Neuro-Oncology 15(9):1212– 1224, 2013.
doi:10.1093/neuonc/not055
Advance Access publication June 3, 2013
N E U RO - O N CO LO GY
MicroRNA-128 coordinately targets Polycomb
Repressor Complexes in glioma stem cells
Pierpaolo Peruzzi, Agnieszka Bronisz, Michal O. Nowicki, Yan Wang, Daisuke Ogawa,
Richard Price, Ichiro Nakano, Chang-Hyuk Kwon, Josie Hayes, Sean E. Lawler,
Michael C. Ostrowski, E. Antonio Chiocca, and Jakub Godlewski
Department of Neurosurgery, Brigham and Women’s Hospital, Boston, Massachusetts (A.B., M.O.N., D.O.,
E.A.C., J.G.); Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery
(P.P., M.O.N., Y.W., D.O., R.P., I.N., C.-H.K., E.A.C., J.G.); Department of Molecular and Cellular Biochemistry
(A.B., M.C.O.); Solid Tumor Program, The Ohio State University Medical Center and James Comprehensive
Cancer Center, Columbus, Ohio (C.-H.K.); Leeds Institute of Molecular Medicine and St. James’s University
Hospital, University of Leeds, Leeds, UK (J.H., S.E.L.)
Background. The Polycomb Repressor Complex (PRC)
is an epigenetic regulator of transcription whose action
is mediated by 2 protein complexes, PRC1 and PRC2.
PRC is oncogenic in glioblastoma, where it is involved
in cancer stem cell maintenance and radioresistance.
Methods. We used a set of glioblastoma patient samples,
glioma stem cells, and neural stem cells from a mouse
model of glioblastoma. We characterized gene/protein
expression and cellular phenotypes by quantitative PCR/
Western blotting and clonogenic, cell-cycle, and DNA
damage assays. We performed overexpression/knockdown
studies by lentiviral infection and microRNA/small interfering RNA oligonucleotide transfection.
Results. We show that microRNA-128 (miR-128) directly
targets mRNA of SUZ12, a key component of PRC2, in addition to BMI1, a component of PRC1 that we previously
showed as a target as well. This blocks the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications.
MiR-128 and SUZ12/BMI1 show opposite expression in
human glioblastomas versus normal brain and in glioma
stemlike versus neural stem cells. Furthermore, miR-128
renders glioma stemlike cells less radioresistant by preventing the radiation-induced expression of both PRC
Received December 20, 2012; accepted March 10, 2013.
Corresponding Authors: Jakub Godlewski, PhD, Harvard Institute of
Medicine, 77 Louis Pasteur Ave., Rm# 921C, Boston, MA 02115
([email protected]); E. Antonio Chiocca, MD, PhD, Harvard
Medical School Neurosurgeon-in-Chief and Chairman, Department of
Neurosurgery Co-Director, Institute for the Neurosciences at the
Brigham Brigham and Women’s/ Faulkner Hospital Surgical Director,
Center for Neuro-oncology Dana-Farber Cancer Institute 75 Francis
Street Boston, MA 02115 ([email protected]).
components. Finally, miR-128 expression is significantly
reduced in neural stem cells from the brain of young, presymptomatic mice in our mouse model of glioblastoma.
This suggests that loss of miR-128 expression in brain is
an early event in gliomagenesis. Moreover, knockdown of
miR-128 expression in nonmalignant mouse and human
neural stem cells led to elevated expression of PRC components and increased clonogenicity.
Conclusions. MiR-128 is an important suppressor of PRC
activity, and its absence is an early event in gliomagenesis.
Keywords: glioblastoma, gliomagenesis, glioma stem
cells, glioma therapy, microRNA.
G
lioblastoma multiforme is the most common and
aggressive intrinsic primary brain tumor in adults.
Approximately 10 000 new cases are diagnosed in
the United States every year, and patients’ median survival
is 14.6 months, even with surgery, radiation, and chemotherapy.1 This tumor presents unique challenges to
therapy due to its location, aggressive biological behavior,
and diffuse infiltrative growth. Despite the development
of new surgical and radiation techniques and the use of
multiple anti-neoplastic drugs, effective long-term treatments remain elusive.2 Numerous recent studies have
demonstrated that glioblastoma cells retain many of the
features of neural progenitor cells, including the ability
to grow as neurospheres in culture and self-renew.
These cells, known as glioma stemlike cells (GSCs), are
a subpopulation of cells in the tumor with self-renewal capacity whose asymmetric division gives rise to the heterogeneous nature of cancer cells that make up the neoplastic
mass. When implanted in immunodeficient mice, as few
as 100 GSCs can give rise to tumors that recapitulate the
histological features and global gene expression patterns
# The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.
All rights reserved. For permissions, please e-mail: [email protected].
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
of the parental patient tumors.3 – 6 These observations
have created novel opportunities for developing antiglioblastoma therapeutics based on targeting the GSC
component of glioblastoma.7
The discovery of microRNAs and the increasing appreciation of their importance in glioblastoma and other
cancers8 – 12 have led to improved understanding of
key mechanisms underlying brain tumors and other
cancer types. MicroRNAs are approximately 20- to
23-nucleotide noncoding RNAs that act as important regulators of gene expression by downregulating expression
of target mRNAs through regions of partial complementarity in their 3′ untranslated regions (UTRs).13 Each
microRNA may have hundreds of targets, and many
genes are targeted by multiple microRNAs, thus leading
to potentially highly complex regulatory networks.
Distinct patterns of microRNA expression have been observed in many cancers, including glioblastomas.8,10,14
In glioblastoma, several deregulated microRNAs have
been strongly implicated in the maintenance of stemness.
Work from our laboratory has established a link between
microRNA-128 (miR-128), which is strongly downregulated in glioblastoma, and loss of GSC self-renewal,
through direct regulation of the neural stem cell (NSC) selfrenewal factor B lymphoma Mo-MLV insertion region 1
homolog (BMI1). We demonstrated that downregulation
of BMI1 is dependent on miR-128 expression, leading to
a decrease in proliferation of glioma cells in vitro and in
vivo and to a reduction of the self-renewal ability of
GSCs.15 BMI1 is a component of the Polycomb Repressor
Complex (PRC), which suppresses expression of key
target genes through chromatin modification. PRC components are commonly upregulated in glioblastoma and play
an important role in normal development,16,17 organogenesis,18 and embryonic stem cell biology.19 The entire PRC is
composed of 2 distinct protein complexes: PRC1 ubiquitinates histone H2A, and PRC2 methylates histone H3.20
These functions generally lead to transcriptional repression
and, in neoplasia, are known to be important in cancer stem
cell self-renewal21 – 23 and epithelial-mesenchymal transition24 by repression of tumor suppressor gene expression.
Many of the protein constituents of PRCs are highly
expressed in glioblastoma compared with normal brain,
including BMI1,25 suppressor of zeste 12 homolog–
Drosophila (SUZ12),26 and others.27 Both PRC1 and
PRC2 have been implicated in GSC maintenance,28,29 and
it has also been suggested that GSCs have acquired an oncogenic addiction to PRC activity.30 Several reports have demonstrated a crucial role for both Polycomb complexes in
maintaining the radioresistance of numerous cancer
models and cancer stem cells,31,32 including glioblastoma.33,34 It is believed that the Polycomb proteins recruit
the DNA damage response machinery and promote doublestrand break repair.35 – 37
Previously, we have shown that BMI1 mRNA expression is downregulated by miR-128 in glioma and GSCs.
However, there have been reports that BMI1 upregulation
by itself does not lead to tumorigenesis.38 This could be
potentially explained by the redundant and complementary functions of PRC components where downregulation of one component leads to upregulation of
other components of PRC. We thus hypothesized that
miR-128 – mediated repression of BMI1, and consequently PRC1 activity, may be only a partial picture of a larger
and more complex role in PRC regulation. In fact, here
we report that miR-128 targets a component of PRC2
as well: SUZ12. It has been shown that the functionality
of PRC2 as a complex depends on the presence of
SUZ12.39 Moreover, upregulation of SUZ12 was associated with the development of cancer stem cells.40
Reestablishment of miR-128 expression blocks PRC
activity, impairs GSC self-renewal, and increases radiosensitivity of GSCs, while loss of miR-128 expression
appears to be an early event in gliomagenesis. These findings emphasize the role of microRNAs as molecules able
to target activity of entire pathways by the simultaneous
inhibition of multiple components, thus preventing their
redundant functions.
Materials and Methods
Human Specimens
All tumor samples were obtained as approved by the institutional review board at The Ohio State University.
Surgery was performed by E.A.C., I.N., and P.P. For
each patient, samples of both tumor and brain devoid of
gross tumor were resected, aliquoted, and processed for
either extraction of total RNA (Trizol, Invitrogen) and
protein (Cell Lysis Buffer, Cell Signaling) or isolation
and establishment of patient-derived GSCs.
Cell Culture
U87 malignant glioma (MG) and U251MG glioblastoma
cells were obtained from the American Type Culture
Collection and cultured in Dulbecco’s modified Eagle’s
medium (DMEM; Invitrogen) supplemented with 10%
fetal calf serum (Sigma). Primary human glioma stemlike
cells GSC84, GSC528, GSC718, GSC816, GSC1123,
AC17, and AC20 were obtained from freshly resected
human MG specimens (GSC84, -528, -718, and -816
are World Health Organization [WHO] grade IV and
GSC1123, AC17, and AC20 are WHO grade III) and
grown, as previously reported,41 in stem cell medium,
consisting of DMEM/F12/Glutamax (Invitrogen) supplemented with 1% N2 and 2% B27 (Invitrogen) and
20 ng/mL epidermal growth factor and fibroblast
growth factor–2 (PeproTech). Cells were grown as adherent monolayers in poly-L-ornithine and laminin-covered
dishes (Invitrogen) as previously described.42 The isolation and characterization of the human fetal NSCs
SCP2743 (a kind gift of Dr Kaspar from Columbus
Nationwide Children’s Hospital) and 16WF44 were reported previously. They were grown as monolayer cultures as we have described. Mouse subventricular zone
(SVZ) cells were obtained by harvesting mouse brain
after euthanasia, isolating the SVZ region, dissociating
tissue with TrypLE Express (Invitrogen), and culturing
cells as neurospheres in stem cell medium.
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Vector Construction
The lentiviral phosphorylated cadherin (pCDH; System
Biosciences) construct carrying miR-128 was previously
described.15 Transducing virus particles were obtained
by co-transfecting human embryonic kidney 293 cells
with pCDH/miR-128 – 1 plasmid DNA along with
Lentiviral Plasmid Packaging Mix (System Biosciences)
in the presence of Lipofectamine (Invitrogen) according
to the manufacturer’s recommendations. For the reporter
assay, pMirTarget-BMI1, a luciferase vector containing
the full-length (1.9 kb) BMI1 3′ UTR was purchased
from Origene. The control vector was obtained by removing the BMI1 sequence by restriction digestion. To obtain
pMirTarget-SUZ12, the full-length SUZ12 3′ UTR
(1.7 kb) was amplified from U87MG genomic DNA and
cloned into pMirTarget control vector. All the vectors
produced in this study were sequenced to confirm sequence identity and in-frame accuracy. Similarly, a
468-bp fragment of the SUZ12 3′ UTR, amplified from
U87 genomic DNA and containing both target sequences
for miR-128, was cloned into pMirTarget control vector
to obtain the part of the 3′ UTR containing pMirTargetSUZ12. The construct was then mutated using the
QuickChange kit (Stratagene), in which the predicted
miR-128 target sequences actgtgg and actgtga were substituted with agtcagg and aaaggct, respectively. The
anti –miR-128 vector (miRZip – miR-128 green fluorescent protein [GFP]) was purchased from System
Biosciences. MiRZip-GFP (negative control) was obtained by removing the anti – miR-128 sequence from
the parental construct by high fidelity PCR-mediated amplification. Sequences of all primers are shown in
Supplementary Table S1.
deregulated microRNAs were used for visualization in a
heatmap and analyzed by principal component analysis45
using dChip software with the Statistical R package. The
array was performed at The Ohio State University
Comprehensive Cancer Center Microarray, Nucleic
Acids, and Proteomic Shared Facilities with their technical assistance. Mature miR-128 expression analysis was
carried out using a microRNA real-time (RT) PCR detection kit (Applied Biosystems), following manufacturer’s
protocol. Quantitative RT-PCR was performed using
the Applied Biosystems Step One Plus PCR apparatus.
U6 small nuclear RNA was used as the endogenous
control. Of note, miR-128 and U6 probe sequences
were identical for both human and mouse transcripts.
Complementary DNA for RT-PCR was synthesized
using iScript (BioRad). Analysis of mRNA expression
was carried out using Power SYBR Green (Applied
Biosystems) with human BMI1, SUZ12, and 18SrRNA
as endogenous control. Sequences of all primers are
shown in Supplementary Table S1. The collection of the
data from The Cancer Genome Atlas (TCGA)46 was compliant with all applicable laws, regulations, and policies
for the protection of human subjects, and necessary
ethical approvals were obtained. Analysis of all data
was done in R project (http://www.r-project.org/).
Agilent 8×15k microRNA expression and gene expression data for 206 glioma samples were downloaded
along with clinical information from TCGA (level 2 normalized data, November 2012, http://tcga-data.nci.nih.
gov/tcga/dataAccessMatrix.htm). Spearman’s rank correlations were calculated for 2 probes for miR-128 – 1 and
miR-128 – 2 against probes for SUZ12.
Cell Studies
Nanostring, Real-time PCR, and Database Analysis
Total RNA was extracted using Trizol (Invitrogen) and
treated with RNase-free DNase (Qiagen) as previously
described.15 In order to identify microRNAs that are specifically deregulated in glioblastoma stem cells, we compared the microRNA expression patterns of 10 samples
(2 nonmalignant NSCs and 8 GSCs). The Nanostring
microRNA technology was used to search for unique
gene signatures linked to glioblastoma stem cells. Total
RNA was used for the nCounter microRNA platform.
All sample preparation and hybridization were performed according to the manufacturer’s instructions.
All hybridization reactions were incubated at 658C for a
minimum of 12 h. Hybridized probes were purified and
counted on the nCounter Prep Station and Digital
Analyzer (Nanostring) following the manufacturer’s instructions. For each assay, a high-density scan was performed. For platform validation using synthetic
oligonucleotides, Nanostring nCounter microRNA raw
data were normalized for lane-to-lane variation with a
dilution series of 6 spikes in positive controls. The sum
of the 6 positive controls for a given lane was divided by
the average sum across lanes to yield a normalization
factor, which was then multiplied by the raw counts in
each lane to give normalized values. All significantly
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Transfection of pre–miR-128 oligonucleotides (Ambion)
at a final concentration of 50 nmol/L and of reporter
plasmids was carried out with Lipofectamine 2000
(Invitrogen), following the manufacturer’s instructions.
To create stable glioblastoma cell lines expressing
miR-128, U87MG and GSC528 cells were infected with
pCDH-128-1-cGFP or pCDH-cGFP and allowed to
grow for 7 days. Stably transfected cells were then
sorted by GFP expression through flow cytometry
(FACSAria III cell sorter, Becton Dickinson). Western
blot analysis was carried out with whole cell protein
lysate. Antibodies used were: BMI1 (Millipore), SUZ12,
p21Waf1/Cip1, H2a-K119ubi, H3-K27me3 and H3 (Cell
Signaling), a-tubulin (Sigma), and CD133 (Amersham).
Horseradish peroxidase– conjugated secondary antibodies against rabbit or mouse immunoglobulin G
(Amersham) were then used. Cell proliferation following
miR-128 transfection/transduction was quantified either
by manual counting after cell dissociation or by the
Click-it ethynyl-labeled (deoxy)uridine (EdU) cell proliferation assay kit (Invitrogen). Briefly, for cell counting,
150 000 cells were seeded on day 0 onto 6-cm well
plates, transfected on day 1, and counted 4 days after
transfection. For EdU studies, 10 000 cells were seeded
onto 48-well plates and allowed to recover overnight.
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
The following day, EdU was added to the medium to a
final concentration of 10 mM. Cells were returned to the
incubator for 3 h before they were fixed with 4% paraformaldehyde, and EdU incorporation was assessed following the manufacturer’s protocol. For the clonogenic assay,
cells were dissociated with TrypLE Express and plated
with complete stem cell medium mixed with 0.4% low
gelling temperature agarose (Sigma) pre-warmed at
378C onto 6-well dishes over a layer of solidified 0.8%
agarose. After allowing cells to settle down to the
0.8%/0.4% agarose interface, the dishes were kept at
room temperature for 10 min to allow the medium to
gel before placing the plates back at 378C for culturing.
Fresh stem cell medium was then used to cover the
agarose and changed every 3 days. For DNA damage
and response experiments, cells were irradiated with 2–
4 Gy x-rays from an RS-2000 Biological Irradiator (Rad
Source). Quantification of apoptosis and necrosis was
carried out with the FITC (fluorescein isothiocyanate)
Annexin V/Dead Cell Apoptosis Kit (Invitrogen) using
a FACSCalibur machine (Becton Dickinson). Briefly,
150 000 cells were plated onto 6-cm dishes and allowed
to recover overnight. The following day, cells were transfected with 50 nmol/L miR-128 or negative control oligonucleotides. After 36 h, cells were irradiated with
2 Gy or 4 Gy (except negative control samples), returned
to the incubator for an additional 48 h, then collected for
analysis following manufacturer’s protocol. Evaluation
of DNA repair was performed using the CometAssay
Kit (Trevigen) according to the manufacturer’s protocol.
Briefly, 100 000 cells were plated onto 6-well plate dishes
and allowed to recover overnight. The following day, cells
were transfected with 50 nmol/L miR-128 or negative
control oligonucleotides and allowed to grow for a
further 36 h. Cells were then either left untreated or irradiated with 2 Gy and then harvested at 30 min and 15 h
after exposure to ionizing radiation. For each sample,
500 dissociated cells were then embedded into agarose
and fixed onto glass coverslips for quantification of
DNA damage.
Immunohistochemistry
Human surgical specimens were fixed in formalin and embedded in paraffin before being sectioned at 4 mM thickness. Ventana BenchMark (Ventana Medical Systems)
was used for staining. Expressions of BMI1 and SUZ12
were optimized at 1:500 and 1:250 dilutions, respectively.
Each sample was counterstained with hematoxylin/
eosin.
Statistical Analysis
Results are expressed as mean + SD, and differences were
compared using the 2-tailed Student’s t-test. Statistical
analyses were performed using Microsoft Office Excel
2010 or GraphPad Prism software. P , .05 was considered statistically significant (indicated by single asterisks
in the Figures), and P , .01 was strongly significant
(indicated by double asterisks).
Results
MiR-128 Targets mRNA Encoding SUZ12, a PRC2
Component
We previously demonstrated that miR-128 targets
BMI1 15 mRNA, a component of PRC1. Based on in
silico analysis, we hypothesized that miR-128 might
also target the 3′ UTR of SUZ12 mRNA, a component
of PRC2 (Fig. 1A). To test this hypothesis, we performed
Western blotting using human glioblastoma cell lines
(U87 and U251) and human GSCs (GSC528). As shown
in Fig. 1B (left panels), transfection of miR-128 precursors
led to a significant reduction in SUZ12 expression in all 3
cells. This was also evident when GSC528 cells were stably
transfected with a lentiviral vector expressing pri–
miR-128 (Fig. 1B, right panel). As a control, miR-128
also led to the expected reduction of BMI1. To demonstrate specific targeting, we used constructs containing a
short fragment of SUZ12 3′ UTR encompassing miR-128
binding sites and showed significantly reduced luciferase
expression upon transfection with miR-128 precursors.
Additionally, mutations introduced into both predicted
miR-128 binding sites of the SUZ12 3′ UTR led to the
restoration of luciferase levels, demonstrating the specificity of miR-128 for these binding sites (Fig. 1C).
Interestingly, mutation of only 1 out of 2 miR-128
binding sites led to only partial restoration of luciferase activity, while mutation of both sites led to full restoration.
Therefore, miR-128 suppression of SUZ12 expression is
most effective when both target sites of the SUZ12
mRNA are targeted. We also demonstrated targeting of
both SUZ12 and BMI1 mRNAs by miR-128 using fulllength 3′ UTRs from both genes (Supplementary Fig. S1A).
We then compared the effect of miR-128 on both genes
with their specific knockdown using small interfering
(si)RNAs (Fig. 1D). Interestingly, siRNA-mediated
knockdown of SUZ12 led to a significant increase in
BMI1 levels in all tested glioblastoma cells, including
GSC528. This result confirmed the previously described
phenomenon of an increase in BMI1 expression after
selective downregulation of PRC2,47 suggesting the existence of a compensatory mechanism that leads to elevation in the levels of one PRC component if another PRC
component is “knocked down” by siRNA. MiR-128
overrides this mechanism as it simultaneously knocks
down both PRCs (Fig. 1B).
Clinical Significance of PRC Expression
Previously, we had shown that there was opposite expression of miR-128 and BMI1 in human glioblastoma samples.15 To examine the clinical significance of
SUZ12 in glioblastoma, we screened for the expression
of SUZ12 in matched (ie, from the same individual) glioblastoma and adjacent brain samples from human subjects. Quantitative RT-PCR showed that the expression
of SUZ12 was significantly upregulated in tumor compared with its adjacent brain tissue that was devoid of
gross tumor (Fig. 2A). The result was confirmed by
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Fig. 1. Identification and characterization of SUZ12 as target of miR-128 in established glioma cell lines and glioma stem cells. (A) Schematic
representation of miR-128 3′ UTR seeding regions. Sequence of both miR-128 target sites within human SUZ12 mRNA 3′ UTR. (B) MiR-128
regulation of BMI1 and SUZ12 protein levels. U87 and U251 glioma cell lines and the GSC528 (528) glioma stem cell line were transiently
transfected with microRNA negative control (NC) or miR-128 precursor (128) or stably infected with either pCDH vector or pCDH vector
expressing miR-128 (pCDH128) (GSC528 cells). Cell lysates were blotted with anti-BMI1 or anti-SUZ12 antibodies; anti–a-tubulin antibody
(aTUB) was used as loading control. (C) Direct targeting of SUZ12 3′ UTR by miR-128. Human embryonic kidney 293 cells were transfected
(n ¼ 3 experiments, each performed in triplicate) with a luciferase reporter vector containing a fragment of SUZ12 wild type (wt) or mutated
in position 1141–1147 (m1) or in both positions 1141–1147 and 1389– 1395 (m1m2) and co-transfected with either microRNA NC or pre–
miR-128 (128). Vector without 3′ UTR insert (no) was used as an additional control. Luciferase levels were quantified as luciferase activity per
milligram of protein. Data are shown as mean relative to controls + SD. RLU, relative luciferase activity. **P , .01. (D) Effect of SUZ12
knockdown on expression of BMI1. U87, U251, and GSC528 cell lines were transiently transfected with either siRNA NC or siRNA against
BMI1 or SUZ12. Cell lysates were blotted with anti-BMI1 or anti-SUZ12 antibody; anti– a-tubulin antibody (aTUB) was used as loading
control. Data were quantified by densitometric analysis using ImageJ software, normalized to loading control. Relative expression to
averaged NC level is shown.
Western blot analysis of protein lysates from 3 of these
samples (Fig. 2B) and immunohistochemistry (Fig. 2C).
As a control, BMI1 also showed the same pattern of expression as previously reported by us (Supplementary
Fig. S2A –C). Therefore, PRC components are highly expressed in human glioma specimens, similar to levels that
had been detected in GSCs and established glioma cancer
cell lines such as U87 and U251 (Fig. 1B).
In contrast, the levels of mature miR-128 in glioblastomas were significantly (8-fold) reduced compared with
those from matched adjacent brain (Fig. 2D). We attempted to correlate miR-128 downregulation with SUZ12
protein upregulation in brain tumor tissue compared
with their matched adjacent tissue. Only 3 samples had
sufficient tissue to extract both mRNA and protein.
We observed a significant inverse correlation between miR-128 and SUZ12 (Supplementary Fig. S2D).
Interestingly, when we queried TCGA46 for human glioblastoma patient survival linked to the level of miR-128
expression, we found that the group with upregulated
miR-128 had a median survival of 620 days, whereas
groups with intermediary and low expression of
miR-128 had a significantly shorter median survival of
only 350 days (Fig. 2E). These results thus validated
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miR-128 expression as significantly downregulated and
SUZ12 and BMI1 as significantly upregulated in human
glioblastoma compared with matched surrounding brain
tissues from the same patient and associated miR-128 expression with improved survivorship. Additionally, we
correlated the expression of SUZ12 and miR-128 based
on the database of TCGA. Spearman’s rank correlations
showed that SUZ12 expression inversely correlated with
expression of both miR-128–1 and miR-128–2 in a significant fashion (Supplementary Fig. S2E). Moreover,
we queried the Repository of Molecular Brain Neoplasia
Data and TCGA to determine the prognostic relevance
of SUZ12 expression and whether there was an inverse
correlation between SUZ12 and miR-128 expression.
We found that human subjects whose gliomas expressed
upregulated SUZ12 had a median survival of only
390 days, whereas those with intermediate expression
of SUZ12 had a significantly longer median survival of
580 days (Supplementary Fig. S2F). To further establish
the importance and selectivity of miR-128 downregulation in the glioblastoma stem cell compartment, we performed a global analysis of microRNA expression in 2
nonmalignant fetal NSC lines and 8 GSC lines derived
from freshly resected human glioblastoma specimens.
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
As shown in Fig. 2F, miR-128 was among the top 4 most
significant differentially expressed microRNAs when
comparing
nonmalignant
NSCs
with
GSCs.
Furthermore, the signature of these 4 microRNAs (including miR-128) was sufficient to discriminate
between NSCs and GSCs (Supplementary Fig. S2G).
We validated this by quantitative RT-PCR analysis, underlining the significant difference in miR-128 expression
between NSCs and GSCs (Fig. 2G). These results show
that miR-128 is downregulated not only in glioma
nonstem cells—a finding that was expected because in
normal brain, miR-128 is expressed preferentially in
neurons48—but also in the GSC compartment.
Phenotypic Changes in GSCs Upon MiR-128 Expression
Fig. 2. SUZ12 deregulation in glioblastoma patients. (A) SUZ12
mRNA level in brain tumor tissue (BT) in comparison with matched
(ie, from the same patient) brain adjacent to the tumor (BAT) from
glioblastoma patients. Expression of SUZ12 was validated by
quantitative (q)RT-PCR (n ¼ 9). Mean relative gene expression
level + SD is shown. (B) SUZ12 protein levels in BT in comparison
with matched (from the same patient) BAT from glioblastoma
patients. Cell lysates were blotted with anti-SUZ12 antibody; anti–
a-tubulin antibody (aTUB) was used as loading control (n ¼ 3). (C)
Number of SUZ12-positive cells in BT in comparison with matched
(from the same patient) BAT from glioblastoma patients. The
percentage of positive cells per random field was determined (left
panel) in BT and BAT sections from 3 matched patient samples;
results are shown as mean + SD on the graph, *P , .05.
Representative images of SUZ12 in immunohistochemistry staining
in paraffin-embedded tissue. Scale bars, 50 mm. (D) Levels of
miR-128 in BT in comparison with matched (from the same patient)
BAT from glioblastoma patients. Expression of miR-128 was
validated by qRT-PCR (n ¼ 9). Mean relative microRNA expression
level + SD is shown. **P , .01. (E) Association of miR-128
expression with patient survival (Kaplan–Meier [KM] plot) in
glioblastoma. Data obtained from The Cancer Genome Atlas.
In order to study the phenotypic effect of miR-128 on
GSCs, we overexpressed it either transiently, by introducing miR-128 precursor oligonucleotides, or stably, by
using a lentiviral construct expressing the primary
miR-128 – 1 sequence (overexpression of miR-128 upon
lentiviral infection in GSC528 and U87 cell lines is
shown in Supplementary Fig. S3A). Both delivery strategies resulted in upregulation of the tumor suppressor
protein p21CIP1 as expected49 (Fig. 3A). PRC1 instigates
ubiquitination of histone 2A at Lys119 (H2A K119ubi),
while PRC2 prompts trimethylation of histone 3 at
Lys27 (H3 K27me3).21 Both PRC-dependent histone modifications were significantly diminished upon enforced
miR-128 expression (Fig. 3A). These molecular changes
also occurred in the established glioblastoma cell lines
U87 and U251 (Supplementary Fig. S3B). Additionally,
stable expression of miR-128 led to a decreased level of
the GSC marker CD133, suggesting reduced “stemness”
of GSC528. As expected, siRNA-mediated knockdown
of SUZ12 resulted in elevation of the PRC1-dependent
H2A K119ubi marker (Fig. 3B), in agreement with specific
knockdown of SUZ12 leading to BMI1 overexpression
(Fig. 1D). Interestingly, siRNA-mediated knockdown of
BMI1 led to an increase in the PRC2-dependent H3
K27me3 marker (Fig. 3B), as shown for another component of PRC1,50 although SUZ12 expression levels did
not seem to change (Fig. 1D). This suggests that there
was functional redundancy or complementation
between PRC1 and PRC2 function. Incorporation of bromodeoxyuridine in U87 and U251 glioblastoma cells and
cell counting assays in GSC528 showed a significant reduction in proliferative potential when miR-128
Upregulated (n ¼ 21), downregulated (n ¼ 20), and intermediary
samples (n ¼ 155) were analyzed. Up- vs downregulated P ¼
.0308; upregulated vs intermediary P ¼ .0018; downregulated vs
intermediary P ¼ .8322. (F) A miR-128– based signature in NSCs
and GSCs. Heatmap showing the top 4 most significantly
downregulated (fold ,0.5, P , .05) microRNAs based on a Nano
String array. (G) MiR-128 expression in GSCs derived from
glioblastoma patients. Expression of miR-128 was validated by
qRT-PCR. Mean relative gene expression level + SD is shown. NSC
n ¼ 2, GSC n ¼ 6.
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Fig. 3. Expression of miR-128 in GSCs alters histone modifications and reduces proliferation and self-renewal. (A) MiR-128 effects on levels of
p21, on histone modifications, and on stemness marker. GSC528 cells (528) were transiently transfected with microRNA negative control (NC) or
miR-128 precursor (128) or stably infected with either pCDH vector or pCDH vector expressing miR-128 (pCDH128). Cell lysates were blotted
with anti-p21 WAF1/CIP1, anti-H2A K119ubi, anti-H3 K27me3, or anti-CD133 antibody; anti–total H3 or anti– a-tubulin (aTUB) antibody was
used as loading control (n ¼ 3). (B) Effect of specific knockdown of SUZ12 and BMI1 on PRC-dependent histone modification. GSC528 cell line
was transiently transfected with either siRNA NC or siRNA against SUZ12 or BMI1. Cell lysates were blotted with anti-H2A K119ubi, anti-H3
K27me3, and anti-SUZ12 and anti-BMI1 antibodies; anti–total H3 or anti– a-tubulin antibodies (aTUB) were used as loading control. Data
were quantified by densitometric analysis using ImageJ software, normalized to loading control. Relative expression to averaged NC level is
shown. (C) MiR-128 effects on the proliferative potential of glioma cells. U251 and U87 cells were transiently transfected with microRNA NC
or miR-128 precursor (128), or GSC528 cells (528) were stably infected with either pCDH vector or pCDH vector expressing miR-128
(pCDH128). Bromodeoxyuridine (BrdU) incorporation in U251 and U87 (top panel) and GSC528 cell number (bottom panel) were
quantified in 3 independent experiments; data are expressed as mean + SD. *P , .05, **P , .01. (D) MiR-128 effects on the self-renewal
capabilities of GSCs. GSC528 cells (528) were stably infected with either pCDH vector or pCDH vector expressing miR-128 (pCDH128).
Representative images of soft-agar colony formation (n ¼ 3) are shown. Results expressed as mean number of colonies per well + SD are
shown on the right. **P , .01.
expression was reestablished (Fig. 3C). SiRNA-mediated
knockdown of SUZ12 and BMI1 also led to decreased
proliferative potential of GSC528 (Supplementary Fig.
S3C). To further examine changes in colony formation
of GSCs, we utilized a soft-agar colony formation assay
with GSC528 stably transduced with miR-128.
MiR-128 expression caused a 2-fold reduction in colony
forming ability compared with controls (Fig. 3D). There
was also a different distribution of colony size. Cells overexpressing miR-128 produced predominantly small and
medium-sized colonies, while control cells produced
larger colonies (Supplementary Fig. S3D). These results
show that miR-128 expression diminished PRCdependent chromatin modifications, thus underscoring
the significance of miR-128 on overall activity of PRCs,
in contrast to siRNA-mediated knockdown of only 1
component leading to functional complementation.
This was also associated with reduced glioblastoma cell
proliferation and GSC colony formation.
Reexpression of MiR-128 in GSC and Glioblastoma
Lines Increases Radiosensitivity
PRCs have been shown to be activated by radiation, as
they play a significant role in DNA repair.31,33,35,37
First, we established that the response of glioblastoma
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cells to radiation involves a dose-dependent increase in
the expression of the Polycomb genes SUZ12 and BMI1
by quantitative RT-PCR (Fig. 4A). The observed increase
in mRNA level in GSC528 cells was accompanied by
increases in SUZ12 and BMI1 total protein levels
(Fig. 4B), and miR-128 expression reduced this increase.
To test whether miR-128 –driven downregulation of
PRC complexes conferred increased radiosensitivity to
GSCs, we determined the effect of radiation on
the clonal growth of miR-128 – expressing GSCs. The
results in Fig. 4C show that miR-128 expression significantly increased the radiosensitivity of GSC528 cells.
A dose of 4 Gy in combination with forced expression
of miR-128 eradicated GSC528 cells. MiR-128– expressing GSC528 plated on laminin-coated dishes irradiated
with 2 Gy showed significant decrease in cell number
when we combined both radiation and miR-128 expression compared with radiation or miR-128 alone (Fig. 4D).
As determined by propidium iodide staining and flow
cytometry, miR-128 alone or radiation alone had little
effect on the percentage of propidium iodide –positive
cells, but combining miR-128 and radiation led to significant cytotoxicity (Fig. 4E). We attempted to determine
the reason for the observed increase in miR-128 –mediated cytotoxicity upon radiation. We demonstrated by a
comet assay that cells expressing miR-128 displayed
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
Fig. 4. Effect of miR-128 expression on radiosensitivity. (A) SUZ12 and BMI1 expression after radiation exposure. U87, U251, and GSC528 (528)
cell lines were untreated or radiated with 2-Gy or 4-Gy doses and harvested 1 h after radiation. Expression of SUZ12 and BMI1was validated by
quantitative RT-PCR (n ¼ 3). Mean relative gene expression level + SD is shown. RQ, relative quantification. (B) MiR-128 effects on SUZ12 and
BMI1 overexpression induced by radiation. GSC528 cells (528) were transiently transfected with microRNA negative control (NC) or miR-128
precursor (128), and after 36 h cells were left untreated or were irradiated using a 2-Gy or 4-Gy dose and harvested 36 h after radiation. Cell
lysates were blotted with anti-SUZ12 and anti-BMI1 antibodies, and anti– a-tubulin antibody (aTUB) was used as loading control (n ¼ 3).
Data were quantified by densitometric analysis using ImageJ software, normalized to loading control. Average of relative expression level is
shown. (C) MiR-128 effects on the clonogenic potential of GSC cells after radiation. GSC528 cells (528) were stably infected with either
pCDH vector or pCDH vector expressing miR-128 (pCDH128), plated in soft agar, and left untreated or irradiated using a 2-Gy or 4-Gy
dose. Representative images of soft-agar colony formation (n ¼ 3) after 14 days are shown (left panels). Data are expressed as mean number
of colonies per well (right panel) + SD. *P , .05. (D) MiR-128 effects on the proliferation potential of GSC cells after radiation. GSC528 cells
(528) were placed on laminin-coated dishes, transiently transfected with microRNA NC or miR-128 precursor (128), and after 36 h cells were
left untreated or irradiated using a 2-Gy dose. Representative images of growing cells (n ¼ 3) after 48 h are shown (left panels). Data are
expressed as mean number of cells per well × 105 (right panel) + SD. *P , .05, **P , .01. (E) MiR-128 and radiation effects on glioma cell
viability. GSC528 cells (528) were transiently transfected with microRNA NC or miR-128 precursor (128), and after 36 h cells were left
untreated or radiated using a 2-Gy dose. After 48 h cell flow cytometry was performed. Data are shown as percentages of propidium iodide–
positive (top) and – negative (bottom) cells staining. (F) MiR-128 effects on the DNA repair capability of radiated cells. GSC528 cells (528)
were transiently transfected with microRNA NC or miR-128 precursor (128), and after 36 h cells were left untreated or radiated using a 2-Gy
dose. A comet assay was performed either with nonirradiated cells or after 30 min and 15 h post-irradiation. Representative images of cells
with fragmented DNA tails are shown (left) under low power and high power (inset) magnification. Data are expressed as mean number of
cells with tails (right) + SD. **P , .01.
weaker DNA repair capability compared with control
cells: 15 h after radiation 50% of miR-128 – expressing
cells had fragmented DNA tails (indicative of damaged
DNA) compared with 25% of control cells (Fig. 4F). In
conclusion, miR-128 appears to prevent irradiated cells
from switching on a DNA repair program mediated by increased expression of Polycomb genes, rendering them
more radiation sensitive.
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Loss of Expression of MiR-128 Occurs Early in
Gliomagenesis
To investigate further the role of miR-128 in glioblastoma biology, we utilized a mouse genetic model of
glioma. We used mut3 mice (hGFAP-cre; Nf1flox/+;
Trp53 2/+ ) and their wild-type counterparts. Mut3
mice represent a genetic model in which initially
healthy mice eventually develop malignant astrocytomas with 100% penetrance by 20 – 30 weeks of
age.51,52 We collected RNA and protein from GSCs obtained from the tumors of glioma-symptomatic, adult
mut3 mice, as well as from the same brain region of
wild-type mice of corresponding age. We were able to
show significant downregulation of MIR128 expression
in GSCs derived from the SVZs of mut3 mice (Fig. 5A).
The loss of MIR128 expression in mut3 GSCs corresponded with elevated levels of SUZ12 and BMI1 in
the same cells (Fig. 5B, quantification shown in
Supplementary Fig. S4A). This confirmed that the
same relationship between MIR128 and PRCs existed
in this mouse genetic model of glioma as existed in
human glioblastoma samples and in human GSCs.
We then tested whether MIR128 expression was
altered in mice before the onset of glioma formation in
the NSC niche, shown to be the glioblastoma cell of
origin in this model.53 We harvested NSCs from the
SVZs of wild-type and mut3 mice at 4 weeks of age,
well before the onset of any glioma-related symptoms or
the appearance of tumors (which usually occur at the
age of 20 – 30 wk), and harvested RNA and protein. As
shown in Fig. 5C, MIR128 was significantly downregulated in the NSCs from the young, presymptomatic
mut3 mice. This downregulation was accompanied by
significantly increased levels of SUZ12 and BMI1 proteins
(Fig. 5D, quantification shown in Supplementary
Fig. S4B) and significantly elevated proliferation compared with wild-type, age-matched NSCs from littermates (Supplementary Fig. S4C). When we knocked
down the expression of MIR128 in wild-type NSCs by introduction of a lentiviral miR-128 – ZIP vector (data
not shown), we observed a significant increase of
SUZ12 and BMI1 levels. Moreover, such cells displayed
increased clonogenicity as measured by increased
colony number in a soft-agar colony formation assay
(Fig. 5E). Also, large colonies were formed almost exclusively by cells infected with a miR-128 – ZIP vector
(Supplementary Fig. S4D).
Finally, to test the relevance of these findings in a
human cell model, we performed similar experiments
using the human fetal NSC line SCP27. Similar to
mouse cells, introduction of a lentiviral miR-128 – ZIP
vector to SCP27 fetal NSCs brought about significant
reduction of miR-128 expression (data not shown) and increased SUZ12 and BMI1 levels, followed by a hyperproliferative phenotype as measured in a soft-agar colony
formation assay (Fig. 5F). These findings thus indicate
that loss of miR-128 expression is an early event in the
course of gliomagenesis in the mouse model, and similar
findings in human fetal stem cells suggest that this conclusion may also be valid for human glioblastoma.
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Discussion
The regulation of PRCs in tumor and glioma pathogenesis
is not well known. Our previously published work uncovered that miR-128 is a regulator of BMI1 gene expression
in gliomas. A number of microRNAs function by repressing the expression of multiple genes in a signaling
pathway. In this instance, we wondered whether miR128 repressed the expression of other components of
PRC, as predicted by target recognition software. Here
we report, for the first time, that (i) miR-128 simultaneously targets important constituents of Polycomb
Repressor Complexes 1 and 2 and that its downregulation
in glioblastoma contributes to their high level of expression compared with normal brain; (ii) specific knockdown
of SUZ12 (PRC2) resulted in elevated expression of the
PRC1 component BMI1 and consequently elevated
PRC1 activity, while specific knockdown of BMI1
(PRC1) resulted in increased activity of PRC2, suggesting
redundancy between both PRCs as postulated previously;54 (iii) miR-128 expression correlates clinically with
improved survivorship in subjects with glioblastoma;
(iv) miR-128 is 1 of 4 components of microRNAs that
are sufficient to discriminate between NSCs and GSCs;
(v) miR-128 expression increases radiosensitivity of
GSCs; and (vi) miR-128 downregulation with corresponding SUZ12 and BMI1 upregulation occurs in
SVZ-derived NSCs in 4-week-old mice genetically engineered to develop gliomas by weeks 20 – 30. Taken in conjunction, these novel findings provide evidence of the
important regulatory role of miR-128 in PRC function
and point to deregulation of this role as an early event in
glioma.
Restoration of miR-128 expression in GSCs is biologically consistent with our understanding of cancer as a
disease of multiple aberrant signaling pathways that
would thus require intervention at multiple levels. The
fact that a single microRNA (such as miR-128) simultaneously downregulates a broad set of mRNA targets with
potential pro-oncogenic properties provides a broad
modulatory mechanism that affects cancers, such as
glioblastomas. Our results also show that miR-128 –
mediated targeting of both PRCs prevents the PRC compensatory mechanism whereby specific knockdown of a
PRC2 component leads to upregulation of PRC1 activity
and vice versa, underlining the unique properties of
microRNA-based interventions and providing the opportunity for the discovery of new therapeutic approaches.
Biologically, evidence for specific inhibitory targeting
between 2 molecules is carried out by rescuing the phenotype inhibited by 1 molecule by overexpression of the targeted molecule. This experiment is unlikely to work for
microRNAs, since each microRNA generally targets multiple mRNAs often linked in several signaling pathways
that all affect a phenotype. Rescue in this case would
involve reexpressing all of the inhibited mRNAs. This is
also true for miR-128: trying to rescue the antiproliferative phenotype observed with miR-128 expression by
overexpression of SUZ12 is unlikely to work because
miR-128 expression also targets multiple other mRNAs
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
Fig. 5. Effect of miR-128 downregulation on clonogenic potential of nonmalignant NSCs. (A) MIR128 expression in GSCs derived from SVZs of
mut3 mice. RNAs were extracted from the cells isolated from SVZs of wild-type (wt) mice (wt NSC, n ¼ 4) or symptomatic (20 –24 wk old) mut3
mice (mut3 GSC, n ¼ 3). Expression of MIR128 was validated by quantitative (q)RT-PCR. Mean relative gene expression level + SD is shown.
(B) SUZ12 and BMI1 protein level expression in GSCs derived from SVZs of mut3 mice. Protein lysates were extracted from the cells isolated from
SVZs of wt mice (wt NSC, n ¼ 3) or symptomatic (20–24 wk old) mut3 mice (mut3 GSC, n ¼ 3). Cell lysates were blotted with anti-SUZ12 or
anti-BMI1 antibodies; anti–a-tubulin antibody (aTUB) was used as loading control. (C) MIR128 expression in NSCs from SVZs of mut3 mice.
RNA was extracted from the cells isolated from SVZs of wt mice (wt NSC, n ¼ 4) or young, presymptomatic (4 wk old) mut3 mice (mut3
NSC, n ¼ 3). Expression of MIR128 was validated by qRT-PCR. Mean relative gene expression level + SD is shown. (D) BMI1 and SUZ12
protein level expression in NSCs from SVZs of mut3 mice. Protein lysates were extracted from the cells isolated from SVZs of young wt mice
(wt NSC, n ¼ 3) or young, presymptomatic (4 wk old) mut3 mice (mut3 NSC, n ¼ 3). Cell lysates were blotted with anti-SUZ12 or anti-BMI1
antibodies; anti– a-tubulin antibody (aTUB) was used as loading control (n ¼ 3). (E) Effects of MIR128 knockdown on target protein levels in
wt NSCs and on clonogenic potential. Protein lysates were extracted from the cells isolated from SVZs of young wt mice. Cells were stably
infected with miR-ZIP control vector (ZIP) or miR-ZIP vector coding hairpin for miR-128 (128-ZIP). Cell lysates were blotted with anti-SUZ12
or anti-BMI1 antibodies; anti–a-tubulin antibody (aTUB) was used as loading control (n ¼ 3) (left panel). Representative images of soft-agar
colony formation (n ¼ 3) after 14 days are shown (middle panels). Results expressed as mean number of colonies per well + SD are shown
on the right. *P , .05. (F) Effects of miR-128 knockdown on target protein levels in human SCP27 NSCs and on clonogenic potential. Cells
were stably infected with miR-ZIP control vector (ZIP) or miR-ZIP vector coding hairpin for miR-128 (128-ZIP). Cell lysates were blotted with
anti-SUZ12 or anti-BMI1 antibodies; anti–a-tubulin antibody (aTUB) was used as loading control (n ¼ 3) (left panel). Representative images
of soft-agar colony formation (n ¼ 3) after 14 days are shown (middle panels). Results expressed as mean number of colonies per well + SD
are shown on the right. *P , .05.
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linked to proliferation (such as BMI1 and others). We thus
believe that the functional readout that we have used
(levels of well-established PRC-specific histone modifications) is more suited to prove a direct link between
miR-128 and the activity of both the PRC1 and PRC2
complexes.
The expression of miR-128 and the PRC components
SUZ12 and BMI1 is inversely correlated in human
glioblastoma patient samples, as we demonstrated on
multiple levels (microRNA/mRNA, proteins, and immunohistochemistry), and relatively higher expression of
miR-128 in patient samples correlated with better survival, while higher expression of SUZ12 correlated with
poorer survival. Additionally, analysis of expression of
miR-128 (along with 3 other microRNAs) was sufficient
to discriminate between human NSCs and GSCs. We
also demonstrated that miR-128 – mediated downregulation of PRC components led to a significant inhibition of
PRC-dependent histone modifications, decreased expression of CD133, derepression of the tumor suppressor p21,
decreased glioma cell proliferation, and decreased GSC
self-renewal. Interestingly, specific, siRNA-mediated
downregulation of SUZ12 (PRC2) or BMI1 (PRC1) led
to increased activity of PRC1 or PRC2, respectively.
GSCs are thought to be characterized by a high degree
of chemo- and radiotherapy resistance.55 – 59 This causes
evasion of these therapeutic modalities, leading to inevitable recurrence and increased resistance to conventional
therapy. We demonstrated that ectopic expression of
miR-128 in GSCs significantly increases their radiosensitivity by preventing the radiation-induced increase of
expression of PRC components, possibly by impaired
DNA repair. Other researchers demonstrated that
DNA damage led to redistribution of PRC components
toward altered chromatin rather than to their transcriptional activation.33 Although miR-128 stops proliferation, a phenotype that is linked to increased
radiosensitivity, its action on inhibition of PRC expression and function known to be linked to DNA repair
after radiation is likely responsible for this otherwise paradoxical effect. MicroRNAs have attracted intense research that points toward therapeutic potential and
immediate targeting for sensitization of glioblastoma
cells to chemo- and/or radiotherapy.60 Our results
present a strong rationale for microRNA-mediated molecular targeting of oncogenic pathways to overcome resistance to conventional therapy.
Recently published findings have provided compelling
evidence that expression of MIR128 is severely impaired
in a mouse genetic model of glioma (H-RasV12,
Trp53+/2 ) and that its reexpression prevents glioma development.61 Our results confirmed loss of MIR128 in a
mouse model of glioma, mut3 (Nf1flox/+; Trp532/+ ),
which is accompanied by elevated levels of SUZ12 and
BMI1. Moreover, we demonstrated that loss of MIR128
expression and the concomitant increase in PRC activity
are observed in young, presymptomatic animals,
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suggesting that this loss is an early event in brain cells
that are poised to become tumorigenic but have not originated a tumor yet. We also showed that knockdown of
miR-128 in mouse and human nonmalignant NSCs led
to increased clonality. Whether this increase leads to enhanced tumorigenesis in vivo will be the subject of
future studies. Additionally, miR-128 has the ability to
target a broad range of oncogenic targets (epidermal
growth factor receptor and platelet derived growth
factor receptor signaling,61 E2F3,62 and p70S6K63
among others). Recently published work has identified
the oncoprotein SNAIL as a direct negative regulator of
miR-128 in breast cancer.64 Interestingly, it was demonstrated that both p53 and neurofibromatosis type 1
repress SNAIL,65,66 providing a plausible explanation
for miR-128 downregulation in mut3 cells. In ovarian
cancer, SNAIL was shown to induce a stemlike phenotype
and radioresistance.67 Finally, SNAIL is targeted by
p53-inducible miR-34,68 which was identified as a
tumor suppressor in glioma cells.69,70 It remains unclear
whether the mechanism of regulation of miR-128 in
glioma cells is the same as in breast cancer.
It has been postulated that microRNA replacement approaches have strong therapeutic potential because of the
fact that single microRNAs can regulate multiple oncogenic pathways that are commonly deregulated in
cancer.71 Using GSCs in this study allowed us to demonstrate that miR-128 is functional both in this subpopulation and in more differentiated glioma cells.
Our report, providing novel evidence that miR-128
is inactivated early in the course of gliomagenesis, simultaneously targets major components of oncogenic PRC
signaling and renders GSCs more susceptible to conventional therapy, pointing toward miR-128 replacement
as an attractive candidate for this therapeutic modality.
Supplementary Material
Supplementary material is available online at NeuroOncology (http://neuro-oncology.oxfordjournals.org/).
Acknowledgments
We thank the members of Dr Arnab Chakravarti’s lab
(OSUMC) for their technical assistance with radiation.
Conflict of interest statement. None declared.
Funding
This work was supported by an Neurosurgery Research
and Education Foundation Research Fellowship
(American Association of Neurological Surgeons)
awarded to P.P. and E.A.C.
Peruzzi et al.: MiR-128 targets Polycomb Repressor Complexes in glioma
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