MiR-145 functions as a tumor-suppressiveRNA by targeting Sox9 and adducin 3in human glioma cells

Sandhya B. Rani†, Sachin Shivaji Rathod†, Shanmuganandam Karthik, Navjot Kaur,Dattatraya Muzumdar, and Anjali S. Shiras

National Centre for Cell Science, Pune, India (S.B.R., S.S.R., S.K., N.K., A.S.); Seth GS Medical College and KEM

Hospital, Mumbai, India (D.M.)

Background. MicroRNAs (miRNAs) are increasinglybeing recognized as being involved in cancer developmentand progression in gliomas.Methods. Using a model cell system developed in our labto study glioma progression comprising human neurogli-al culture (HNGC)–1 and HNGC-2 cells, we report herethat miR-145 is one of the miRNAs significantly downre-gulated during malignant transformation in glioblastomamultiforme (GBM). In a study using tumor samplesderived from various glioma grades, we show that expres-sion of miR-145 is decreased in a graded manner, withGBM patients showing lowest expression relative tolower-grade gliomas (P , .05) and normal brain tissues(P , .0001). Functional studies involving ectopic expres-sion of miR-145 in glioma cells had a negative impact oncell proliferation and tumor development, as well as inva-sion and induced apoptosis, providing further support tothe concept that inactivation of miR-145 is important forglioma disease pathogenesis. More notably, thesegrowth-suppressive effects of miR-145 are mediatedthrough its target proteins Sox9 and the cell adhesion-associated molecule adducin 3 (ADD3).Results. Inhibiting Sox9 and ADD3 rescued effects ofmiR-145 loss. Interestingly, miR-145 loss in glioma cellsled to overexpression of molecules involved in cell prolif-eration, like cyclin D1, c-myc, and N-myc, as well as en-hanced expression of cell adhesion- and invasion-relatedmolecules N-cadherin and E-cadherin, an effect whichwas again restored upon miR-145 overexpression inglioma cells. The miR-145 promoter was methylated at

its cytosine–phosphate–guanine (CpG) islands in theglioma cell lines studied.Conclusion. Our study demonstrates that miR-145 has atumor-suppressive function in glioblastoma in that itreduces proliferation, adhesion, and invasion of glioblasto-macells, apparentlyby suppressing the activityofoncogenicproteins Sox9 and ADD3. Reduced levels of miR-145 maylead to neoplastic transformation and malignant progres-sion in glioma due to unregulated activity of these proteins.

Keywords: adducin 3, glioma stem cells, invasion,neoplastic transformation, promoter methylation, Sox9.

Malignant gliomas are the most common tumorsof the CNS, with dismal prognosis. Amonggliomas, glioblastoma multiforme (GBM;

World Health Organization [WHO] grade IV) is one ofthe most aggressive CNS tumors and is unresponsive tomost modalities of treatment. The chemo- and radiore-sistance of GBM tumors stems from high levels of hetero-geneity displayed by the tumor, often attributed to a smallsubpopulation of tumor-initiating cells driving tumorgrowth.1 These tumor-initiating cells or glioma stemcells display the ability to self-renew, yielding progenycomprising additional stem cells as well as cells differenti-ating into various nontumor cells within the tumor.2 It isnot evident whether glioma arises from neural stem cells,from differentiated progenitors, or fromfully differentiat-ed glial cells themselves.

The high level of molecular diversityand heterogeneityin GBM have led to its classification into 4 subtypes basedon the gene-expression data derived from The CancerGenome Atlas. The heterogeneity possibly arises fromcomplex interactions of the tumor-initiating cells withthe host microenvironment. Hence, it is critical to identifyspecific molecular signaling pathways activated in gliomastem cells or nontumor stem cell population regulat-ing processes like cell survival, proliferation, and angio-genesis. This will help outline novel approaches to target

†These authors contributed equally to this work.

Corresponding Author: Anjali Shiras, MSc, PhD, Scientist-F, National

Centre for Cell Science (NCCS), NCCS Complex, University of Pune

Campus, Ganeshkhind, Pune 411007, Maharashtra, India

(anjalishiras@nccs.res.in; anjalishiras@gmail.com).

Received August 9, 2012; accepted May 5, 2013.

Neuro-Oncologydoi:10.1093/neuonc/not090 NEURO-ONCOLOGY

#The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rightsreserved. For permissions, please e-mail: journals.permissions@oup.com.

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resistance mechanisms as well as define strategies for drugdelivery across the blood–brain barrier. Most of the GBMtumors exhibit abnormalities in pathways regulating thecell cycle, leading to failureofgrowtharrest andsenescencedue to defects in core signaling pathways like p53, retino-blastoma, and receptor tyrosine kinase.3

Importantly, aberrations in core signaling pathwaysidentified through genome-wide screening studies are fre-quently driven by abnormal expression of microRNAs(miRNAs).SeveralmiRNAsare implicated inalmostallbi-ological processes by virtue of their role in regulating geneexpression. Using phenomena of RNA interference, thesemiRNAs induce gene silencing of their cognate targetgenes, through either translational repression or mRNAdecay. The distinctpatternsofaberrant DNA methylation,histone modifications, and varied miRNA expression pro-files reiterate the heterogeneity evident in glial tumors.Interestingly, either overexpression of miRNAs withpro-oncogenic functions or downregulation of miRNAswith growth-suppressive effects contributes to tumorige-nicityand invasiveness inglioma.Recent studieshavedem-onstrated that miR-483-5p,4 miR-137,5 miR-128,6 andmiR-1247 are significantly downregulated in gliomatumor tissues and cell lines, while miR-34a is specificallyrepressed in the proneural subtype ofgliomas.8 Incontrast,miRNAs like miR-21 and the miR-221/222 cluster areoverexpressed in glioma cells and exert their effects by af-fecting multiple genes associated with glioma cell prolifer-ation. The role of microRNAs like miR-143 and miR-145assuppressorsof tumormetastasisand invasion is reportedin several colon and ovarian cancer tissues and celllines,9,10 prostate cancers,11 and several cervical cancertissues.12 The role of miR-145 in glioma is not clearly un-derstood. While there are studies indicating its role as atumor suppressor,13–16 few studies suggest it to be associ-ated with an invasive phenotype in glioblastoma celllines.17 We here provide evidence for the role of miR-145as one of the tumor-suppressive miRNAs that is frequentlydownregulated in a graded manner during malignanttransformation in glioblastomas. Next, we demonstratethat miR-145 affects GBM tumorigenesis through itstargets Sox9 and adducin 3 (ADD3). Sox9, a member ofthe Sry-related high-mobility group box family, functionsas a transcription factor that plays a significant role in thedevelopment and differentiation of multiple cell lineagesand has recently been reported to be overexpressed ingliomas.18 Interestingly, ADD3, a membrane protein in-volved in stabilization of epithelial junctions, is presumedto have a role in epithelial cancers19 but is not reported inbrain tumors. In this study, we demonstrate that downre-gulation of miR-145 leads to activation of its targetsSox9 and ADD3 in GBM, causing pro-invasive and malig-nant characteristics in GBM.

Materials and Methods

Clinical Samples

The use of human tumor tissues in the present study wasapproved by the Institutional Ethics Committee of the

National Centre for Cell Science (NCCS), Pune, andKEM Hospital, Mumbai, India. Tumor tissue sampleswere collected from KEM Hospital and assigned specifictumorstagesandpathologicalgradesbyaneuropathologistaccording toWHOcriteria forgliomas.20 Signedconsent touse tissues for research purposes was obtained from pa-tients prior to surgery. Human brain tumor biopsies weretaken during standard neurosurgical resection of braintumors. A total of 29 glioma tumor tissue samples and 5normal brain tissues were collected and processed for ex-traction of RNA, and a part of the remaining tissue wasused for generation of long-term glioma cultures.

Cell Culture

The development of the human neuroglial culture(HNGC) stem cell lines HNGC-1 and HNGC-2 fromglioma tissue has been described.21,22 The cell linesHNGC-1 and HNGC-2 and other long-term glioma cul-tures generated from tumor tissue specimens(Supplementary Table S1) were cultured in Dulbecco’smodified Eagle’s medium (DMEM)/Ham’s F12medium (1 : 1; Invitrogen) with 1× B27 supplement(Invitrogen), epidermal growth factor (10 ng/mL;Invitrogen) and basic fibroblast growth factor (20 ng/mL; Invitrogen), 1× nonessential amino acids(Invitrogen), and 1× Glutamax (Invitrogen) at 378Cwith 5% CO2 in a humidified incubator. The stem cellline NSG-K16, derived from GBM tumor tissue, was es-tablished as a neurosphere culture and maintainedunder serum-free conditions.23 Human glioblastomacell lines LN-18 and LN-229 were obtained from theAmerican Type Culture Collection and maintained inDMEM (Sigma-Aldrich) supplemented with 5%(volume/volume) fetal bovine serum (FBS; Gibco),50 U/mL penicillin (Sigma-Aldrich), and 50 mg/mLstreptomycin (Sigma-Aldrich) and incubated in 5%CO2. The glioma cell line U373MG (American TypeCulture Collection) was grown in minimum essentialmedium with sodium pyruvate in 5% FBS.

Real-time PCR

TotalRNAwasextracted fromtissues and cellswithTrizolReagent (Invitrogen) in accordance with the protocol spec-ified by the manufacturer, and its quality was assessedwith a BioPhotometer (Eppendorf). 100 ng of total RNAwas reverse transcribed into cDNA using the MirVanaMiRNA Detection Kit (Ambion). Expression of maturemiR-145 was quantified using SYBR green master mix(Applied Biosystems) with the 7500 Fast Real Time PCRSystem (Applied Biosystems). The amplification reaction(10 mL) included2× SYBRgreenmastermix,gene-specificprimers, and cDNA. Fold changes in gene expression werecalculated using the 22DDCt method with 18S rRNAserving as an internal control.

Construction of Plasmids

The pre–miR-145 lentiviral vector was a kind gift fromDr Yin-Yuan Mo (Southern Illinois University). It was

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constructed by ligating the miR-145 precursor (1.5 kb)into the EcoR1 and NotI sites of the pCDH-CMV-MCS-EF1-copGFP lentivector.24 The lentivector waspackaged using PACKH1 Packaging Plasmid mix(System Biosciences) and transiently cotransfected intoHEK-293 cells to generate recombinant virus particles.After 48 h of infection, lentivirus in the supernatant wastransduced into glioma cells—HNGC-2, NSG-K16, andLN-229—using 8 mg/mL of polybrene (Sigma-Aldrich).Stable clones were maintained on 10 mg/mL of puromy-cin (Sigma-Aldrich). The control lentiviral vectorpCDH-CMV-MCS-EF1-copGFP stably expressed greenfluorescent protein (GFP), while the miR-145 lentiviralvector, Lenti-GFP-miR-145, stably expressed GFP andmiR-145. In our experiments, cells were divided into 2groups, designated the EV group (empty vector; trans-duced with vector only) and the miR-145 group (trans-duced with miR-145). The luciferase-3′ untranslatedregion (UTR) reporter plasmid was constructed by intro-ducingSox9andADD33′UTR regions carrying a putativemiR-145 binding site into luciferase plasmid pMIR-REPORT miRNA Expression Reporter Vector (LifeTechnologies). We amplified Sox9 and ADD3 3′UTR se-quences from U87MG DNA using PCR primers(Supplementary Table S2). The PCR products were geleluted and ligated into pMIR-REPORT Luciferaseplasmid. The specificity of PCR products was confirmedby DNA sequencing.

MTT Assay

Proliferationpotential of cells wasdetermined byassaybyMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide] .25 Briefly, cells were seeded into 96-wellplates (BD Biosciences) at a cell density of 1 × 103 cells/well in growth medium. Cell growth was assayed by addi-tion of 20 mL of MTT (5 mg/mL; Sigma-Aldrich) to eachwell, and the plate was incubated at 378C for 4 h. Theproliferation assay was performed for 3 days and cellgrowth was assayed at every 24 h interval. Later, the reac-tion was stopped by addition of 200 mL dimethyl sulfox-ide (Sigma-Aldrich). Optical density was measured at570 nm with a microplate reader (Bio-Rad).

Cell Cycle Analysis

Gliomacell linesHNGC-2,NSG-K16,andLN-229 trans-fected with EV and miR-145 were used for determinationof cell cycle kinetics using flow cytometry. EV cell lineswere also transfected with small interfering (si)RNAs toSox9 or ADD3 (50 ng/mL; Santa Cruz Biotechnology)with Lipofectamine 2000 reagent (Invitrogen). Afterovernight transfection, cells were incubated for an addi-tional 36 h. Later, the siRNA-transfected cells as well asthe EV and miR-145 stable glioma cell lines were harvest-ed, washed twice in phosphate buffered saline (PBS), andfixed with 75% ethanol for 30 min on ice. After washingin cold PBS 3 times, cells were resuspended in 500 mL PBSwith 50 mg/mL of propidium iodide (PI) solution and10 mg/mL of DNase-free RNase A for 30 min at 378C.

Samples were analyzed for DNA content byFACSCalibur (Becton Dickinson) using a blue 488 nmlaser for excitation and an FL2 detector 585+21 nmfor emission. DNA histograms were analyzed withCellQuest Pro analysis software 5.2.1 (BD Bioscience).

Apoptosis Assay

Apoptosis occurring via active caspases was detected inthe EV and miR-145 glioma cells using the SR-FLICA[Fluorescent Labeled Inhibitor of Caspases] PolyCaspases Assay kit (ImmunoChemistry Technologies).Inbrief, cellsweregrownoncoverslipsand1× FLICAsol-ution was added to the cells for 1 h at 378C. Nuclei werestained with 4,6-diamidino-2-phenylindole (DAPI;Sigma-Aldrich) for 10 min and mounted in medium con-taining fixative. Confocal images were acquired with aLeica TCS SP5II microscope.

Analyses of caspase 3, caspase 9, and poly(ADP-ribose) polymerase (PARP) activities were per-formed using an apoptosis sampler kit (Cell SignalingTechnology) per manufacturer’s protocol. Briefly, cellswere fixed, permeabilized, and blocked in blockingbuffer (2% bovine serum albumin in 1× PBS) for60 min followed by 1 h incubation with unconjugatedcaspase 3, caspase 9, or PARP antibodies separately (1 :100 dilutions). Later, the cells were washed in blockingbuffer followed by 30 min incubation with Alexa Fluor594 anti-rabbit antibody (1 : 200; Invitrogen). The cellswere resuspended in 0.5 mL 1× PBS and data were ac-quired on a FACSCalibur flow cytometer (BDBiosciences) and analyzed with CellQuest Pro analysissoftware 5.2.1.

Confocal Microscopy

Glioma cells (EV and miR-145 transfected) were grownon coverslips, fixed, permeabilized, and blocked with5% bovine serum albumin in 1× PBS for 1 h at room tem-perature. Cells were incubated with Ki67 antibody (1 :100; Chemicon International) for 2 h at room tempera-ture and washed thrice with PBS, followed by incubationfor 1 h with Alexa Fluor 594 anti-rabbit antibody (1 :200; Invitrogen). Nuclei were stained with DAPI(Sigma-Aldrich) for 10 min. Confocal images were ac-quired with a Leica TCS SP5II microscope.

Western Blot Analysis

Cells were lysed in 1× radioimmunoprecipitation assaylysis buffer (Thermo Scientific) with protease inhibitorcocktail (1×; Sigma-Aldrich). Homogenates were clari-fied by centrifugation at 12 000 rpm for 15 min at 48C.Cell lysates (40 mg) were loaded on sodium dodecylsulfate–polyacrylamide gel electrophoresis gels andtransferred onto polyvinylidene difluoride membranes(Millipore). The blots were incubated with primary anti-bodies to ADD3 (Santa Cruz), Sox9 (Santa Cruz), N-myc(Santa Cruz), cyclin D1 (Abcam), c-Myc (Santa Cruz),E-cadherin (Cell Signaling Technology), or N-cadherin

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(1 : 1000 dilution; Cell Signaling Technology), followedby incubation with horseradish peroxidase–conjugated,species-specific secondary antibodies (1:2000 dilution;Thermo Scientific). The specific proteins were detectedusing a Super Signal protein detection kit (ThermoScientific). Protein levels were normalized by reprobingblots with b-actin antibody (MP Biomedicals).

Luciferase Assay

EVandmiR-145stablyexpressinggliomacellswereseededinto 24-well plates (1 × 104 cells/well). After 24 h, thecells were cotransfected with 200 ng per well ofpMIR-REPORT Luciferase reporter vector (AppliedBiosystems) or pMIR-REPORT vector containing eitherSox9 or ADD3 3′UTR variants. Luciferase activity wasmeasured 48 h posttransfection using the Dual-GloLuciferase Assay System according to the manufacturer’sinstructions (Promega). Foreach sample, Renilla luciferaseactivity was normalized to firefly luciferase expression.

Matrigel Invasion Assay

Invasion assay in vitro was performed in 24-well cellculture plates using Matrigel Invasion Chambers of8.0 mm pore size membrane using manufacturer’s proto-col (BD Biosciences) and the method described by Albiniet al.26 Briefly, 5 × 103 cells of either EV or miR-145glioma cells in 0.5 mL serum-free DMEM were seededin the upper compartment of each well of the test andcontrol inserts. DMEM with 10% FBS served as a chemo-attractant in lower compartments of both inserts. Theplate was incubated for 22 h. After incubation, the nonin-vading cells on the upper surface were mechanicallyscrubbed and removed, and the filters were fixed andstained with 0.1% (weight/volume) crystal violet(Sigma-Aldrich) for 15 min and washed with 1× PBS toremove excess stain. Migrated cells on the bottom sideof the membrane were counted in 10 different fields toacquire the average number of migrating cells per cellline with an Olympus IX51 fluorescent microscope andquantified with cellSens 1.3 software (Olympus).

In vivo Tumor Growth Assay

The animal studies were approved by the InstitutionalAnimal Ethics Committee of NCCS and experiments wereperformed in accordance with the Animal Ethics guidelinesof NCCS. For in vivo tumorigenicity assays, 6-week-oldnonobese diabetic severe combined immunodeficient(NOD-SCID) mice were used. Briefly, 1 × 104 EV cellsand miR-145 transfectant glioma cells were injected subcu-taneously in NOD-SCID mice (n ¼ 18), who were periodi-cally observed for tumor development. Tumor volumeswere determined using the formula: 4/3p (

pmajor axis/

2 × minor axis/2). Tumors were fixed with formalin andanalyzed by hematoxylin and eosin (H&E) staining.

Orthotopic Grafts With GFP Transfected Cells

Into the striatum of NOD-SCID mice (n ¼ 0.5) were in-tracranially implanted unilaterally 1 × 104 EV cells and

miR-145 transfected glioma cells in 1× PBS. The micewere euthanized after 4 weeks. The brain tumor tissuewas analyzed for tumor foci under the Olympus IX51fluorescent microscope followed by H&E staining of thebrain tumor sections.

Methylation Assays

Genomic DNA was isolated using the Gen Elute mamma-lian DNA isolation kit (Sigma-Aldrich). About 2 mg ofgenomic DNA was subjected to bisulphite modificationand purified using the Epitect Bisulphite kit (Qiagen) ac-cording to the manufacturer’s instructions. A primerpair targeting a 0.3 kb fragment relative to the start siteof the primary transcript of miR-145 (SupplementaryTable S2) was designed using Methprimer software.27

Using bisulphite modified genomic DNA as a template,PCR was performed with the designed primer pair, andthe products were thymine-adenine cloned in pGEM-Tvector (Promega). About 6 positive clones per templatewere sequenced using the Applied Biosystems PRISMDNA sequencer, and the data were analyzed usingBismark software.28 For inhibition of DNA methylation,cells were treated with 5 pmol/mL 5-azacytidine(Sigma-Aldrich) for 4 days at 378C. Cells were fed withfresh medium containing 5-azacytidine every 24 h.Control cells were treated with 1× PBS without the de-methylating agent 5-azacytidine and underwent freshmedia change in parallel with the treated group. Aftertreatment, genomic DNA and total RNA were isolatedfor further analyses.

Bioinformatics

Candidate miR-145 targets were generated using the pub-licly available algorithms TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/),PicTar (pictar.mdc-berlin.de), and miRDB (http://www.mirdb.org).

Statistical Analysis

Data are presented as mean+ SEM from at least 3 inde-pendent experiments. Statistical analysis was performedusing a 1-way ANOVA followed by either Student’st-test or Fisher’s exact test. Differences between groupswere considered to be statistically significant at P ≤ .05.Graphs were made in GraphPad Prism 5.01.

Results

MiR-145 Is Downregulated in Glioblastomasand Glioma Stem Cells

Our gene expression microarray data for global miRNAanalysesof gliomacells HNGC-2 compared with the non-tumorigenic neural stemlike cells HNGC-1 indicated thatmiR-145 was one of the miRNAs predominantly down-regulated in HNGC-2 cells (Supplementary Fig. S1).

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To determine whether miR-145 levels were similarly lowin glioma tumor tissues and GBM cell lines, we performedexpression analyses using stem-loop quantitative realtime (qRT)–PCR. For this, we quantified miR-145 ex-pression in 29 glioma tumor tissue samples derived fromall 4 grades, 5 established glioma cell lines, and 5normal brain tissues. As shown in Fig. 1A, miR-145levels were significantly downregulated in higher-gradegliomas and established GBM cell lines, compared withnormal brain tissues. The expression levelsnegatively cor-related to glioma grades, with lower grades (n ¼ 2) exhib-iting downregulation .5-fold and higher grades (n ¼ 17)demonstrating a .50-fold decrease in miR-145 expres-sion. These data provided us with experimental evidencethat miR-145 expression was downregulated in a gradedmanner in gliomas, with higher grades showing lowest ex-pression. Similarly, the various GBM cell lines analyzed(LN-18, U373MG, LN-229, HNGC-2, and NSG-K16)showed a .100-fold decrease in miR-145 levels(Fig. 1B) compared with expression of miR-145 normal-ized to one with respect to expression in the nontrans-formed neural stemlike cell line HNGC-1.

Because qRT-PCR data demonstrated that miR-145levels were downregulated in higher-grade gliomas andGBM cell lines, we sought to understand the biologicalrole of miR-145 by performing miR-145 overexpressionstudies. For this, we chose to use HNGC-2, NSG-K16,

and LN-229 GBM cell lines, as they showed low expres-sion of miR-145. We generated miR-145 overexpressingstable GBM cell lines that displayed increased expressionof miR-145 by .500-fold in HNGC-2 and NSG-K16cells (Fig. 1C) and by �5000-fold in LN-229 cells(Supplementary Fig. S3A) compared with correspondingEV cells. Here, EV cells were glioma cell lines transfectedwith vector alone.

MiR-145 Overexpression in Glioma Cells InducesGrowth Arrest

Effects of miR-145 on cell growth, tumorigenicity, and in-vasion ingliomacell linesoverexpressingmiR-145using aplethoraof in vitroand in vivo assays. The MTTassaydis-played a remarkable inhibition in cell growth due tomiR-145 were studied overexpression in HNGC-2,NSG-K16 (Fig. 2A), and LN-229 (Supplementary Fig.S3B) cells compared with EV cells. Further, staining forKi67, a proliferation marker, confirmed inhibitoryeffects of miR-145 on glioma cell growth. The EVgroups comprising HNGC-2 and NSG-K16 cells (trans-fected with EV) showed most of the cells positive forKi67, whereas glioma cells expressing miR-145 werealmost devoid of Ki67 positivity (Fig. 2B), confirminggrowth-suppressive effects of miR-145 on cell prolifera-tion.Amarked inhibition incell growthwasalsoapparent

Fig. 1. MiR-145 levels are downregulated in higher-grade gliomas and upregulated after transduction of cells with Lenti-GFP-miR-145 (A)

Real-time quantitative RT-PCR assay for expression of miR-145 in low-grade and high-grade glioma tissues, compared with expression in

normal brain tissues. The qRT-PCR assay revealed downregulation of miR-145 in glial tumors in a graded manner compared with normal

brain tissue. ***P , .0001 vs control; (B) qRT-PCR analyses of miR-145 in glioma cell lines. The data were normalized with respect to

expression of miR-145 in nontumorigenic neural stem cell line HNGC-1. Expression of miR-145 was downregulated in GBM cell lines.

***P , .0001 vs control. (C) The miR-145 level was significantly upregulated after transduction of HNGC-2 and NSG-K16 glioma cells with

Lenti-GFP-miR-145, compared with EV-GFP groups. *P , .05 vs control. Transcript levels were normalized with respect to 18S rRNA

expression. Each bar represents the means+SEM of 3 independent experiments, with P-values vs controls generated using Student’s t-test.

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at the morphological level, wherein HNGC-2 andNSG-K16 cells expressing miR-145 showed sparsegrowth in culture compared with EV cells seededequally over a 3-day period (Supplementary Fig. S2A).Further, we investigated whether overexpression ofmiR-145 in GBM cells affected cell cycle progression byflow cytometry. As shown in Fig. 2C, compared withEV cells, introduction of miR-145 into both HNGC-2and NSG-K16 cells significantly arrested cells at the G0/G1 transition, evidenced by a marked accumulation ofcells in the G0/G1 peak. There was an increase in thenumber of cells arrested in G0/G1 phase by about34.6% in HNGC-2 and by 30% in NSG-K16 cells com-pared with EV cells (Fig. 2C). A similar effect occurredin LN-229 cells, wherein there was significant increasein the number of cells arrested in the G0/G1 phase(Supplementary Fig. S3C). These results indicated thatmiR-145 overexpression produced a consistent reductionin cell proliferation together with defects in cell cycle pro-gression leading to accumulation of cells in G0/G1 andextensive decrease in percentage of cells in S phase.

Thegrowth-suppressiveeffectsongliomacells ledus toanalyze whether these effects were an outcome of the po-tential of miR-145 to induce apoptosis. To determine the

numberof cells that were driven to apoptosis by miR-145,we performed annexin V and PI staining in EV cellsand the miR-145 group. For this, HNGC-2 cells transfect-ed independently with the miR-145 construct or EV werecostained with annexin V/PI. Interestingly, the HNGC-2and NSG-K16 miR-145 cells possessed an increasednumber of cells in apoptotic phase compared with EVcells (data not shown). To investigate whether apoptosisinduced by miR-145 in glioma cells was mediatedthrough caspase-dependent mechanisms, we stainedcells for polycaspases. We found a significantly highernumber of miR-145-expressing HNGC-2, NSG-K16(Fig. 3A), and LN-229 (Supplementary Fig. S3D) cellsstained for polycaspases, whereas therewere no apoptoticcells in EV cells. Next, by flow cytometry we analyzed ex-pression of proteases, caspase 3, caspase 9, and PARP,which are involved in initiation and execution of apopto-sis. As shown in Fig. 3B, 3 distinct peaks were obtained inmiR-145–overexpressing HNGC-2 and NSG-K16 cells.The first cluster was overlaid with an EV cell population,while the second and third peaks represent cells in earlyand late apoptotic phases, respectively (Fig. 3B). Thedata showing number of apoptotic cells are representedas a histogram and show percentage positivity for

Fig. 2. Overexpression of miR-145 inhibits cell proliferation and induces G1/S transition arrest in vitro. (A) Effects of miR-145 overexpression on

proliferation of HNGC-2 and NSG-K16 cells compared with EV–control group cells as measured by MTTassay. MTT data are representative of 3

independent experiments, presented as mean+SEM of triplicates. OD, optical density. (B) Immunofluroscence staining of proliferation marker

Ki67 inEVandmiR-145–overexpressinggliomacells anddetectedusinganti-rabbitAlexaFlour595(red).Nuclei arestainedwithDAPI (blue). (C)

Representative cell cycle distribution determined by flow cytometric analysis after staining with PI and showing distribution of cells in G0/G1, S,

and G2/M phases in HNGC-2 and NSG-K16–EV and miR-145 cells (upper panel). MiR-145 overexpression promoted accumulation of cells in

G0/G1 phase and resulted in G1/S transition arrest compared with EV-control cells. The lower panel represents data showing percentage

distribution of cells in various phases of cell cycle generated from 3 independent experiments of cell numbers in various phases and

represented as means+SEM (n ¼ 3).

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caspase 3, caspase 9, and PARP in HNGC-2 (Fig. 3Ca)and NSG-K16 cells (Fig. 3Cb). The same data representedquantitatively as a bar diagram in Fig. 3C shows a largepercentage of glioma cells undergoing apoptosis due tomiR-145 in both HNGC-2 (Fig. 3Ca) and NSG-K16(Fig. 3Cb) cells. These studies confirmed that one of themechanisms through which miR-145 functions as agrowth suppressor is induction of apoptosis in a caspase-dependent manner.

MiR-145 Inhibits Tumor Formation and Invasionin Glioma Cells

The dramatic effects of miR-145 overexpression leadingto effects on cell cycle progression and apoptosis analyzedat in vitro levels prompted us to investigate effectsof miR-145 on tumor development and xenograft for-mation in NOD-SCID mice. The EV cells introduced sub-cutaneously generated rapidly growing tumors within amonth (Fig. 4Aa), while the miR-145–expressing cells in-troduced similarly were unable to form tumors even uponperiodic inspection for up to 2 months. The mice were

euthanized after this period. The tumors formed by EVcells on excision displayed an average tumor volume of4356 mm3 for HNGC-2 and 2459 mm3 for NSG-K16cells within 30 days (Fig. 4Ab). More notably, mice on in-tracranial implantation of GFP-labeled HNGC-2 EV andNSG-K16 EV cells demonstratedGFP-positive tumor fociwithin the brain tissue (Fig. 4Ba). The brain tumor tissueson histopathological analyses and H&E staining wereconfirmed to be glioma mainly on the basis of theirhighly infiltrative and necrotic nature (Fig. 4Bb). In con-trast, the miR-145 glioma cells failed to get implantedwithin the brain parenchyma and colonize the braintissue. Since invasion plays a critical role in glioma path-ogenesis, we analyzed the role of miR-145 overexpressionon cell migration and invasion using transwell invasionchambers. A profound decrease in invasion potentialwas induced due to miR-145 in HNGC-2 and NSG-K16cells as shown in Fig. 4Ca and quantified in Fig. 4Cb, sig-nifying the role of miR-145 in invasion. These experimen-tal findings demonstrate the role of miR-145 as a tumorsuppressor, the loss of which in glioma cells contributesto enhanced cell growth, proliferation, tumorigenicity,and invasion.

Fig. 3. MiR-145 sensitizes glioma cells to caspase-dependent apoptosis. HNGC-2 and NSG-K16 cells stably expressing miR-145 or cells

transfected with EV alone were analyzed for (A) fluorescence detection of polycaspases using FAM (carboxyfluorescein)-FLICA reagent and

(B) measuring positivity for cleaved caspase 3, caspase 9, and PARP by flow cytometry. Iso-type control is represented as a filled plot. An

unfilled histogram indicates staining for all 3 caspases in miR-145–overexpressing cells. (C) Quantitative representation of fluorescence

activated cell sorting data showing relative expression of cleaved caspase 3, caspase 9, and PARP in HNGC-2 (a) and NSG-K16 (b) miR-145

transfected and control cells. Each bar represents the means+SEM of 3 independent experiments. *P , .05, ***P , .0001 vs EV cells

generated using t-test.

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Sox9 and ADD3 Are Direct Targets of MiR-145

To identify novel targets for miR-145, we searchedmiRNA target prediction databases that includedmiRanda, PicTar, miRDB, and TargetScan. Using a mul-tiplicity of these prediction algorithms, we were able topredict 51 common targets. On the basis of them, we de-termined that the seed sequence of miR-145 matched the3′UTR of Sox9 and ADD3 mRNA (Fig. 5A). Further, weperformed luciferase reporter assays to determinewhether Sox9 and ADD3 were direct targets ofmiR-145 in glioma cells. For this, we cloned the 3′UTRof these genes downstream of the luciferase promoterand performed luciferase activity assays (Fig. 5Ba).MiR-145 significantly suppressed expression of Renillaluciferase reporter carrying the wild-type putativetargets of Sox9 and ADD3 in HNGC-2 and NSG-K16(Fig. 5Bb) and LN-229 cells (Supplementary Fig. S3F)compared with control cells transfected with their respec-tive wild-type 3′UTR constructs. Further, we confirmedthat expression of Sox9 and ADD3 was indeed regulatedby miR-145 by analyzing whether miR-145 overexpres-sion into GBM cells reduced their endogenous protein ex-pression by Western blotting. Consistent downregulationin Sox9 and ADD3 protein levels were found in

miR-145–overexpressing HNGC-2 (Fig. 5Ca),NSG-K16 (Fig. 5Cb), and LN-229 (Supplementary Fig.S3Ea) cells compared with their respective EV cells. Thesame data were quantified by densitometry (Fig. 5Ca, band Supplementary Fig. S3Eb). These experiments con-firmed that Sox9 and ADD3 genes were functionaltargets of miR-145, as their binding to miR-145 specifi-cally inhibited their expression in glioma cells.

Inhibition of Sox9 and ADD3 Mimics MiR-145 Effects

Next, we determined whether glioma tumor tissuesamples that showed downregulation of miR-145 in agraded manner expressed correspondingly higher levelsof its targets Sox9 and ADD3. For this we performedqRT-PCR, wherein we individually analyzed low-grade(n ¼ 12) and high-grade gliomas (n ¼ 17) along withnormal brain tissues (n ¼ 5) for expression of Sox9and ADD3. These same glioma tumor tissues werequantified earlier for expression of miR-145 (Fig. 1A;Supplementary Table SI). Expectedly, Sox9 was overex-pressed to a �10-fold greater extent in low-gradegliomas and by �500-fold in GBM tumor samples com-pared with normal brain tissues (n ¼ 5; Fig. 6Aa).

Fig. 4. Overexpression of miR-145 inhibits xenograft growth in vivo and suppresses cell invasion in vitro. (A) HNGC-2 cells (1 × 104) stably

transduced with EV (right) or stably expressing miR-145 (left) were injected subcutaneously in NOD-SCID mice (n ¼ 18 mice).

Representative picture of mice bearing tumors with HNGC-2-EV cells, while the HNGC-2 expressing miR-145 cells failed to form tumors (a).

The HNGC-2 and NSG-K16 EV mice on bearing tumors (�4 wk) were sacrificed and the tumors were dissected (b) and quantitated for

tumor volumes (c). (B) NOD-SCID mice were injected intracranially with each of 1 × 103 EV and miR-145 HNGC-2 and NSG-K16 GFP cells

(n ¼ 5 mice/group). GFP-positive tumor focus seen within brain tissue (a). The H&E image shows tumor tissue (T) within the whole brain

tissue section (b). (C) Invasion assay showing migrating HNGC-2 and NSG-K16–EV and miR-145 cells stained with crystal violet and

observed at 10× magnification (a) and quantified (b). The percentages of invading cells were determined by considering migration of

HNGC-2 and NSG-K16–EV cells as 100%. Data are shown as mean number of cells in triplicate wells. Data represent means+SEM (n ¼ 3),

***P , .0001.

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Similarly, ADD3 levels increased with grades of gliomamalignancy, wherein ADD3 levels were .5-fold in low-grade gliomas and .500-fold in higher-grade tumorscompared with normal brain tissues (n ¼ 5; Fig. 6Ab).The luciferase reporterassaysandWesternblottingexper-iments provided experimental evidence that Sox9 andADD3 were indeed targets for miR-145. Further, we ana-lyzed whether Sox9 and ADD3 were responsible formiR-145–mediated effects on proliferation and

tumorigenicity of glioma cells. For this, we used siRNAsto knock down endogenous ADD3 and Sox9 expressionfrom EV cells and determined whether that led to effectson cell proliferation and cell cycle progression that weresimilar to those seen with miR-145 restoration. Forrescue experiments, siRNAs were transiently transfectedinto 3 glioma cell lines—HNGC-2, NSG-K16, andLN-229—and cells were analyzed for proliferation byMTT assay and for cell cycle kinetics by flow cytometry.

Fig. 5. (A) Sox9 and ADD3 are direct primary targets of miR-145. SOX9 and ADD3 possess binding sites for miR-145 in their 3′UTRs. (B)

Schematic depiction of Renilla luciferase reporter vectors containing wild-type Sox9 and ADD3 3′UTRs (a); CMV, cytomegalovirus. MiR-145

directly downregulates expression of Sox9 and ADD3 in luciferase assay. Relative luciferase values were determined with dual-luciferase

assay of HNGC-2 and NSG-K16 cells cotransfected with reporter constructs and miR-145 vector (b). Renilla luciferase activity is normalized

to firefly luciferase activity as control for transfection efficiency. Columns: mean of triplicate experiments; bars: SEM. *P , .05, ***P ,

.0001, the significant difference from EV. (C) Total cell lysates were used to determine levels of Sox9 and ADD3 by Western blotting in

HNGC-2 (a), NSG-K16 (b)–EV, and miR-145 cells. Beta-actin served as loading control. The band intensities of Sox9 and ADD3 were

quantified by densitometry using ImageJ software.

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Fig. 6. Sox9 and ADD3 are functional targets of miR-145. Total RNA was extracted from tissues by Trizol Reagent and converted into cDNA using a

cDNAsynthesiskit. (A)QuantitativeRT-PCRanalysisofSox9andADD3expression inglial tumors.The lowgrade(n¼ 12) represents samplesderived

fromgrades I and IIglial tumors,whereashighgrade (n¼ 17) representsgrades IIIand IVtumors.RNAexpression levelswerenormalizedwith respect

to levels in normal brain tissue samples (n ¼ 5) collected from frontal lobectomy surgeries (Supplementary Table S1). Data represent means+SEM

from3independentexperiments.*P , .05,***P , .001. (B)MTTassayofHNGC-2andNSG-K16gliomacells stablyexpressingEV,miR-145,orcells

transfectedwithsiRNAstoADD3orSox9.InhibitionofSox9andADD3usingspecificsiRNAsmimicsthegrowth-suppressivefunctionofmiR-45.Data

represent means+SEM (n ¼ 3); OD, optical density. (C) Representative plots showing cell cycle analyses of HNGC-2 cells stably expressing EV,

miR-145, or cells transfected with siRNA’s to ADD3 or Sox9 and stained with PI after 72 h transfection (a). Cell cycle distribution is represented as

a table showing distribution of cells in 3 different phases: G0/G1, S, and G2/M (b). Data are shown as mean+SEM (n¼ 3), P , .0001.

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The MTT assay demonstrated that while the HNGC-2and NSG-K16 EV cells displayed exponential growth,the Sox9 and ADD3 siRNA transfected cells were retard-ed in their growth potential and displayed similar growthas did miR-145–overexpressing HNGC-2, NSG-K16(Fig. 6B), and LN-229 glioma cells (Supplementary Fig.S3G). The effects of these siRNA’s on glioma cells werealso evident at levels of cell cycle kinetics assayed byflow cytometry. A marked increase in percentage of cellsin G0/G1 phase and decrease in S-phase cells occurredon treatment of HNGC-2 cells with miR-145. ThesiRNA’s to Sox9 and ADD3 mirrored the effects ofmiR-145 on glioma cells. The Sox9 and ADD3 siRNAtreated cells showed similar distribution of cells in eachof the 3 phases as was displayed by miR-145–overex-pressing HNGC-2 cells and LN-229 cells (Fig. 6C,Supplementary Fig. S3H). These studies indicate thatdownregulation of the miR-145 targets Sox9 and ADD3in glioma cells generates cells with growth characteristicssimilar to those manifested by miR-145–expressing cells.

MiR-145 Is Regulated by Promoter Hypermethylation

To determine the DNA methylation status of miR-145promoter in glioma cells, we performed bisulphite

genome sequencing. The distribution of cytosine–phos-phate–guanine (CpG) dinucleotides is shown for a0.3 kb region upstream of the miR-145 promoter(Fig. 7A). Hence, we designed primers that spanned a1.5 kb region upstream of the start site to the miR-145promoter and cloned this region in a pcDNA vector fol-lowed by bisulphite DNA sequencing in glioma cells.Among the multiple positive clones obtained, we screened6 clones of each cell line for miR-145 promoter analysis atthe CpG islands. The data generated from bisulphitegenome sequencing for each of these clones were analyzedusing Bismark tools and are represented in Fig. 7, whereinDNA hypermethylation is represented by red and bluecolor and depicts hypomethylation of the CpG islands.In all 6 clones analyzed for each of these cell lines, the ma-jority of CpG islands showed hypermethylation ofmiR-145 promoter, indicating that it is epigenetically si-lenced in HNGC-2 and NSG-K16 (Fig. 7A) glioma cells.To further corroborate that miR-145 is epigenetically si-lenced in these glioma cell lines, we treated HNGC-2and NSG-K16 cell lines with 5-azacytidine (5 mM), apotent inhibitor of DNA methyltransferases, for 24 h.Later, cells were harvested and analyzed for expressionof miR-145 by RT-PCR. We found that 5-azacytidinetreatment reversed the repression of miR-145 in theHNGC-2 and NSG-K16 (Fig. 7B) cell lines. Together

Fig.7. CpG islandhypermethylationcauses silencingofmiR-145 ingliomacell lines. (A)Schematic representationofhsa-miR-145genomic locus

on chromosome 17. Bisulphite genome sequence analyses for miR-145 CpG islands in glioma cell lines. The distribution of CpG dinucleotides is

shown for a 0.3 kb region upstream of miR-145 promoter. Presence of methylated (red squares) or unmethylated CpG islands (blue squares) in

each of the 6 clones for HNGC-2 and NSG-K16 cells is shown. (B) Expression of mature miR-145 by qRT-PCR in cells treated with DNA

demethylation agent 5-aza-2-deoxycytidine (AZA). cDNA synthesized from glioma cells was used for expression of miR-145 using PCR

conditions as specified in Fig. 1A and primer sequences as specified in Supplementary Table S2. U6 was used as an internal control, and

values were normalized to one with respect to cells not treated with AZA. The use of the DNA methylation inhibitor restores miRNA

expression in the methylated glioma cell lines. Data represent means+SEM, P , .05.

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these results indicated that miR-145 promoter was epige-netically regulated in the glioma cell lines HNGC-2 andNSG-K16 studied by us.

Interaction of MiR-145 With Its Targets

The in vitro and in vivo data exemplify the role ofmiR-145 as a tumor suppressor in gliomas. The effectsof miR-145 overexpression on inhibiting cell cycle pro-gression from G0/G1 to S phase and adhesion suggest in-teraction of this miRNA with molecules involved in cellcycle progression, proliferation, and invasion. An analy-sis of cell cycle regulatory molecules like c-myc, N-myc,and cyclin D1 by Western blotting demonstrated theirsubstantial downregulation in miR-145–overexpressingglioma cells HNGC-2 and NSG-K16 compared with EVglioma cells (Fig. 8). Importantly, molecules importantfor invasion and metastasis like E-cadherin andN-cadherin were also downregulated in HNGC-2 andNSG-K16 miR-145 glioma cells compared with thecontrol group (Fig. 8). These findings indicate that the cel-lular signaling effects mediated by miR-145 are manifest-ed through direct and indirect effects of miR-145 on genesinvolved in cell cycle progression, adhesion, and invasion.

Discussion

MiRNAs are small (�21 nucleotides) noncoding RNAsthat are evolutionarily conserved and encoded withinthe genomes of almost all eukaryotes. MiRNAs posttran-scriptionally regulate protein synthesis by base pairing topartially complementary sequences in the 3′UTRs oftarget mRNAs, leading to gene repression.29 Essentially,miRNAs execute their functions in a dynamic andcontext-dependent manner by affecting diverse

downstream target genes from transcriptional factors toepigenetic regulators.30 With a large number ofmiRNAs known to be involved in regulating cell growthand survival, they play a pivotal role as modulators of car-cinogenesis.31 While many oncogenic miRNAs are in-volved in pathogenesis of solid tumors, a generaldownregulation in their expression compared withnormal tissues is reported in several cancers, suggestingthat the majority of miRNAs are tumor suppressors.32,33

The current hypothesis regarding cell of originfor gliomas proposes that these tumors arise from a pop-ulation of cells that share with normal neural stem cellsa requirement for self-renewal and multilineage differen-tiation.34 Thus, identification of miRNAs regulating mul-tiple steps of neurogenesis, from neural stem cellproliferation to neuronal differentiation and maturation,is vital for understanding mechanisms of cellular signal-ing and gene regulation. In glioblastomas, miRNAs likemiR-21 are overexpressed, which causes tumor growthand survival in vivo, an effect mediated by downregulat-ing several tumor suppressors like phosphatase andtensin (PTEN) homolog and promoting migration bytargeting pro-invasion genes.35

Glioma stem cells or brain tumor–initiating cells aresmall populations of cells within a tumor that drivetumor growth, propel progression, enhance invasion,and induce therapeutic resistance.36 Recent studiessuggest thatmiRNAs are involved in regulatingpropertiesof these cells through their targets, like genes essential forpluripotency and stem cell function.37 The present studywas aimed at identifying miRNAs critically involved intransformation of nontumorigenic neural stem cells tobrain tumor stem cells (BTSCs). For this we used a cellculture model developed by us to study glioma progres-sion comprising the cell lines HNGC-1, representing anontumorigenic neural stem cell line, and HNGC-2, dem-onstrating features of cancer stem cells.21 The extent andkind of differential expression of miRNAs in this modelwere achieved through a microarray service wherein wecompared miRNA profiles of nontumorigenic HNGC-1cells with BTSCs like HNGC-2. Similar studies were per-formed with another newly developed BTSC culture,NSG-K16. Among the several differentially expressedmiRNAs, miR-145 was most discretely represented inthis cell system as a tumor-suppressive RNA.38 Studies in-dicate thatmiR-145 causes growth inhibition by targetingoncogenes like c-Myc and represses proliferationand pro-motes differentiation by regulating pluripotency factorsOct4, Sox2, and Kruppel-like factor 4 (KLF4).39

MiR-145, while not significantly affecting tumorgrowth, slowed potential to inhibit both migration and in-vasion in breast cancer cell lines, possibly related to itsability to inhibit tumorcell invasionandmetastasisby tar-geting Mucin1 and Fascin1.40 MiR-145 blocks migrationof vascular smooth muscle cells by inhibiting Fli1, whilemiR-143 targets versican protein, which is involved in mi-gration and is induced by platelet-derived growth factor(PDGF).41 The specific expression of miR-145 in peri-cytes, wherein it targets the hematopoietic transcriptionfactor Fli1, causes inhibition of migration in responseto growth factor gradients and supports its role in

Fig. 8. Western blot analysis. Decreased expression of molecules

involved in cell growth (c-myc, N-myc, cyclin D1) and adhesion and

invasion (E-cadherin, N-cadherin) in miR-145–HNGC-2 and

NSG-K16 cells compared with EV cells. Beta-actin served as loading

control. Values represent relative band intensity normalized to actin

using ImageJ software 1.44p.

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vascularizationandangiogenesis.42 MiR-145, a regulatorof the mammalian target of rapamycin (mTOR) pathway,directly targets p70S6K1 and downregulates the expres-sion of hypoxia-inducible factor–1a (HIF-1a) and vascu-lar endothelial growth factor (VEGF), thus inhibitinginvasion and angiogenesis. Similar to embryonic stemcells, 2 specific miRNAs expressed from the samegenetic locus, miR-143 and miR-145, downregulateSox2, and Sox2 downregulates miR-143 and miR-145,suggesting that Sox2 and these miRNA’s form a double-negative feedback loop in GBM cells; however, their rela-tionship to GBM has not been studied.43 There have beenrecent reports about involvement of miR-145 as a tumorsuppressor in gliomas with a more prominent role inglioma invasion mediated through its targets, likeADAM17 (a disintegrin and metalloproteinase metallo-peptidase domain 17), a member of the Zn-dependentmetalloproteinase family,14 or through NEDD9 (neuralprecursorcell expressed, developmentally downregulated9), a scaffold protein involved in invasion in high-gradegliomas.16 In contrast, miR-145 is regarded as a positiveregulator of glioblastoma invasion, a variation from itstumor-suppressor role, attributed mainly to the slowergrowth rate of glioma cells during invasion.13,17 Ourstudy demonstrates the potential of miR-145 as amiRNAwith tumor-suppressive, antimigratory, and anti-invasive potential in glioma cells as well as in glioma stemcells. By qRT-PCR analyses, we obtained .50-folddownregulation of miR-145 in most of the GBM tumorsas well as in glioma stem cells. However, the tumor-suppressive effects were not specific to a stem cell popula-tion, as they were also evident in other established gliomacell lines, like LN-229 (Supplementary Fig. S3).

We further established that miR-145 directly targetsSox9 and ADD3. These genes are related to cell prolifera-tion and invasion and were identified by a bioinformaticsapproach using various algorithms and validated by3′UTR luciferase assays. Higher-grade gliomas withminimal expression of miR-145 showed overexpressionof Sox9 and ADD3. The levels of Sox9 were found to beoverexpressed in several brain tumors that includedsonic hedgehog–dependent medulloblastomas andgliomas, as well as GBM samples, indicating that highSox9 mRNA expression was a prognostic factor forglioma patients.18,42 Human malignant glioma and prim-itive neuroectodermal tumor samples, as well as gliomacell lines, showed strong nuclear positivity for Sox9.44

Interestingly, there is a role for Sox9 in PDGF signaling,as Sox9 together with Sox10 regulate PDGF receptor-aexpression in oligodendrocyte progenitors of the spinalcord.45 Sox9 is also present in the neural crest, neuralstem cells, and neurospheres46 and is coexpressed withSox1 and Sox2 in brain regions that harbor neural stemcells.47 When Sox9 in U87MG cells was reduced bysiRNA treatment, there was retardation in cell prolifera-tion. Our studies demonstrating Sox9 as a target formiR-145 specify its role as an oncogene in glioma and apotential drug target. There are few studies proposingmiR-145 as an important regulator of human chondro-cyte function, wherein levels of miR-145 inversely corre-late with Sox9 expression during human chondrocyte

dedifferentiation, thereby inducing characteristicchanges, asoccur inosteoarthritis.48 Importantly,overex-pression or suppression of miR-145 resulted in inhibitingor promoting, respectively, chondrogenic differentiation,suggesting that miR-145 acts as a key mediator to antag-onize early chondrogenic differentiation via attenuatingthe effect of transcription factor Sox9.48 There arepresently no reports specifying ADD3 as a target tomiR-145, though there are reports linking it tomiR-143.49 Ours is the first study to validate ADD3 as agenuine target to miR-145 using both Western blottingand luciferase assay. Adducins are heteromeric proteinscomposed of different subunits referred to as adducinalpha, beta, and gamma encoded by distinct genes andbelong to a family of membrane skeletal proteins involvedin the assembly of a spectrin-actin network in erythro-cytes and at sites of cell–cell contact in epithelialtissues.50,51 Alternatively spliced adducin gamma tran-scripts encoding different isoforms are recognized,though with unknown functions. The role of ADD3 ingliomas is not known, although there may be some asso-ciations derived from the infiltrative growth behavior ofGBM. Since the neural cell membrane lacks typical extra-cellular matrix proteins like collagen, fibronectin, andtype II laminin, migrating glioma cells may invade neigh-boring brain tissue by coordinated cross-bridgingbetween nonmuscle myosin II and actin. The involvementof miR-143 in cardiac myogenesis is known and occursthrough direct repression of ADD3, which encodes anF-actin capping protein. MiR-143 and ADD3 act cell au-tonomously in zebrafish to control F-actin dynamics andcell morphology.49 Our data suggest that upregulationof ADD3 is one of the mechanisms contributing to the in-filtrative behavior of gliomas. Additional studies are re-quired to establish the role of this molecule in gliomainvasion.

The growth-suppressive effects of miR-145 possiblystem from its potential to inhibit cell cycle progression,evident in terms of decreased levels of proteins likec-Myc, N-myc, and cyclin D1, as well as genes importantto invasion and epithelial-mesenchymal transition likeE-cadherin and N-cadherin in miR-145–overexpressingglioma cells. These findings point to a regulatory frame-work centered on miR-145 in glioma cells. MiR-145 co-ordinately downregulates expression of oncogenes, suchas Sox9 and ADD3. The role of Sox9 in proliferation, sur-vival, and invasion in gliomas is well documented andis reported in phenomena like epithelial-mesenchymaltransition. Interestingly, molecules like N-cadherin,E-cadherin, N- myc, C-myc, and cyclin D1, downregu-lated in response to miR-145 overexpression, are eitherdownstream targets or interacting partners of Sox9.Although our study does not signify a direct correlationbetween Sox9 and these genes, we hypothesize thatmiR-145 executes its tumor-suppressor function by in-hibiting Sox9, causing downregulation of these prosur-vival genes. The direct function of ADD3 has not yetbeen addressed in glioma biology, but microarray-basedprofiling has shown it’s overexpression in various malig-nancies, like small cell lung cancer.52 As ADD3 is an im-portant regulator of both the spectrin-based membrane

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skeleton and the actin cytoskeleton, we envisage that itfunctions along with other cytoskeletal components,like N-cadherin and E-cadherin. Another miRNA,miR-195, downregulated during GBM progression, isshown to function through its target genes E2F3 andCCND1, which are involved in cell cycle progression.53

Based on our data, we propose a model highlighting thepossible mediators and signaling molecules throughwhich miR-145 may exert its growth- and tumor-

suppressive effects (Fig. 9). Hence, our study demon-strates that interaction of miR-145 with its targets Sox9and ADD3 has crucial ramifications on tumor progres-sion and invasion in glioma cells. Our studies showingthe role of miR-145 as a tumor suppressor propose it tobe an important therapeutic target for glioblastomas.

Supplementary Material

Supplementary material is available online at Neuro-Oncology (http://neuro-oncology.oxfordjournals.org/).

Acknowledgments

We are grateful to Dr Avinash Pradhan, KEM Hospital,Pune, for his expert help in histopathogical analyses ofglioma tissue sections. We are thankful to M. L. Shaikhfor his technical help in mice tumorigenicity experiments.

Conflict of interest statement. None declared.

Funding

This work was supported by grants from Department ofBiotechnology (DBT), Ministry of Science andTechnology, Government of India, India and NationalCentre for Cell Science (NCCS), Pune, India.

Grant support for carrying out research was providedby Stem Cell Task Force, Department of Biotechnology(DBT) (grant no. BT/PR10557/MED/31/29/2008),Government of India, New Delhi, India, and NationalCentre for Cell Science (NCCS), Pune, India. The fellow-ship of S.R. was supported by ICMR, New Delhi.Fellowship funding for S.R and K.S. was provided byDBT, and the fellowship for N.K. was provided byCouncil of Scientific and Industrial Research (CSIR),New Delhi, India.

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