 1–5mM
were prepared in DMEM+5% dialyzed serum media
from concentrated stock (8.821M) purchased from
Cell Biolabs, San Diego, CA, USA (Part No. 234102).
A quanrity of 7ml of the media containing respective
amounts of H2O2 were then added to the plates and
cells were incubated for 1, 5, 12 and 24h.
RNA isolation and qRT–PCR analysis
Total RNA was isolated from the samples using
mirVanaTMmiRNA isolation kit, Life Technologies,
qRT–PCR analysis of the Sfmbt2 gene and miR-466h-5p
were performed in Prism 7900H Sequence Detector
(Applied Biosystems, Carlbad, CA, USA) with 40 ampli-
fication cycles according to manufacturer’s protocols. The
Sfmbt2 gene quantification was done using TaqMan?
Mm00616783_m1) and normalized to 18S levels (Life
Technologies, Assay ID: Hs99999901_s1) in the respective
sample. The mmu-miR-466h-5p and miR-669c quantifica-
tion was done with TaqMan?microRNA assays (Life
ID:AM002646), normalized to mmu-let-7c levels (Life
Technologies, Assay ID: AM00379) and analyzed as pre-
viously described (2).
7292Nucleic Acids Research, 2012,Vol.40, No. 15
Intracellular ROS detection
Intracellular ROS concentration was measured using
OxiSelectTMIntracellular ROS Assay Kit with green fluor-
escence (Cell Biolabs, San Diego, CA, USA; Cat. No.
STA-342). The cells were exposed to glucose-free media
as described above for a specified time. The cells were
harvested, washed with PBS, resuspended in 1ml PBS
and incubated with and without 20,70-dichlorodihydro-
fluorescin diacetate (DCFH-DA) probe (20mM) for
45min at 37?C with gentle vortexing in a thermal
mixer. Non-fluorescent DCFH-DA probe diffuses to
the cells and gets cleaved to non-fluorescent DCFH,
which is rapidly oxidized to highly fluorescent 20,70-
dichlorofluorescin (DCF) by ROS. Cells were then
washed and resuspended
analysis was done using ExpressPlus assay in Guava
Easycyte 5HT (Millipore, Billerica, MA, USA). Cells
were excited with blue laser at 488nm and green fluores-
cence was determined. Dead cells and debris were gated
out by forward and side scatter. The signal was first
adjusted with unlabeled cells to fluorescence values
below 10 (red markers in figure 4). The mean fluorescence
intensity (MFI) of DCF in the labeled cells (green marker
in Figure 4) corresponded to the respective levels of intra-
in PBS.Flow cytometry
Estimation of intracellular reduced GSH concentration
Cells were exposed to glucose-free media as described
above. GSH levels were measured with GSH detection
kit (Millipore Cat.No.
monochlorobimane (MCB) dye that fluoresces blue upon
thiol binding and has a high affinity to GSH. The treated
cells were collected, washed with ice-cold Wash Buffer,
resuspended in Lysis buffer, incubated on ice and
centrifuged. The lysates were mixed with MCB in
96-well plates and incubated at room temperature for
1.5h. The florescence was read in the SPECTRAmax
GEMINI-XS (Molecular Devices, Sunnyvale, CA, USA)
spectrofluorometer using the 380-/460-nm filter set.
Measurement of HDACs activity
Following incubation in glucose-free media, the cells were
collected and washed with PBS. Nuclear extracts were
prepared using the NE-PER?extraction kit (Thermo
Scientific, Rockford, IL, USA; Cat. No. 78833) and
protein levels were quantified by the bicinchoninic acid
(BCA) protein assay (Thermo Scientific, Cat. No.23227).
HDACs activity was measured using a colorimetric assay
kit (Enzo Life Sciences, Farmingdale, NY, USA; Cat. No.
BML-AK501). The kit uses Color de Lys substrate con-
taining deacetylated lysine side chain and is incubated
with the nuclear extract. Deacetylation sensitizes the sub-
strate and it produces a yellow chromophore upon treat-
ment with a developer. A standard curve was first
prepared using known amounts of the deacetylated
standard included in the kit. The nuclear extracts (25mg
of protein) were incubated with 0.5mM substrate in
half-volume 96-well plates for 1h at 37?C with gentle
mixing. Developer solution was then added to the plate
and the plate was incubated at 37?C for additional 15min.
Absorbance was analyzed with the SPECTRAmax 190
(Molecular Devices, Sunnyvale, CA, USA) plate reader
at 405nm and the values were related to known amount
of deacetylated standard (Figure 3). HeLa cells nuclear
extract was used as a positive control.
Western blot analysis of HDAC2
Following incubation in glucose-free media, the cells were
collected and washed with PBS. Cells were lysed with
RIPA buffer (Sigma, St. Louis, MO, USA; Cat. No.
R0278) and protein levels were quantified by the BCA
protein assay (Thermo Scientific). Protein samples (25mg
for each sample) were run on SDS–PAGE with b-actin
used as the loading control. HDAC2 and b-actin were
first detected with primary antibodies (Santa Cruz bio-
technology, Santa Cruz, CA, USA; Cat. No. sc7899 and
Abcam, Cambridge, MA, USA; Cat. No. ab6276, respect-
ively) and then visualized using secondary antibodies
conjugated with horseradish peroxidase.
Chip assay was performed using the SimpleChiPTM
Enzymatic Chromatin IP Kit (magnetic beads) from Cell
Signaling Technology, Danvers, MA, USA (Cat. No.
9003) according to the manufacturer’s protocol. The
cells were exposed to glucose deprivation for indicated
time, collected, cross-linked with fresh formaldehyde
(MG Scientific, Pleasant Prairie, WI, USA; Cat. No.
2106-01), and treated with glycine to stop the cross-
linking. At least 15 million of viable cells were used for
each time point. Cells were resuspended and chroma-
tin was fragmented by partial digestion with micrococ-
cal nuclease. Nuclei were then broken by sonication
using Ultrasonic Liquid Processor XL-2020 (Qsonica,
Newtown, CT, USA) equipped with 1/16-inch probe at a
setting of 2.5 and 3 sets of 15-s pulses, the lysates were
centrifuged and the supernatant was collected. The chro-
matin concentration was determined after DNA purifica-
tion from supernatant sample; its size was verified by
gel electrophoresis to be between 1 and 5 nucleosomes
(150–900bp). The equivalent of ?15mg of chromatin
was diluted and incubated with either non-specific
negative control normal rabbit IgG (Cell Signaling, Cat.
No. 2729), positive control histone H3 rabbit mAb
(pc-H3) (Cell Signaling, Cat. No. 4620) or anti-acetyl-
histone H3 rabbit polyclonal Ab (Millipore, Cat. No.
17-615) overnight at 4?C with rotations. A quantity of
2% of each sample was removed before Ab incubation.
Chromatin was then eluted, reverse cross-linked and DNA
was purified. DNA from the 2% sample was also purified
and used as control for quantitative qRT–PCR analysis.
Quantitative analysis was done using SYBR detection in
Prism 7900H Sequence Detector (Life Technologies) with
SYBR?Green PCR Master Mix (Life Technologies, Cat.
No. 4309155) according to manufacturer’s protocol.
Primers for qRT–PCR detection were selected within the
predicted miR-466h transcription start site (25) and
yielded 101bp PCR product (forward: 50-GTAGACTTG
GTGAGTTAGAAGGCT-30, reverse: 50-GTGTGCAAT
Nucleic Acids Research, 2012,Vol.40, No. 157293
CTTCTCTTTCCAAGG-30). The fraction of DNA in the
sample that was immunoprecipitated with Anti-Ac-H3
was normalized to the amount of total DNA in the
sample (2% sample) and the amount of DNA was
immunoprecipitated with non-specific negative control
Ab (nc-Ab) as previously described (12). The pc-H3
mAb was used as an additional positive control for
histone H3 detection and for qRT–PCR stringency verifi-
miR-466h promoter areas were consistent with the
amounts of total DNA in the respective samples.
DNAamounts in the
Activation of miR-466h-5p and the Sfmbt2 gene by
histone deacetylases inhibition
The expression of several microRNAs in mammalian cells
has been shown to correlate with DNA methylation and
histone modifications (11–13). It was, therefore, intriguing
to test the effect of the DNA-demethylating agent 5-aza-
20-deoxycytidine (Aza) and the histone deacetylases
(HDACs) inhibitor, 4-phenylbutyric acid (PBA) on the ac-
tivation of miR-466h-5p. Given that intronic miRNA(s)
(or miRNA clusters) are known to be co-regulated with
the embedding gene, the activation of Sfmbt2 was also
examined. Figure 1 shows the expression profiles of
miR-466h-5p and Sfmbt2 gene following treatment with
PBA and Aza. The expression of Sfmbt2 and miR-466h-5p
was significantly increased by PBA, whereas it was not
significantly affected by Aza alone or in combination
with PBA (Figure 1A and B). About 1 and 3mM
of PBA in the media resulted in 3.5- and 8.1-fold
induction of Sfmbt2, respectively, and 1.3- and 3.1-fold
induction of miR-466h-5p. It is, therefore, possible
that reduced levels of HDACs caused the increased
increased histone acetylation and transcriptional activa-
and Sfmbt2 through
Effects of glucose deprivation on miR-466h-5p expression
and HDACs activity
In our previous studies, we showed that miR-466h-5p was
nutrient-depleted conditions (2). In order to examine if
altered glucose metabolism played a role in the activation
of miR-466h-5p, the levels of this molecule were measured
following glucose deprivation in mouse cells. Shown in
Figure 2A is the time-dependent profile of miR-466h-5p
during exposure to glucose-free media; this miRNA was
up-regulated after 25 h of glucose-free media exposure.
The 3.7- to 4.4-fold up-regulation of miR-466h-5p after
45 h of glucose-free media exposure was comparable
with its activation by 3mM of PBA (Figure 1B). Shown
in Figure 2B is the time-dependant profile of another
member of the miR 297-669 cluster, miR-669c, during
exposure to glucose-free media. The up-regulation of
miR-669c (3.8- to 4.3-fold after 45 h) and Sfmbt2 was
similar to the miR-466h-5p activation profile during
glucose deprivation (Figure 2B and C).
cells wereexposed to
To determine if the reduction of HDACs activity cor-
relates with miR-466h-5p induction by glucose depriv-
ation, HDACs activity and the protein levels of HDAC2
were measured during the glucose deprivation. Shown in
Figure 3A is the time-dependant reduction of HDACs
activity in cells exposed to glucose-free media. HDACs
activity was reduced by >2-fold in mouse cells at and
after 45 h of glucose-free media exposure compared with
its activity in glucose-containing media. The protein levels
of HDAC2 were also reduced following glucose depriv-
ation (Figure 3B). This suggests that the reduction of
HDACs activity during glucose deprivation can cause an
increased histone acetylation and transcriptional activa-
tion, which leads to miR-466h-5p induction.
Accumulation of ROS during glucose deprivation
Glucose deprivation has been shown to induce cytotox-
icity and metabolic oxidative stress as a result of increased
concentrations of pro-oxidants and decreased intracellular
levels of reduced GSH (16,18,19,27). It was shown previ-
ously that limited intracellular glucose caused the accu-
mulation of reactive oxygen species (ROS) such as
H2O2,which can mediate glucose
deprivation-induced oxidative stress (18). To examine
the effect of glucose depletion on the mouse cells studied
here, the concentrations of both ROS and reduced GSH
time-dependent screens of intracellular ROS in glucose-
depleted media conditions. The MFI of the DCF-
labeled population of the mouse cells (shown in green)
was time-dependent and directly proportional to intracel-
lular levels of ROS. The 2.1- and 2.8-fold increase of intra-
cellular ROS levels was observed at 45 and 50 h of glucose
depletion, respectively compared with fresh media growth
Depletion of GSH in the mouse cells as a result of
exposure to glucose-free media is shown in Figure 5.
The time course levels of intracellular GSH levels
(3.4- to 15.2-fold reduction) inversely correlated with the
accumulation of the intracellular ROS (Figure 4) and the
activation of miR-466h-5p (Figure 2A). These findings
suggest that exposure of mouse cells to glucose-free
media resulted in onset of oxidative stress shown by
increased intracellular ROS generation and decreased
rate of their removal likely due to GSH depletion.
?OH, and O2
in Figure4 arethe
Activation of miR-466h-5p by cells treatment with H2O2
As was shown above, glucose deprivation led to intracel-
lular ROS accumulation (Figure 4) and to miR-466h-5p
induction (Figure 2A). To verify if ROS alone can cause
miR-466h-5p activation, mouse cells were treated with the
pro-oxidant, H2O2, which is known to induce oxidative
stress in mammalian cells, (15,17,22) and miR-466h-5p
up-regulation of miR-466h-5p was observed after 1,
5 and 12h of cell treatment with 1–5mM of H2O2(data
not shown). However, miR-466h-5p expression levels were
significantly increased (1.8- to 5.3-fold) when the cells were
exposed to 3mM and higher concentrations of H2O2for
24 h (Figure 6). Furthermore, activation of miR-466h-5p
7294Nucleic Acids Research, 2012,Vol.40, No. 15
by 5mM of H2O2 was comparable with its activation
by PBA and glucose deprivation at later times (50 h).
The exogenous oxidative stress induction with 5mM
H2O2 for 24h also reduced the HDACs activity to
levels comparable with its activity at 45 and 50 h of
glucose-free media exposure (data not shown). Hence,
the induction of miR-466h-5p can be linked to decreases
in HDACs activity associated with ROS during the oxi-
Increased acetylation of histone 3 is associated with
miR-466h-5p promoter area
As the acetylation of histone H3 is associated with tran-
scriptional activation and gene expression (23,26,28) the
acetylation of histone H3 around the predicted transcrip-
tion start site of miR-466h-5p (25) was evaluated. Shown
in Figure 7 is the relative amount of histone H3 acetyl-
ation associated with the miR-466h-5p promoter region as
Figure 1. Expression of Sfmbt2 gene and miR-466h-5p after 24h of treatment with 1mM and 3mM of PBA and/or 1mM and 3mM of Aza.
(A) Relative expression of Sfmbt2 gene with 18S as a control for 2???Ctanalysis. (B) Relative expression of miR-466h-5p with let-7c as a control
Nucleic Acids Research, 2012,Vol.40, No. 157295
a function of the time the cells were exposed to
glucose-free media. No significant changes in histone H3
acetylation of the miR-466h-5p promoter region was
observed within the first 25 h of glucose-free media
exposure. However, compared with fresh media growth
conditions, acetylation of histone H3 was increased by
31, 44 and 59% after 35, 45 and 50 h exposure to
glucose-free media, respectively (Figure 7). HDACs
activity is known to be inhibited by oxidative stress and
ROS accumulation (20–22,29). It is, therefore, possible
that glucose deprivation-induced oxidative stress inhibits
HDACs activity leading to increases in the acetylation of
histone H3 in the miR-466h-5p promoter area and
enhanced gene transcription and up-regulation of this
miRNAs were found to be involved in regulation of
multiple processes including cell differentiation, prolifer-
ation, metabolism and death (30,31), and alteration of
their expression has been linked to cancer development
and progression. Some studies have shown that miRNAs
are frequently down-regulated or deleted in different
cancers, an indication of their role as tumor suppressors
(30,32,33), while other miRNAs have an oncogenic role
and their inhibition can serve as a possible therapeutic
controlling miRNAs’ expression at different physiological
and pathophysiological conditions will lead to a better
understanding of the utilization of these molecules in
controlling of cellular processes.
DNA modifications have been shown to have significant
effects on development of human diseases (23) and several
molecules that alter DNA methylation or modifications
of histones are being evaluated as therapeutic agents
(23). As several miRNAs were previously shown to be
up-regulated by the inhibition of DNA methylation and/
or inhibition of HDACs activity (11–13), we first tested
Treatments of mouse cells with 3 mM of PBA (HDACs
inhibitor) resulted in up-regulation of miR-466h-5p and its
embedding gene, Sfmbt2 (3.2- and 8.1-fold, respectively).
Inhibitors of HDACs (HDACi) have been explored for
clinical applications as anti-cancer agents due to their
ability to induce cell cycle arrest, cells differentiation and
apoptosis (36–38). However, even though these agents are
known to activate genes transcription, they are cytotoxic
Figure 2. Time course expression of miR-466h-5p, miR-669c and Sfmbt2 gene as a function of exposure to glucose-free media. (A) and (B) Relative
expression of miR-466h-5p and miR-669c, respectively with let-7c as a control for 2???Ctanalysis. (C) Relative expression of Sfmbt2 gene with 18S
as a control for 2???Ctanalysis.
7296 Nucleic Acids Research, 2012,Vol.40, No. 15
to the cells and there is no clear understanding of the
extent of their activity (23). Understanding the role that
HDACi play in miRNAs expression may enable better
design of therapeutic tools for targeting specific diseases.
In the next step, we proposed the successive molecular
events that caused the activation of the miR-466h-5p in
mouse cells exposed to glucose deprivation. We evaluated
the specific effect of glucose-free media exposure on the
expression of miR-466h-5p; following 50-h exposure of
miR-466h-5p and its embedding gene, Sfmbt2, were
up-regulated 4.4-and 15-fold, respectively together with
reductionof HDAC2 concentration
decrease of HDACs activity. Then, we showed that the
HDACs inhibition led to 59% increase of histone H3
acetylation in the miR-466h-5p promoter area, which
resulted in activation of this miRNA. We also showed
that exogenous induction of oxidative stress by H2O2led
to activation of miR-466h-5p and HDACs inhibition and
verified that glucose deprivation caused the metabolic oxi-
dative stress onset via accumulation of ROS (2.8-fold
increase) and depletion of GSH (15.2-fold decrease).
Shown in Figure 8 is a general outline of the suggested
miR-466h-5p activation: transcriptional activation of this
miRNA followed the reduction of HDAC2 activity and
increased acetylation in miR-466h-5p promoter region
caused by glucose deprivation-induced metabolic oxida-
tive stress. Although we showed that glucose deprivation
activated miR-466h-5p, more research needs to be con-
ducted to determine the specific activation mechanism. It
may be worthwhile to investigate the activation of specific
kinases by ROS leading to post-translational modifica-
tions and the inhibition of specific HDACs involved in
silencing of miR-466h-5p promoter region.
Several members of the miR 297-669 cluster, including
miR-466h-5p, were also shown to be induced by acet-
aminophen cytotoxicity (7) which, together with our ob-
servation of miR-466h-5p induction by oxidative stress,
indicates a possible role of this cluster during accumula-
tion of toxic metabolites. The up-regulation of this
miRNA cluster may be an indication of reduced cellular
detoxification capacity and may be explored as biomarker
for drugs-induced cellular injuries (7). As miR-466h-5p
Figure 3. Time course activity of histone deacetylases and HDAC2 protein levels as a function of exposure to glucose-free media. (A) Histone
deacetylases activity. Fluorescence values at all time points were related to the values of Color de Lys deacetylated standard. (B) HDAC2 proteins
Nucleic Acids Research, 2012,Vol.40, No. 157297
Figure 4. Intracellular levels of ROS as a function of exposure to glucose-free media. (A) Fluorescence-activated cell sorter (FACS) image of
fluorescence intensity distribution of DCF-labeled cells (green marker) exposed to glucose-free media for indicated time points. Mean fluorescent
intensity value (MFI) is proportional to intracellular levels of ROS. (B) Accumulation of ROS with time of glucose-free media exposure.
7298 Nucleic Acids Research, 2012,Vol.40, No. 15
was previously shown to have a pro-apoptotic role by
down-regulating several anti-apoptotic genes including
bcl2l2, birc6, dad1, smo and stat5a, (2) and given the fact
that clustered miRNA are usually transcribed together as
polycistronic transcripts to regulate genes with similar
functions (31,39), the miR 297-669 cluster may be
induced in mammalian cells exposed to cytotoxic condi-
tions. This induction could be inhibited during oncogenic
transformation as cancer development is known to result
from the loss of oxidative stress defense mechanisms
(27,40,41). As the predicted transcription start sites for
all members of the miR 297-669 cluster is located
directly upstream of Sfmbt2 gene (25), all members of
this miRNA cluster may be co-transcribed in response to
cytotoxic stimuli; the up-regulation of both miR-466h-5p
and miR-669c by glucose deprivation (Figure 2A and B)
supported this assumption. The Sfmbt2 gene belongs to
the Scm-family of Polycomb transcriptional repressor
genes and has four mbt domains known to have tumor
suppressor activity (42,43). This gene has been implicated
in development of brain tumor and neurological disorders
(42,44). Although the inhibition of some Polycomb group
Figure 6. Relative expression of miR-466h-5p after treatment with H2O2for 24h.
Figure 5. Time course levels of GSH levels as a function of exposure to glucose-free media.
Nucleic Acids Research, 2012,Vol.40, No. 157299
proteins was shown to be associated with the inhibition of
HDACs activity (45), the Sfmbt2 gene was previously
shown to be activated by valproate, the mood stabilizer
known to increase acetylation of histone H3 in mouse
brain cells (43,46), and to selectively reduce the levels of
HDAC2 (47). Further studies need to be conducted to
verify the activation of the other miR 297-669 cluster
members and to elucidate the role of Sfmbt2 gene in this
Our study is a novel approach to link the miRNA acti-
vation to a known physiological condition and it shows
the importance of understanding the mechanisms of
Figure 8. Suggested mechanism of miR-466h-5p activation. Glucose depletion leads to accumulation of ROS and inhibition of HDACs activity that
increases histone H3 acetylation and activates miR-466h-5p (figure was created using ‘pathway designer’ tool in IPA Ingenuity Software).
Figure 7. Acetylation of histone H3 in miR-466h-5p promoter area as a function of time of exposure to glucose-free media. The amounts of DNA
bound to Ac-H3 were related to its amount in fresh media grown cells.
7300 Nucleic Acids Research, 2012,Vol.40, No. 15
miRNAs activation. Together with previous reports, it
suggests a potential role of miR 297-669 cluster and its
embedding gene, Sfmbt2, in development of various
The authors would like to thank Dr. J. Hanover for
critical review of the manuscript and Mrs. D. Livnat for
critical editorial assistance.
Funding for open access charge: Intramural program of
the National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health.
Conflict of interest statement. None declared.
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