BMC Medicine

BioMed Central

Research article

Open AccessmiR-124 and miR-137 inhibit proliferation of glioblastoma
multiforme cells and induce differentiation of brain tumor stem
cells
Joachim Silber1, Daniel A Lim†1, Claudia Petritsch†1, Anders I Persson†2,3,
Alika K Maunakea1, Mamie Yu1, Scott R Vandenberg4, David G Ginzinger5, C
David James1, Joseph F Costello1, Gabriele Bergers1, William A Weiss2,3,
Arturo Alvarez-Buylla1 and J Graeme Hodgson*1
Address: 1Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA, 2Department of Neurology,
University of California San Francisco, San Francisco, CA, USA, 3Department of Pediatrics, University of California San Francisco, San Francisco,
CA, USA, 4Department of Pathology, University of California San Francisco, San Francisco, CA, USA and 5Applied Biosystems, Foster City, CA, USA
Email: Joachim Silber - Joachim.Silber@ucsf.edu; Daniel A Lim - LimD@neurosurg.ucsf.edu; Claudia Petritsch - Claudia.Petritsch@ucsf.edu;
Anders I Persson - anders.persson@ucsf.edu; Alika K Maunakea - Alika.Maunakea@ucsf.edu; Mamie Yu - myu@cc.ucsf.edu;
Scott R Vandenberg - Scott.Vandenberg@ucsf.edu; David G Ginzinger - dginz@yahoo.com; C David James - david.james@ucsf.edu;
Joseph F Costello - Joseph.Costello@ucsf.edu; Gabriele Bergers - gabriele.bergers@ucsf.edu; William A Weiss - weiss@cgl.ucsf.edu;
Arturo Alvarez-Buylla - ABuylla@stemcell.ucsf.edu; J Graeme Hodgson* - ghodgson@cc.ucsf.edu* Corresponding author †Equal contributors

Published: 24 June 2008
BMC Medicine 2008, 6:14

doi:10.1186/1741-7015-6-14

Received: 9 October 2007
Accepted: 24 June 2008

This article is available from: http://www.biomedcentral.com/1741-7015/6/14© 2008 Silber et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor
despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecularand cellular mechanisms that drive GBM formation are required to improve patient outcome.
MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, andhave been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM
remains poorly understood. In this study, we investigated the role of microRNAs in regulating thedifferentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.
Methods: We used quantitative RT-PCR to assess microRNA expression in high-grade
astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs
in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells
and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining,and cellular proliferation was determined using fluorescence-activated cell sorting.
Results: Our studies revealed that expression levels of microRNA-124 and microRNA-137 weresignificantly decreased in anaplastic astrocytomas (World Health Organization grade III) andglioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain
tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neuralstem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to
12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation
with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced
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morphological changes and marker expressions consistent with neuronal differentiation in mouseneural stem cells, mouse oligodendroglioma-derived stem cells derived from S100β-v-erbB tumorsand cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969).
Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 andSF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclindependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.
Conclusion: microRNA-124 and microRNA-137 induce differentiation of adult mouse neuralstem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiformederived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest thattargeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells
may be therapeutically efficacious for the treatment of this disease.

Background
MicroRNAs (miRNAs) are a class of small non-coding
RNAs that regulate diverse cellular processes through RNAinterference-based mechanisms. miRNAs are transcribedas primary RNA transcripts (pri-miRNAs), processed in
the nucleus to smaller precursor hairpin structures (premiRNAs), and then exported to the cytoplasm where they
are processed further by the Dicer nuclease to become
mature, functional miRNAs approximately 21 nucleotidesin length. Mature miRNAs, the endogenous equivalent ofshort interfering RNAs (siRNAs), are then incorporated
into the RNA-induced silencing complex, which facilitates
their interaction with, and inhibition of, target messenger
RNAs (mRNAs) by translational repression or message
cleavage (as reviewed in [1]).
Since the initial discovery of miRNAs as developmental
mutants in Caenorhabditis elegans, their role as important
regulators of stem cell division and development in evolutionarily divergent organisms has become increasingly
apparent. For example, functional ablation of the RNaseIII enzyme Dicer, resulting in the ablation of miRNAbiogenesis, disrupts the division of insect germline stem
cells [2] and mouse embryonic stem cells [3], and impairs
early embryonic development in zebrafish [4] and mice[5]. It is also becoming increasingly evident that miRNAs
play important roles in cancer etiology. For example,
expression of the mir-17–92 miRNA cluster augments
tumor angiogenesis [6] and accelerates c-Myc-induced Bcell lymphoma development in mice [7], and the let-7
miRNA transcriptionally regulates the ras oncogene [8]and inhibits growth of lung adenocarcinoma cells [9].
Further, impairment of miRNA processing enhances cellular transformation and tumorigenesis [10], consistent
with observations that global down-regulation of miRNAs
occurs in multiple tumor types compared with normal tissues [11].
Most recently, specific miRNAs have been implicated in
the differentiation of cultures derived from mouse embryonic stem (ES) cells and mouse tumors. For example,

expression of miR-124 and miR-9 increases during differentiation of mouse ES cell-derived neural progenitors, andexperimental manipulation of miR-124 and miR-9
expression affects neural lineage differentiation in the EScell-derived cultures [12]. Up-regulation of miR-124 also
induces neuronal differentiation of mouse neuroblastoma cell lines CAD and Neuro2a and the mouse embryonal tumor cell line P19 [13]. These results suggest thatmiRNAs may be valuable therapeutic agents if they similarly promote differentiation of human tumor cells andtumor stem cells (TSCs).
The discovery of a rare, highly tumorigenic, self-renewing
sub-population of glioblastoma multiforme (GBM) cells
that express the cell surface marker cluster of differentiation (CD) 133 (see [14,15]), the so-called GBM stem cell
population, suggests that therapeutic approaches that
effectively inhibit or kill CD133+ GBM stem cells maylead to marked improvements in patient outcome. To thisend, it has recently been demonstrated that induction ofdifferentiation of CD133+ GBM cells by bone morphogenetic protein 4 can effectively inhibit intracerebral GBM
tumor growth in mice [16]. In light of the growing bodyof evidence supporting a role for miRNAs in promoting
stem cell differentiation, we investigated the role of miRNAs in differentiation and proliferation of human GBM
stem cells, mouse oligodendroglioma tumor stem cells(mOSCs) and normal adult mouse neural stem cells(mNSCs), putative progenitors of adult gliomas [17]. Our
results show that miR-124 and miR-137 can induce neuronal differentiation of OSCs and GBM stem cells andinhibit proliferation of GBM cell lines. These results suggest that miR-124 and miR-137 may be useful therapeutic
agents for the treatment of GBMs.

Methods
Primary human tissues
Fresh frozen primary human tissues were acquired fromthe Brain Tumor Research Center tissue core at the University of California San Francisco (UCSF) in accordancewith the Committee on Human Research approved procePage 2 of 17(page number not for citation purposes)

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dures. All samples were thoroughly reviewed by a neuropathologist(S
Vandenberg),
andanaplastic
astrocytomas (AAs) and GBM tumors were confirmed tocontain at least 90% tumor. Non-neoplastic brain tissues
were derived from the temporal lobes of epileptic patient
surgeries and comprised primarily cortex with mild tomoderate reactive astrocytosis and neurons. For further
details about the samples see Additional file 1.
Quantitative reverse transcriptase polymerase chain
reaction
Total RNA was extracted using the miR-Vana RNA isolation system (Ambion, Austin TX). Expression of 192
human miRNAs was quantitated in human tissues using
TaqMan® miRNA assays human panel-early access kit(Applied Biosystems, Foster City CA). Expression of thesix high-grade astrocytomas (HGA)-miRNAs during NSC
differentiation was quantitated using individual TaqMan®
MicroRNA Assays. The comparative Ct (ΔΔCt) method
was used to determine the expression fold change.
Statistical analyses
Statistical analysis of miRNA expression in primary tissues
was performed on log2 transformed fold change datausing freely available R language. The limma package in
Bioconductor was used to compare the three types of primary tissues (glioses, AAs and GBMs). Moderated t-statistics were obtained as described elsewhere [18] and Pvalues were adjusted for multiple comparisons by controlling the false discovery rate. Changes were considered significant if the false discovery rate was less than 0.05.
Demethylation and deacetylation experiments
U87 and U251 glioma cell lines were seeded at 1 × 105
cells per well of a six-well plate, incubated for 24 hours in
Dulbecco's Modified Eagle's Medium (DMEM) high glucose 10% serum, and then supplemented with fresh
media containing 5-aza-dC (1 or 5 μM; Sigma-Aldrich) for
72 hours or trichostatin A (TSA) (100 ng/ml; SigmaAldrich) for 12 hours. For the combination study, 1 or 5
μM 5-aza-dC was present for 72 hours and TSA was added
for the last 12 hours. The media containing drugs werechanged every 24 hours.
miRNA oligonucleotides
miRIDIAN miRNA mimic negative control (cel-miR-67)
and miRIDIAN miRNA mimics (mmu-miR-124, mmumiR-137) were purchased from Dharmacon (Lafayette,CO) and validated using the pMIR-REPORT miRNA
Expression Reporter Vector System (Ambion, Austin, TX).
For results, see Additional file 2.
CDK6-3'UTR miR-137 reporter assays
Cyclin-dependent kinase 6 (CDK6)-3'UTR reporter assays
were performed in U251 cells. pMIR-REPORT vectors har-

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boring CDK6-3'UTR sequences with wild type (WT) miR137 binding sites or mutated (MUT) miR-137 bindingsites were generated by cloning the following oligonucleotides into the HindIII and SpeI restriction sites of pMIRREPORT: CDK6-UTR-WT fw 5'-AGCTTGATCACAGAAATATTGCTAGCTGATACATATTATTGCATTTCATAAAACTA
CDK6-UTR-WT
rv
5'CTAGTAGTTTTATGAAATGCAATAATATGTATCAGCTAGCAATATTTCTGTGATCA CDK6-UTR-MUT fw 5'-AGCTTGATCACAGAAATTAACGAAGCTGATACATATTATTGCATTTCATAAAACTA CDK6-UTR-MUT rv 5'-CTAGTAGTTTTATGAAATGCAATAATATGTATCAGCTTCGTTAATTTCTGTGATCA Cells were transfected with (1) miR-137 or celmiR-67-negative-control mimics (50 nM), (2) pMIRREPORT vectors containing WT or MUT miR-137 bindingsites (400 ng) and (3) pRL-SV40 (Promega) expressing
Renilla luciferase (400 ng) for normalization. Cells weregrown in high-glucose DMEM supplemented with 10%
fetal bovine serum, and luciferase measurements were
performed 48 hours post-transfection using the DualLuciferase Reporter Assay System (Promega).
Establishment and transfection of mouse subventricularzone-NSCs
Adult mouse subventricular zone (SVZ)-NSC cultures
were derived and grown as described previously [19] witha few modifications. SVZ microdissections from 2-monthold CD-1 mice (Charles River Laboratories) were dissociated to a single cell suspension with 0.25% trypsin, 0.5
mM ethylene diamine tetraacetic acid (EDTA) and gentle
trituration. Cells were cleared on a 22% Percoll (Sigma)
step-gradient (2) and grown in proliferation medium(DMEM/F12/N2), 5% fetal calf serum (FCS), 20 ng/ml
epidermal growth factor (EGF), 20 ng/ml basic fibroblast
growth factor (bFGF) and 35 μg/ml bovine pituitary
extract (all media and supplements from Invitrogen, Inc.).
Non-attached cells were collected after 1 day and replated
into a 35-mm tissue culture dish (Corning). After 7 to 10
days, the plate was hyperconfluent with SVZ-NSCs andthese were routinely passaged 1:2 with 0.25% trypsin and
0.5 mM EDTA. Cells were passaged at least six times
before use in experiments. Media was half-changed every
2 days and completely changed every 4 days. Differentiation of SVZ-NSCs for the miRNA expression time course
was induced by removing EGF, FGF and FCS from themedia [19].

For transfection of miR-124/137 into SVZ-NSCs, 50,000
cells were plated into eight-well culture slides (BD Falcon
Biosciences) pretreated with 0.1 mg/ml poly-D-lysine(Sigma) and 10 μg/ml laminin (Invitrogen) 24 hours
prior to transfection. A total of 100 nM miRIDIAN miRNA
mimics (50 nM each for miR-124 and miR-137 co-transfections) were complexed with LipofectAMINE 2000 (Invitrogen) and added directly to cells growing inPage 3 of 17(page number not for citation purposes)

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proliferating medium. Transfection and proliferating
medium was removed 12 to 24 hours post-transfection
and cells were induced to differentiate as described above.

to a pre-separation filter. Fluorescence-activated cell sorter
analyses confirmed a pure CD133- fraction and a highly
enriched CD133+ fraction.

Growth and transfection of S100βv-erbB mouse tumor
stem cells
Adult tumor stem cells were derived from a low-grade oligodendroglioma of a 120-day-old FVB/N transgenic
mouse expressing the v-erbB transgene under control of
the S100β promoter [20]. Tumor tissue was microdissected from the surrounding normal brain and dissociatedto a single cell suspension with papain, gentle triturationand filtration through a 40-μM mesh screen (Falcon).
Neurospheres were grown from single cells in Neurobasal
medium (Invitrogen) supplemented with 20 ng/ml EGF(Sigma), 20 ng/ml bFGF (Peprotech) and B27 (Invitrogen) on low-adherent tissue-culture dishes (Corning).
After four passages, neurospheres were dissociated andreplated into 10-cm culture dishes (Corning) in proliferation medium (see above). These TSCs are self-renewing
and multi-potent and express markers for astrocytes (glial
fibrillary acidic protein, GFAP), neuronal progenitors(Tuj1) and oligodendrocytic progenitors (NG2) under
differentiating conditions.

For transfections, both CD133+ and CD133- cells wereplated (20,000 cells per well) in 24-well plates coatedwith polyornithine and laminin. Cells were transfectedwith miR-124 and/or miR-137 (100 nM) or a negative
control oligonucleotide for 4 hours using lipofectamine.
Cells were then washed and cultured for 10 days in NBE
media without growth factors.

For miRNA transfections, 25,000 cells were plated into
eight-well pre-coated culture dishes (Nunc), 24 hours
prior to transfection. Transfections and differentiation
procedures were performed as described for SVZ-NSC cultures.
Growth, CD133 sorting and transfection of early passage
human GBM cells (SF6969)
Human GBM tissue was acquired by surgical removal after
informed consent at UCSF and washed with Hank's buffered saline solution without magnesium and calcium.
Tumors were then enzymatically dissociated with papain(Worthington) for 30 minutes at 37°C. After centrifugation and one wash with phosphate buffered saline, pH
7.4, cells were transferred to NBE media consisting of neurobasal media without retinoic acid (Invitrogen), N2 andB27 supplements (0.5× each; Invitrogen), 20 ng/ml
human recombinant bFGF (Peprotech) and 20 ng/ml
human recombinant EGF (Sigma-Aldrich). Cells wereplated in ultra-low adherent plates (Corning). Medium
was changed every 3 to 5 days.

Cells cultured in suspension were dissociated using
accutase (Innovative Cell Technologies) for 30 minutes at
37°C. After one wash in RinseMACS buffer (Miltenyi Biotech), cells were incubated with magnetic beads conjugated with an antibody against the CD133/1 epitope. The
cells were incubated with beads for 30 minutes at 4°C.
Thereafter, cells were washed with 20× RinseMACS buffer,centrifuged and added on to large cell columns connected

Immunocytochemistry
Stem cell cultures were fixed, washed and preblocked
prior to incubation with primary antibodies (Tuj1, 1:500,
Covance Inc.; GFAP, rabbit polyclonal, 1:500, Dako Inc.;
microtubule-associated protein 2 (MAP2) ab, 1:500,
Sigma). Cells were then stained with Alexa488- orAlexa594-conjugated secondary antibodies and the nuclei
counterstained with Hoechst 33258 (Molecular Probes)
or DAPI (Sigma).
Cell cycle analysis
Cell cycle analyses were conducted using the fluorescein
isothiocyanate BrdU Flow Kit following manufacturer's
recommendations (BD Pharmingen, San Diego, CA).
Immunoblotting
Immunoblotting was performed using standard protocols
with antibodies CDK6 (1:1000; Cell Signaling, Temecula,CA), Phospho-Rb (1:1000; Cell Signaling Technology,
Temecula, CA), and β-actin (1:5000; Sigma, St Louis,MO).

For more detailed information regarding experimental
methods, see Additional file 3.

Results
miR-124 and miR-137 are down-regulated in high-grade
gliomas and up-regulated during adult NSC differentiation
To identify deregulated miRNAs that have not been previously implicated in GBM cells [21,22], we used quantitative reverse transcriptase polymerase chain reaction (RTPCR; Taqman) to measure expression of 192 mature
miRNA sequences in human non-neoplastic brain tissues(glioses), AAs (World Health Organization (WHO) gradeIII) and GBMs (WHO grade IV). The comparative Ct(ΔΔCt) method was used to determine the expression foldchange of each miRNA in tumor samples relative toglioses (see Additional file 4). Briefly, the ΔCt of eachmiRNA was determined relative to let-7a and miR-16,
endogenous control miRNAs that were robustly andinvariantly expressed across all samples (see Additional
file 5) and the average ΔCt of the four glioses was used as
the calibrator for the tumor samples. Consistent with pre-Page 4 of 17(page number not for citation purposes)

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vious observations in GBMs, we observed recurrent upregulation of miR-10b [22] and miR-21 [21] in our sample set; miR-10b was up-regulated more than 100-fold in
two out of four AA and two out of four GBM tumors; miR21 was up-regulated 5- to 30-fold in two out of four AA
and all four GBM tumors. Also consistent with previous
studies on other tumor types [11], we observed a global
decrease of expression in AA and GBM tumors relative to
non-neoplastic brain tissue.
We next performed statistical analyses of our miRNA
expression data to identify novel miRNAs of interest in
HGAs (GBM and AA). For a summary of these analyses see
Additional file 6. As shown in Table 1, we found 35 miRNAs that were significantly deregulated (P < 0.05) in AA or
GBM tumors. Thirteen (37%) of these miRNAs were differentially expressed in both tumor classes relative toglioses, 16 (45%) were differentially expressed in GBM
tumors only, and 6 (17%) were differentially expression
in AA tumors only. We identified six miRNAs of particularinterest, miR-7, miR-124, miR-129, miR-137, miR-139
and miR-218, which were down-regulated in both AAs
and GBMs (Figure 1A, Additional file 8 and Table 1) at amore stringent level of significance (P ≤ 0.01). We hereafter refer to these six miRNAs as HGA-miRNAs.

We observed that the majority of the HGA-miRNAs show
expression changes during, or have been implicated in,differentiation of various cell lineages: miR-7 during photoreceptor differentiation [23]; miR-124 and miR-137
during erythropoiesis [24]; miR-124 and miR-218 during
neuronal differentiation of embryonal carcinoma cell differentiation [25]; miR-124 during neuronal differentiation of ES cells [12]. To test whether expression of HGAmiRNAs was altered during differentiation of adult NSCs,
putative precursor cells of high-grade gliomas [17], we
established early passage (passage 6) cultures of SVZ-NCS
as described [19]. This is a monolayer NSC culture system
in which growth factor withdrawal rapidly (within 2 to 4days) induces a large number of neuroblasts that constitute approximately 50% of the total cells after 3 to 4 daysof differentiation. Consistent with previous studies [19],we observed a steady increase in the number of Tuj1+ neuroblasts over a 5-day differentiation time course (Figure2A). In parallel cultures, we measured miRNA expression
every 24 hours for 5 days (Figure 2B). Expression of miR124 and miR-137, respectively, increased up to 8- and 24fold, expression of miR-129 and miR-139, respectively,
decreased up to 2- and 4-fold, and expression of miR-7
and miR-218 did not change appreciably.
Our differentiation studies in mNSCs suggested thatgrowth factor signaling, which is recurrently activated inTable 1: Differentially expressed microRNAs in anaplastic astrocytoma and/or glioblastoma multiforme tumors relative to nonneoplastic brain tissue

Glioblastoma multiforme and anaplastic astrocytoma
Fold change
glioblastoma
multiforme

Fold change
anaplastic
astrocytoma

Down-regulated
miR-7
miR-31
miR-107
miR-124
miR-124b
miR-129
miR-137
miR-138
miR-139
miR-187
miR-203
miR-218

34
17
14
57
45
43
63
27
66
6
13
40

13
9
10
26
27
27
66
12
34
5
4
16

Up-regulated
miR-10b3575

Glioblastoma multiforme only

Ref

Fold change
glioblastoma
multiforme

Down-regulated
miR-101
miR-128amiR-132
miR-133a
miR-133b
miR-149
miR-153[21] miR-154*
miR-185
miR-29b
miR-323
miR-328
miR-330

4
27
7
17
14
9
12
7
5
6
17
8
18

[22] Up-regulated
miR-21
miR-155
miR-2107
87Anaplastic astrocytoma only
Fold change
anaplastic
astrocytoma

Ref

Down-regulated
miR-125b
miR-126
miR-127
miR-134

153
7
149
9

[22]

Up-regulated
miR-296
miR-320

11
5

Ref

[22]

[21,22]

P-values for all miRNAs are P < 0.05 for comparisons of miRNA expression in tumors versus glioses; for miRNAs in bold P < 0.01. Referencesindicate high-grade astrocytoma miRNA expression studies in which miRNA has been described previously.

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Figure 1and multiforme
miR-124
glioblastoma
miR-137 arecell
down-regulatedlines followingintreatment
anaplasticwith
astrocytomas
DNA demethylating
and glioblastoma
agents multiformes and are up-regulated inmiR-124 and miR-137 are down-regulated in anaplastic astrocytomas and glioblastoma multiformes and are
up-regulated in glioblastoma multiforme cell lines following treatment with DNA demethylating agents. (A)Expression of high-grade astrocytomas-microRNAs in individual tumor samples measured relative to let-7a (black dots) andmiR-16 (white dots). Sample classes are glioses, anaplastic astrocytomas and glioblastoma multiformes. (B) Glioblastoma multiforme cell lines (U87 and U251) were treated with 5-aza-dC at 1 μM (Aza.1) or 5 μM (Aza.5) alone, trichostatin A (100 ng/ml)
alone, or combinations of both agents. MicroRNA expression was measured relative to let-7a and normalized to vehicle control (dimethyl sulfoxide). Error bars represent standard deviation of triplicate polymerase chain reactions from a single experimental set. Similar results were obtained in independent experiments (see Additional file 7).

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Figure 2expression during differentiation of subventricular zone-neural stem cells
MiRNA
MiRNA expression during differentiation of subventricular zone-neural stem cells. (A) Marker expression during
adult neural stem cell neurogenesis. Photomicrographs are shown of proliferating subventricular zone-neural stem cells cultures in proliferation conditions (A)-(D), and following 1 day (E)-(H), 2 days (I)-(L), 3 days (M)-(P) and 4 days (Q)-(T) of
mitogen deprivation. Phase images (A), (E), (I), (M), (Q) are shown with corresponding epifluorescent images showing 4'-6-diamidino-2-phenylindole-stained nuclei (B), (F), (J), (N), (R) and Tuj1 expression (C), (G), (K), (O), (S). Glial fibrillary acidic protein expression (D), (H), (L), (P), (T) is shown in parallel cultures. (B) Expression analysis of high-grade astrocytomamicroRNAs during a 5-day differentiation time-course of subventricular zone-neural stem cells.

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HGAs, suppresses expression of miR-124 and miR-137. It
has also been shown that miR-124 expression is epigenetically suppressed in a number of tumor types includingcolorectal and breast cancers [26]. Further, miR-137 isclosely associated with a large CpG island [27], suggestingthat it may also be epigenetically silenced in tumors. We
tested, therefore, whether expression of miR-124 andmiR-137 could be activated in GBM cell lines following
treatment with 5-aza-2'-deoxycytidine (5-aza-dC), a DNA
methylation inhibitor and/or TSA, a histone deacetylase
inhibitor. MiRNA-124 expression increased around 2-foldin U251 and U87 cells following combined treatment
with 5-aza-dC (5 μM) and TSA (Figure 1B and Additional
file 8). MiRNA-137 expression increased up to 8-fold in
GBM cell lines treated with 5-aza-dC, and up to 12-fold incells treated with both 5-aza-dC and TSA (Figure 1B and
Additional file 8). Expression of both miRNAs remained
relatively unchanged in cells treated with TSA alone (Figure 1B and Additional file 8). These data suggest that epigenetic modification of regulatory sequences in CpG
islands may contribute to miR-124 and miR-137 silencing
in GBMs.
miR-124 and miR-137 promote neuronal differentiation of
adult NSCs
To test whether up-regulation of miR-124 and miR-137
promote differentiation of adult mNSCs, we transfected
proliferating mNSCs with double-stranded RNA oligonucleotides corresponding to the mature sequences of eachmiRNA. In each experiment, at least 80% to 90% transfection efficiencies were achieved. NSCs were maintained inproliferation medium during transfection in which cells
generally have a spindle, non-neuronal morphology, withhigh expression of GFAP, a stem cell and astrocyte marker,
but low expression of the neuronal marker Tuj1 (Figure2A). Growth factors were withdrawn 12 to 24 hours following transfection and cells were allowed to differentiate
for 72 hours. Transfection of either miR-124 or miR-137
resulted in a 5-fold increase in the numbers of cells
stained with the neuronal marker Tuj1 relative to controls(Figure 3A, B and 3C). Distinct morphological changes
were also apparent for each miRNA; miR-124 induced
neuritic branching of the cells whereas miR-137 induced
a rounded or trapezoidal cellular appearance with no neuritic outgrowth (Figure 3A and 3B). Co-transfection of
miR-124 and miR-137 resulted in a near 2-fold increase in
Tuj1+ cells relative to miR-124 or miR-137 transfections
alone but did not promote neuronal morphologic characteristics (Figure 3C). Finally, transfection of miR-124, but
not miR-137, resulted in a 2-fold decrease in the numbersof GFAP-positive cells (Figure 3A and 3C). Thus, overexpression of miR-124 and miR-137 enhances neuronal-like
differentiation of adult NSCs in vitro.

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miR-124 and miR-137 promote neuronal differentiation ofbrain TSCs
As we observed that expression of miR-124 and miR-137
is reduced in HGAs and that miR-124 and miR-137 promote differentiation of non-neoplastic adult mNSCs, wetested next whether up-regulation of miR-124 and miR137 could promote differentiation of brain tumor-derived
stem cells. We first assessed differentiation of mOSCs
derived from S100β-v-erbB transgenic mouse oligodendrogliomas [20]. MiRNA-124 is down-regulated inhuman oligodendrogliomas [28], and both miR-124 andmiR-137 are down-regulated over 10-fold in S100β-v-erbB
tumor stem cells relative to mNSCs (Additional file 7).
Consistent with our observations in mNSCs, we observed
a significant increase in the numbers of cells that express
the neuronal marker Tuj1 following transfection with
miR-124, miR-137 or a combination of both miRNAs(Figure 4A). Transfection with either miR-124 or miR-137
resulted in rounded or trapezoidal cellular morphology of
Tuj1-positive cells with reduced neuritic outgrowth. We
also observed that transfection of miR-124 and miR-137
reduced the numbers of GFAP-positive mOSCs (Figure
4A).

We tested next whether miR-124 and miR-137 could promote differentiation of human GBM stem cells. GBM cells
were isolated from a primary tumor (SF6969) andexpanded as tumor spheres in nonadherent plates. Cells
were sorted using magnetic beads conjugated to an antibody against CD133, a putative marker of GBM stem cells[14,15]. Both CD133+ and CD133- cells were transfectedwith miR-124 and/or miR-137 and then cultured for 10
days in NBE media without growth factors. Transfectionof miR-124 and/or miR-137 dramatically increased thepercentage of Tuj1-positive cells, and reduced the percentage of GFAP-positive cells and in both CD133+ andCD133- GBM cell fractions (Figure 4B). Tuj1 was
expressed in cells with a neuronal morphology but also inrounded cells and cells undergoing mitosis. The expression of GFAP-positive cells was confined to cells displaying typical morphology of type I and type II astrocytes.
To further investigate the role of miR-137 in neuronal differentiation of GBM cells, we assessed expression of anadditional neuronal marker, MAP2, following overexpression of miR-137. Unsorted SF6969 GBM cells were transfected with miR-137 and cultured for 10 days in NBE
media without growth factors. In addition to the expected
increase of cells positive for Tuj-1 after 10 days, we also
observed an evident increase in MAP2-postive cells following transfection of miR-137 (Figure 4C). Again, as inmNSCs and oligodendroglioma tumor spheres, miR-137
induced rounded morphology with little evidence of neuritic outgrowth (Figure 4C). Collectively, our results showthat in the absence of growth factor signaling, miR-124

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Figure 3and miR-137 promote neuronal differentiation of subventricular zone-neural stem cells
miR-124
miR-124 and miR-137 promote neuronal differentiation of subventricular zone-neural stem cells. (A) Epifluorescent images of subventricular zone-neural stem cells 72 hours after transfection with miR-124, miR-137 and control oligonucleotide. Cells were immunostained with Tuj1 and glial fibrillary acidic protein antibodies, nuclei were counterstained with 4'-6diamidino-2-phenylindole- and images are merged. Scale bar is 10 μm. (B) Phase contrast images of subventricular zone-neuralstem cells 48 hours post-transfection by miR124 and miR137 and Tuj1 immunostaining of the same cultures 72 hours posttransfection. (C) Quantification of percentage of Tuj1+ cells, Tuj1+ cells with neuronal morphology and glial fibrillary acidic
protein+ cells 72 hours after transfection with miR-124, miR-137, both miR-124 and miR-137, control oligonucleotide ortransfection reagent.

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Induction
Figure 4 of neuronal differentiation of tumor-derived neural stem cells by miR-124 and miR-137
Induction of neuronal differentiation of tumor-derived neural stem cells by miR-124 and miR-137. (A) Epifluorescent images of tumor-derived neural stem cells 72 hours after transfection with miR-124, miR-137, control oligonucleotides
and lipofectamine reagent alone. Cells were immunostained with Tuj1 and glial fibrillary acidic protein antibodies and DNA wasstained with Hoechst 33258 reagent. The percentage of Tuj1- and glial fibrillary acidic protein-positive cells was quantified ineach sample after transfection and staining and plotted against the total number of counted cells (n = 450). (C) Quantificationof Tuj1+ and glial fibrillary acidic protein+ cells in primary glioblastoma multiforme cultures 10 days after transfection of miR124, miR-137 or control oligonucleotides. The inset shows a Tuj1+ cell with neuronal morphology from a miR-124 and/or
miR-137 cotransfection. (D) Immunostaining with neuronal markers Tuj1 and microtubule-associated protein 2 10 days after
miR-137 or negative control miR-transfections in glioblastoma multiforme lines maintained as neurospheres.

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and miR-137 enhance neuron-like differentiation of oligodendroglial and GBM TSCs.
miR-124 and miR-137 inhibit proliferation of GBM celllines
Since exit from the cell cycle is required for induction ofdifferentiation, we tested whether miR-124 and miR-137
inhibit proliferation of GBM cells. Relative to control oligonucleotides, transfection of miR-124 or miR-137
resulted in a marked reduction in the number of cells in
the S-phase of the cell cycle and a marked increase in thenumber of cells in G0/G1 in U251 GBM cells (Figure 5A)
and early passage GBM cells derived from a newly diagnosed human GBM (Figure 5B). No reproducible differences were observed for cells in G2/M of the cell cycle, orin cells undergoing apoptosis (sub G1) in any of the celllines examined (data not shown). Our data suggest thatmiR-124 and miR-137 induce G0/G1 cell cycle arrest in
GBM cells.

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miR-124 and miR-137 inhibit CDK6 expression andphosphorylated retinoblastoma levels in GBM cells
To ascertain the molecular mechanisms by which miR124 and miR-137 induce G0/G1 cell cycle arrest in GBM
cells, we assessed expression of CDK6, a regulator of thecell cycle and differentiation (reviewed in [29]), following
transfection of these miRNAs to U251 cells. CDK6 is anestablished target of miR-124 in HCT-116 colon cancer
cells [26], a predicted target of miR-137 (TargetScan and
PicTar), and has been functionally implicated in thedevelopment of multiple malignancies. In independent
experiments we observed marked reductions of CDK6transcript (Figure 6A) and CDK6 protein (Figure 6B) in
response to miR-124 and miR-137 transfection. Levels ofphosphorylated retinoblastoma (RB) (pSer 807/811), a
known target of CDK6 [30], were also reduced in responseto miR-124 and miR-137 transfection (Figure 6B).

To validate that the 3' UTR of CDK6 is a direct target ofmiR-137, we used a luciferase reporter system in which

FiguremiR-1245and miR-137 inhibit proliferation of glioblastoma multiforme stem cells and induce cell G0/G1 cycle arrest
miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme stem cells and induce cell G0/G1 cycle
arrest. Cell cycle analysis was conducted by fluorescence-activated cell sorter at 48 hours after transfection of 100 nM (final
total microRNA concentration) miR-124, miR-137, miR-124 and miR-137 together or negative control oligonucleotides(neg#1, neg#2) to U251 (A) and SF6969 (B) glioblastoma multiforme cells. Cells were treated with bromodeoxyuridine for 30
minutes, fixed, treated with fluorescein isothiocyanate-labeled antibromodeoxyuridine antibody and the DNA stain 7-aminoactinomycin D and subject to flow cytometry. Values represent mean ± standard deviation of replicate experiments; *P < 0.05.

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CDK6
Figureexpression
6
is inhibited by miR-124 and miR-137 in glioblastoma multiforme cells
CDK6 expression is inhibited by miR-124 and miR-137 in glioblastoma multiforme cells. (A) Transfection of 100
nM miR-124 or miR-137 reduces cyclin-dependent kinase 6 mRNA transcript levels by 50% in U251 cells at 48 hours relative
to cells transfected with 100 nM control oligonucleotide. Cyclin-dependent kinase 6 expression was determined by TaqMan
relative to control genes Gus (black bars), GAPDH (gray bars) and 18S (white bars). Values represent average +/- standard deviation of independent experiments. (B) Cyclin-dependent kinase 6 protein expression is dramatically reduced as determined bywestern blotting following transfection of miR-124 or miR-137. Levels of phosphorylated RB (pSer 807/811) are also markedly
reduced in response to miR-124 or miR-137 transfection. (C) miR-137 sequence in relation to the pMIR-REPORT vector containing the predicted cyclin-dependent kinase 6 miR-137 binding site (wild type). Mutated bases (underlined) were also introduced into the miR-137 seed region (boxed) of the cyclin-dependent kinase 6-3'UTR (mutated). Vertical lines denote WatsonCrick base pairing. (D) Relative luminescence of U251 cells following transfection miR-137 or negative control microRNA in
conjunction with wild type or mutated cyclin-dependent kinase 6 reporter constructs. *P < 0.0001.

the predicted miR-137 binding site of CDK6 was cloned
downstream of luciferase. A control reporter vector was
also developed in which the seed region of the miR-137
binding site was mutated (Figure 6C). Co-transfection of
U251 cells with a WT CDK6-3'UTR (CDK6-WT) reporterand the miR-137 mimic resulted in a significant decreasein luminescence (P < 0.0001) relative to cells co-transfected with CDK6-WT and a negative control miRNA
mimic (Figure 6D). Mutation of the CDK6 miR-137 seedregion rendered the reporter construct insensitive to inhibition by miR-137 (Figure 6D). Therefore, miR-137, in
addition to miR-124, is a direct inhibitor of CDK6.

Discussion
Identification of miRNAs in high-grade gliomas
To identify miRNAs that are recurrently deregulated inhigh-grade gliomas, we used quantitative RT-PCR to pro-file expression of 192 miRNAs in human non-neoplastic
brain tissues, AAs and GBMs. From our analyses, we identified a number of miRNAs that have been described previously in GBM tumors such as miR-10b (see [22]) andthe apoptosis regulator miR-21 (see [21,22,31]). We also
identified a number of miRNAs, including miR-124 andmiR-137, which have not been described in prior GBM
profiling studies. It remains unclear why miR-124 andmiR-137 were not detected previously in GBM tumors,particularly in light of our results that show dramatic
expression decreases of miR-124 and miR-137 in GBMs
(and AAs) relative to non-neoplastic brain tissue, andresults that show clear down-regulation of miR-124
expression in human oligodendrogliomas [28], humanastroblastomas [32] and GBM cell lines [32,33].

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Another notable discrepancy is that of miR-221 expression, which was not overexpressed in any of the tumors
tested in our study, but was shown to be overexpressed infive out of nine of GBMs studied by Ciafre et al. [22], andhas been shown to inhibit expression of the cell-cycle
inhibitor p27(Kip1) in GBM cells [34]. The most obvious
differences between our study and previous GBM profiling studies are: (1) the control tissues, adult glioses fromepileptic surgeries, a routinely used control tissue for GBM
molecular profiling studies, versus normal human fetal
brain and macroscopically characterized surgical specimens from the tumor periphery [21,22]; (2) profilingtechnologies, TaqMan versus micro-array [21,22]. Future
profiling studies on larger numbers of patient samples
will help to resolve these apparent discrepancies.

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We also note that in this study we analyzed 192 of the 533
known human miRNAs that are currently described in
miRBase, release 10.0 (see [35]), which reflects the rapid
pace of miRNA discovery since the inception of ourmiRNA expression studies. It is likely that miRNAs of
potential significance to brain tumor biology have notbeen assessed here. Examples of such miRNAs include
those that show enriched expression in brain tissue suchas miR-451 and miR-488 (see [32]) and miRNAs that havebeen implicated in the etiology of other tumor types, suchas miR-346 in follicular thyroid carcinoma [36]. Therefore, comprehensive miRNA expression studies are warranted in large HGA tumor sets that are linked to clinicaldata, such as survival and therapeutic response in order togenerate an in-depth assessment of the role of miRNAs inbrain cancer etiology and therapy.

Our expression analyses revealed a total of 35 miRNAs
that were differentially expressed between high-grade gliomas and non-neoplastic brain tissue (P < 0.05, Table 1).
A vast majority of these miRNAs were down-regulated (29out of 35; 83%), which is consistent with observations
that miRNA expression is globally down-regulated in multiple tumor types [11]. Of the 35 miRNAs, we identified
six HGA-miRNAs, which were down-regulated in both AA
and GBM tumors at a more stringent degree of significance (P < 0.01): miR-7, miR-124, miR-129, miR-137,
miR-139 and miR-218. Although we restricted further
analyses of these six miRNAs to miR-124 and miR-137
because of their elevated expression during adult NSC differentiation (Figure 1B), assessments of the other HGAmiRNAs may lead to novel insights into the biology ofhigh-grade gliomas. Similarly, assessments of the miRNAs
that were differentially expressed in AA tumors only or
GBM tumors only (Table 1) may shed light on the biological differences underlying these different tumor grades.

Regulation of miR-124 and miR-137 expression
Our results reveal two potential mechanisms by which
miR-124 and miR-137 may be suppressed in stem cells
and/or tumor cells. The first mechanism is growth factor
signaling: removal of EGF, and FGF from the culture
media resulted in robust increases in miR-124 and miR137 expression in adult NSCs. Given that activation of
EGF [37], PDGF [38] and FGF [39] signaling pathways
have each been implicated in gliomagenesis, it is reasonable to speculate that one mechanism by which growth factor signaling promotes brain tumor formation is throughsuppression of miR-124 and/or miR-137 expression and
NSC/TSC differentiation. Further analyses are required todetermine the relative contributions of EGF-, FGF- and
PDGF-induced signaling on suppression of miR-124 andmiR-137 transcription in adult NSCs and GBM tumor
stem cells.

It is important to note that our miRNA expression profiling studies were conducted at the tissue level, not at thecellular level, which has important implications for theinterpretation of our results. In particular, prior work [28]has shown that miR-124 is only expressed in the neuronsof adult human brains, which indicates that our observed
decrease in miR-124 expression in HGAs is a likely consequence of there being relatively fewer neurons in tumor
tissue compared with non-neoplastic glioses controls.
While this does not change our conclusions that miR-124
and miR-137 can induce mNSC-, mOSC- and human
GBM-derived stem cell (hGSC)-differentiation, it indicates that in situ expression analyses of miRNAs in HGAs,
non-neoplastic adult brain tissue, and during fetal- andpost-natal development of the mammalian central nervous system will be an important component of studies
aimed at investigating the functions of miRNAs during
normal brain development and tumorigenesis.

The second mechanism by which miR-124 and miR-137
expression may be suppressed in GBM stem cells is via
epigenetic modification of their transcriptional regulatory
sequences. Indeed, epigenetic modification of specificmiRNAs in other tumor types has been reported recently.
For example, miR-127, which is down-regulated in prostate, colon and bladder tumors relative to matched normal tissues, is up-regulated in cell lines derived from thesetumor types following inhibition of DNA demethylationand histone deacetylase [40]. Of particular interest to ourstudies, miR-124 is hyper-methylated in over one-third ofcolon, breast, lung, lymphoma and leukemia primarytumors, and is up-regulated in breast (MCF-7) and colon(HCT-116) cancer cell lines following DNA demethylation [26]. We observed that miR-137 expression increasedin GBM cell lines U87 and U251 following treatment with
the DNA demethylating agent 5-aza-dC (Figure 1B). Interestingly, we did not observe an increase in miR-124
expression in either cell line following 5-aza-dC treatment. Further analyses of miR-137 and miR-124 pro-Page 13 of 17(page number not for citation purposes)

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moter sequence methylation in primary tumors, TSCs and
NSCs are warranted to establish the degree to which epigenetic mechanisms contribute to suppression of thesemiRNAs in HGAs.

GBM and oligodendroglioma-neurosphere lines will be
required to address the general applicability of our results
in relation to the biology and therapeutics of these diseases.

Regulation of differentiation and the cell cycle by miR-124
and miR-137
Previous studies have demonstrated that miR-124 is upregulated during development of the rodent nervous system [41,42], and during neuronal differentiation ofmouse ES cells [12], and mouse and human embryonalcarcinoma cells [25]. Further, neuronal differentiation isenhanced following ectopic overexpression of miR-124 inmouse ES cells [12], mouse neuroblastoma cells [13], andmouse embryonal carcinoma cells [13]. Our results indicate that overexpression of either miR-124 or miR-137
promotes neuron-like differentiation of non-neoplastic
adult (mNSCs), mOSCs and CD133+ hGSCs. Thus, ourstudy is the first to implicate miR-124 in neuronal differentiation of post-natal NCSs and brain TSCs.

Recent studies have begun to shed light on the molecularmechanisms by which miR-124 regulates differentiationand proliferation. For example, miR-124 directly targets
PTBP1 (PTB/hnRNP I) mRNA, a global repressor of alternative pre-mRNA splicing in non-neuronal cells, resulting
in the transition from non-neuronal- to neuronal-specificalternative splicing patterns [13]. miR-124 also directly
targets and suppresses expression of small C-terminal
domain phosphatase 1 (SCP1), an inhibitor of neuronal
gene expression [44]. Finally, miR-124 overexpression in
HCT-116 colon cancer cells inhibits the expression of
CDK6, an established target of miR-124 (see [26]). Ourstudies revealed that miR-137, as well as miR-124, inhibited expression of CDK6, a predicted target of both miRNAs. Further, as with miR-124a (see [26]), our results
show that miR-137 is a direct inhibitor of CDK6. Overexpression of miR-124 or miR-137 also reduced the expression of phosphorylated RB (Figure 6B), a downstream
target of CDK6 [30]. It is interesting to note that CDK6 isknown to regulate both cell cycle progression and differentiation (reviewed in [29]), suggesting that mir-124- andmiR-137-mediated inhibition of CDK6 may, in part,
account for the observed effects on GBM cell proliferationand differentiation in this study. Further investigations areneeded to define the relationship between CDK6 downregulation and cell cycle arrest and/or differentiation in
GBM stem cells, and to identify and characterize additional miR-124 and miR-137 target genes.

The ability of miR-124 to induce robust stem cell differentiation appears to be dependent on cell type, developmental timing and other, as yet unidentified, factors. For
example, in mouse neuroblastoma cell lines CAD and
Neuro2a, ectopic up-regulation of miR-124 alone is sufficient to induce neuron-like differentiation, whereas inmouse embryonic carcinoma cells (P19), miR-124
enhances neuronal differentiation only in the presence ofretinoic acid, an established inducer of P19 neuronal differentiation [13]. Investigations of miR-124 expression
and function during development of the embryonic chickspinal cord have determined that the proneural activity of
miR-124 is, at best, subtle [43,44], suggesting that additional factors- and/or signals are required for robust neurogenesis at this developmental stage. Our studies showthat miR-124 and miR-137 enhance neurogenesis of
mNSCs, mOSCs and hGSCs in the absence of growth factor signaling. Although we have not tested whether miR124 and miR-137 alone can induce differentiation of thevarious stem cells tested in this study, transfection of miR124 or miR-137 alone was sufficient to induce G1 cell
cycle arrest in standard GBM cell lines (Figure 5A). However, cell cycle arrest was more pronounced in miR-124and miR-137-transfected GBM cells (SF6969) that weredeprived of growth factors (Figure 5B). Overall, the mostrobust effects of miR-124 and miR-137 overexpression on
cellular differentiation and proliferation were observed in
growth factor-deprived human cells (Figures 4B and 5B).
Collectively, our results suggest that while miR-124 andmiR-137 have the capacity to induce alone cell cycle arrestand differentiation in human GBM cells and stem cells,
abrogation of growth factor signaling enhances theircapacity to do so. Additional studies will be required toaddress this hypothesis, and incorporation of additional

Therapeutic potential of miR-124 and miR-137
The ability of miR-124 and miR-137 to induce potent
antiproliferative and prodifferentiation effects in CD133+
and CD133- human GBM cells suggests their potential
value for treatment of this disease. RNAi-based therapeutics holds great promise for the development of entirely
novel therapeutic strategies for disease treatment [45],and early phase clinical trials using siRNAs are currently
underway [46]. While delivery of siRNAs or miRNAs tothe central nervous system is particularly challenging
because of the blood brain barrier, a number of promisingstrategies have been developed recently to circumvent thisproblem. These include intranasal delivery of oligonucleotides [47], lipid encapsulation and targeted delivery of
nucleic acids [48,49], and direct administration of therapeutic agents to brain tumor tissues by convectionenhanced delivery [50,51]. Further testing of miR-124 andmiR-137 in pre-clinical models of GBM [52,53] in conjunction with various delivery strategies will help define
their ultimate therapeutic potential for treatment of GBM.

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Conclusion
We have investigated the role of miRNAs in adult human
HGAs and hGSCs and in adult mNSCs and mOSCs. Ourstudies showed, for the first time to the best of the authors'knowledge, that miR-124 and miR-137: (1) are expressedat significantly lower levels in GBM tumors relative to
non-neoplastic brain tissue; (2) are up-regulated during
neuronal differentiation of adult mNSCs induced by
growth factor withdrawal; (3) promote neuronal-like differentiation of growth-factor-deprived mNSCs, mOSCs
and hGSCs; (4) promote G0/G1 cell cycle arrest in GBM
cells and growth-factor-deprived hGSCs; (5) inhibit
expression of CDK6 mRNA, CDK6 protein and phosphorylated RB in GBM cells. These results suggest that targeted
delivery of miR-124 and/or miR-137 to GBM tumor cells
may be therapeutically valuable for GBM disease treatment.

List of abbreviations
AA: anaplastic astrocytomas; bFGF: basic fibroblast
growth factor; CD: cluster of differentiation; CDK6: cyclin-dependent kinase 6; DMEM: Dulbecco's Modified
Eagle's Medium; EDTA: ethylene diamine tetraacetic acid;
EGF: epidermal growth factor; ES: embryonic stem; FCS:
fetal calf serum; FGF: fibroblast growth factor; GBM:
glioblastoma multiforme; GFAP: glial fibrillary acidic protein; HGA: high grade astrocytoma; hGSC: human GBMderived stem cell; MAP2: microtubule-associated protein
2; miRNA: microRNA; mNSC: mouse neural stem cells;
mOSC: mouse oligodendroglioma tumor stem cells;
mRNA: messenger RNA; MUT: mutated; NCS: neural stem
cells; PDGF: platelet-derived growth factor; RB: retinoblastoma; RT-PCR: reverse transcriptase polymerase chain
reaction; siRNA: short interfering RNA; SVZ: subventricular zone; TSA: trichostatin A; TSC: tumor stem cell; UCSF:
University of California San Francisco; WHO: World
Health Organization; WT: wild type.

Competing interests
David Ginzinger, a collaborator and co-author of themanuscript, was an employee of Applied Biosystems at
the time the experiments were conducted. The miRNA
TaqMan assays were provided by David Ginzinger.

Authors' contributions
JS performed the miRNA expression studies, proliferation
assays in GBM cells, CDK6 and Rb Western blotting, luciferase reporter assays, developed the miRNA transfection
protocols and helped to draft the manuscript. DAL
designed, implemented, interpreted and described experiments involving SVZ-NSCs. CP designed, implemented,
interpreted and described experiments involving mOSCs.
AIP designed, implemented, interpreted and described
experiments involving hGSCs. AKM designed, implemented, interpreted and described demethylation andhttp://www.biomedcentral.com/1741-7015/6/14

deacetylation experiments. MY assisted with design,
implementation and interpretation of miRNA expression
studies. SRV provided a histopathological review ofhuman tissues and critical revision of the manuscript.
DGG provided design and support for the miRNA expression analyses. CDJ provided overall conception anddesign support and critical revision of the manuscript. JFC
provided design and analyses support for the demethylation and deacetylation experiments and critical revision of
the manuscript. GB provided design and analyses support
for the differentiation studies of mOSCs and critical revision of the manuscript. WAW provided design and analyses support for the differentiation studies of hGSCs andcritical revision of the manuscript. AA–B provided design
and analyses support for the miRNA expression and differentiation studies of SVZ-NSCs and critical revision of
the manuscript. JGH conceived the study, participated inits design and coordination and drafted the manuscript.
All authors read and approved the final manuscript.

Additional material
Additional file 1
Characteristics of the patient tissues used in this study.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S1.txt]

Additional file 2
Validation of miR-124 expression and function in glioblastoma multiforme cells.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S2.pdf]

Additional file 3
Supplementary methods. This pdf file provides further details related tothe methods used in this study.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S3.pdf]

Additional file 4
Log2 fold change of microRNAs in non-tumor brain samples (glioses),
anaplastic astrocytomas and glioblastoma multiforme tumors.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S4.txt]

Additional file 5
Validation of let-7a and miR-16 as appropriate control miRNAs in primary tumor samples and neural stem cells.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S5.pdf]

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9.

Additional file 6
Statistical comparisons of miRNA expression in glioblastoma multiforme
and anaplastic astrocytoma tumors versus non-tumor brain tissue (gliosis).
Click here for file
[http://www.biomedcentral.com/content/supplementary/17417015-6-14-S6.txt]

Additional file 7

12.

Additional file 8

14.

Acknowledgements
The authors would like to thank Jane Fridlyand and Ritu Roydas-Gupta of
the UCSF Comprehensive Cancer Center Biostatistics Core for statisticalanalyses of the primary tumor miRNA expression data, and Cynthia Cowdrey of the neurological surgery tissue bank for distribution of primary
human tumor samples. Grant support was provided by: National Institutesof Health CA101777 (JGH), CA097257 (JGH), NS28478 (AAB); UCGenentech Discovery Grant (CDJ, WAW, JGH); Pediatric Brain Tumor
Foundation (CDJ, WAW, JGH); Goldhirsh Foundation (AAB); Danish Cancer Society (JS); Danish Research Agency (JS); National Brain Tumor Foundation (WAW); Burroughs Wellcome Fund (WAW); AANS NREF award(DAL); Sandler Opportunity Research Award (GB, CP); AAB holds the
Heather and Melanie Muss Endowed Chair; Swedish Medical ResearchCouncil and Swedish Society for Medical Research (AIP).

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