MiR-196a exerts its oncogenic effect in glioblastoma multiforme by

Neuro-Oncology Neuro-Oncology 16(5), 652 –661, 2014 doi:10.1093/neuonc/not307 Advance Access date 23 January 2014

MiR-196a exerts its oncogenic effect in glioblastoma multiforme by inhibition of IkBa both in vitro and in vivo Guang Yang†, Dayong Han†, Xin Chen†, Daming Zhang, Lu Wang, Chen Shi, Weiguang Zhang, Chenguang Li, Xiaofeng Chen, Huailei Liu, Dongzhi Zhang, Jianhao Kang, Fei Peng, Ziyi Liu, Jiping Qi, Xin Gao, Jing Ai, Changbin Shi, and Shiguang Zhao Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 15001, China (G.Y., D.H., X.C., D.Z., W.Z., C.L., X.C., H.L., D.Z., J.K., F.P., Z.L., S.Z.); Institute of Brain Science, Harbin Medical University, Harbin, China (G.Y., D.H., X.C., D.Z., W.Z., C.L., X.C., H.L., D.Z., J.K., F.P., Z.L., S.Z.); Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, China (L.W., J.A.); Department of Neurosurgery, New York University Langone Medical Center and School of Medicine, New York, New York (C.S.); Department of Pathology, The First Affiliated Hospital of Harbin Medical University, Harbin, China (J.Q.); Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia (X.G.); Section of Neurosurgery, Department of Surgery, The University of Chicago, Medical Center and Pritzker School of Medicine, Chicago, Illinois (C.S.) Corresponding author: Shiguang Zhao, MD, PhD, Department of Neurosurgery, The First Affiliated Hospital of Harbin Medical University, Youzheng Street 23, Nangang District, Harbin, Heilongjiang Province, 150001, China ([email protected]). †

These authors contributed equally to this work.

Background. Recent studies have revealed that miR-196a is upregulated in glioblastoma multiforme (GBM) and that it correlates with the clinical outcome of patients with GBM. However, its potential regulatory mechanisms in GBM have never been reported. Methods. We used quantitative real-time PCR to assess miR-196a expression levels in 132 GBM specimens in a single institution. Oncogenic capability of miR-196a was detected by apoptosis and proliferation assays in U87MG and T98G cells. Immunohistochemistry was used to determine the expression of IkBa in GBM tissues, and a luciferase reporter assay was carried out to confirm whether IkBa is a direct target of miR-196a. In vivo, xenograft tumors were examined for an antiglioma effect of miR-196a inhibitors. Results. We present for the first time evidence that miR-196a could directly interact with IkBa 3′ -UTR to suppress IkBa expression and subsequently promote activation of NF-kB, consequently promoting proliferation of and suppressing apoptosis in GBM cells both in vitro and in vivo. Our study confirmed that miR-196a was upregulated in GBM specimens and that high levels of miR-196a were significantly correlated with poor outcome in a large cohort of GBM patients. Our data from human tumor xenografts in nude mice treated with miR196 inhibitors demonstrated that inhibition of miR-196a could ameliorate tumor growth in vivo. Conclusions. MiR-196a exerts its oncogenic effect in GBM by inhibiting IkBa both in vitro and in vivo. Our findings provide new insights into the pathogenesis of GBM and indicate that miR-196a may predict clinical outcome of GBM patients and serve as a new therapeutic target for GBM. Keywords: apoptosis, glioblastoma, IkBa, miR-196a, tumor growth.

Glioblastoma multiforme (GBM) is the most common and deadliest primary malignant brain tumor.1 Despite intense multimodality therapies including surgical excision, radiotherapy, and chemotherapy, the prognosis of GBM patients still remains unfavorable.2 Thus, there remains a critical need to develop novel predictive and more effective therapeutic strategies against this malignancy. MicroRNAs (miRNAs) are a class of endogenous, small noncoding RNAs that regulate gene expression by inducing translational inhibition or direct degradation of target mRNAs through base

pairing to partially complementary sites.3 Alteration of miRNA expression and activity has been associated with a range of human diseases, including cancer.3 – 5 For example, miR-196a is one of the most important miRNAs involved in cancer.6,7 Its aberrant expression has been observed in a varietyof solid tumors.8 – 10 Recent studies revealed that miR-196a was upregulated in GBM and correlated with the clinical outcome of patients with GBM.11 However, its potential regulatory mechanisms in GBM have never been reported.

Received 15 January 2013; accepted 19 November 2013 # The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected].

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Yang et al.: MiR-196a exerts its oncogenic effect in GBM

Thus, the aim of this study was to explore the potential regulatory mechanisms of miR-196a in GBM and to identify a potential predictive biomarker for the treatment of GBM patients. We showed for the first time that upregulation of miR-196a could promote proliferation of and suppress apoptosis in GBM cells both in vitro and in vivo through direct suppression of IkBa expression. Furthermore, we described that overexpression of miR-196a is strongly associated with high risk and poor prognosis in a large cohort of GBM patients at a single institution.

Materials and Methods Patients with Glioblastoma Multiforme and Their Surgically Excised Specimens Tumor specimens were collected from 132 GBM patients who underwent surgery in the Department of Neurosurgery at the First Affiliated Hospital of Harbin Medical University in China between 2005 and 2009. Tissue samples were routinely processed for histological diagnosis in strict accordance with World Health Organization criteria. Normal brain tissues (NBTs) were obtained from normal adjacent tissues away from tumor tissues or non-neoplastic brain diseases and were histologically confirmed to be free of any pathological lesions. The follow-up data were available for 114 cases. The study was approved by the Institutional Review Board of Harbin Medical University, and the participants gave informed consent.

Cell Culture and Transfection The human glioblastoma U87MG and T98G cells and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection (ATCC). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). MiR-196a mimics, miR-196a inhibitors (anti-miR-196a antisense oligodeoxyribonucleotide, AMO-miR-196a), negative control miRNA (NC) (GenePharma), reporter plasmid, or pc-IkBa plasmid were transfected into cultured cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.

RNA Extraction and Quantitative Real-time Polymerase Chain Reaction Total RNA was isolated from cultured cells using a Trizol standard protocol (Invitrogen). For formalin-fixed, paraffin-embedded (FFPE) GBM samples, total RNA was extracted from 5 to 10 of 10 mm-thick tissue sections using the Ambion RecoverAll kit (Ambio) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate in the ABI 7500HT fast real-time PCR System (Applied Biosystems) and normalized with U6 and glyceraldehyde 3phosphate dehydrogenase (GAPDH) endogenous control. Total RNA from NBTs was used as a control. MiR-196a and U6 levels were measured with the TaqMan microRNA assay kit, and endogenous mRNA levels of IkBa and GAPDH were detected using SYBR Green PCR Master Mix kit in accordance with the manufacturer’s instructions (Applied Biosystems). The RT-PCR primers for IkBa and GAPDH are listed in Supplementary Table 1.

Construction of IkBa 3 ′ Untranslated Region (3 ′ UTR) Reporter Plasmid and pc-IkBa Plasmid The IkBa-3′ UTR was amplified using the primers listed in Supplementary table 1 and cloned into the psi-CHECK-2 vector (Promega) at 2 restriction sites for XhoI and EcoRI. Mutations were introduced by site-directed mutagenesis into putative binding sites in the 3′ UTR of IkBa gene for miR-196a using the TaKaRa MutanBEST Kit (Takara). To construct IkBa expression plasmid, the full-length cDNA was first amplified using the primers listed in Supplementary table 1 and then cloned into the pcDNA3.1 vector (Invitrogen) at 2 restriction sites for HindIII and EcoRI.

Luciferase Assays HEK 293Tcells were cotransfected on 24-well plates by Lipofectamine 2000 reagent (Invitrogen) with 0.5 mg of reporter plasmid and miR-196a mimics or control miRNA at a final concentration of 50 nM. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. All experiments were repeated at least 3 times with duplicate samples.

Protein Extraction and Western Blot Cell Viability Assays U87MG or T98G cells were seeded onto 96-well plates and transfected as described above. On each of 3 consecutive days, 20 mL of dimethyl thiazolyl diphenyl tetrazolium (MTT; 5 mg/mL; Sigma) was added to each well. The reaction was then stopped by lysing cells with 200 mL of dimethyl sulfoxide. Optical density measurements were obtained at a wavelength of 570 nm using spectrophotometric analysis (Tecan).

Hoechst 33258 Staining After transfection of fluorescent dye labeled FAM-AMO-miR-196a or FAM-NC (GenePharma) for 24 hours, cells were washed with phosphatebuffered saline, fixed with 4% paraformaldehyde for 15 minutes, and then stained with 50 mM of Hoechst 33258 (Sigma) for 10 minutes in the dark. Nuclear morphological change in GBM cells undergoing apoptosis were detected by fluorescence microscopy (Nikon).

Apoptosis Assay The apoptosis ratio was analyzed using the Annexin VApoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. Apoptotic cells were examined and quantified using flow cytometry (Becton Dickinson). All experiments were repeated in triplicate.

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The proteins were extracted from human GBM cells and specimens. Lysate was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gel was blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked in 5% nonfat milk, and then incubated with either rabbit anti-human NF-kB p65 (1:200; Santa Cruz), Caspase-3 (1:200; Santa Cruz), Bcl-2 (1:200; Santa Cruz), Bax (1:200; Santa Cruz), mouse anti-human IkBa (1:1000; Cell Signaling Technology), or b-actin (1:1000; Santa Cruz). After washing, the membrane was incubated with the fluorescence-conjugated anti-mouse or anti-rabbit IgG (1:4000; Invitrogen). The bound secondary antibody was quantified using the Odyssey v1.2 software (LI-COR) by measuring the band intensity (area×optical density) for each group and then normalized with b-actin. The final results are expressed as fold changes by normalizing the data to control values.

Immunohistochemistry Paraffin-embedded sections of excised GBM specimens were immunostained for IkBa protein. Staining was performed with the streptavidin– biotin peroxidase complex method according to the manufacturer’s recommendation (Dako). Mouse anti-human IkBa primary antibody (1:50; Cell Signaling Technology) was administered, followed by secondary goat antimouse IgG (Dako). Negative controls were performed throughout the entire immunohistochemistry procedure. All of the immunostained sections were reviewed in blinded fashion by 2 investigators.

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Tumorigenicity Assays in Nude Mice 6

5 ×10 U87MG or T98G cell suspension was subcutaneously injected into the flank of 5-week-old female athymic BALB/c nude mice (Shanghai SLAC Laboratory Animal Company). At 10 days after the implantation, the mice were treated with AMO-antagomiR-196a or scrambled control antagomiR (GenePharmaa) in 10 mL Lipofectamine through local injection of the xenograft tumor at multiple sites. (AMO-antagomiR is a chemically modified antisense oligonucleotide, which could be used to inhibit endogenous miRNA in animal research.)12,13 The treatment was performed once every 3 days for 18 days. The tumor volume was measured with a caliper once every 3 days using the following formula: volume ¼ length× width2/2. Growth rates were determined by measuring tumor size over time. All animal procedures were approved by the Harbin Medical University Animal Committee.

Statistical Analysis Statistical analysis was performed with SPSS13.0 software. Student t test, ANOVA, or chi-square analysis were applied, where appropriate. Survival rates were estimated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Survival data were evaluated by using univariate and multivariate Cox regression analyses. A probability of ,.05 (*) or ,.001 (**) was considered significant.

Results MiR-196a Upregulation Correlates with Clinical Outcome of Human Glioblastoma Multiforme It has recently been reported that high levels of miR-196a in 39 human GBM specimens were significantly correlated with the malignant progression of gliomas and poor survival rates.11 To further confirm those findings, we detected the expression levels of miR-196a in U87MG and T98G cells and a larger cohort of 132

FFPE GBM specimens by qRT-PCR. Our data showed miR-196a levels were significantly higher in GBM cell lines and specimens as compared with those in NBT samples (P , .001, Fig. 1A and Supplementary Fig. 1A). We observed high variability in miR-196a expression in GBM specimens as compared with NBT samples. Moreover, the expression levels of miR-196a were significantly correlated with patient survival. Patients with miR-196a expression levels above the median showed a shorter overall survival when compared with patients in the low-expression group measured by Kaplan-Meier survival curve analysis with a log-rank comparison (P , .001; Fig. 1B). The median survival time of patients whose tumors had low-level expression of miR-196a was 12 months (95% CI, 10.07–13.93), whereas the median survival time of those with high expression levels of miR-196a was only 7 months (95% CI, 4.95 –9.05). The log-rank test showed a statistically significant difference in the median survival (P ¼ .001). Subsequently, we determined the correlation of miR-196a expression with clinical variables (sex, age, KPS, tumor size, and extent of resection) using the Cox proportional hazard regression model. Univariate and multivariate analysis showed that expression levels of miR-196a were an independent and significant predictor of overall survival in GBM patients (P ¼ .001; HR ¼ 2.326; Table 1), which is consistent with previous studies.11

Role of miR-196a in Cell Proliferation and Apoptosis in Vitro To explore the potential biological significance of miR-196a in tumorigenesis, we transfected U87MG or T98G cells with miR196a mimics, inhibitors (anti-miR-196a antisense oligodeoxyribonucleotide, AMO-miR-196a), or negative control miRNA. MiR-196a mimic-transfected cells showed a significant increase in miR-196a expression, while AMO-miR-196a-transfected cells demonstrated a significant decrease in miR-196a expression when compared

Fig. 1. Clinical significance of miR-196a in GBM patients. (A) miR-196a expression in 132 FFPE GBM specimens. NBTs refer to normal brain tissues. (B) Correlation of miR-196a expression with overall survival in GBM patients.

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Table 1. Univariate and multivariate Cox regression analysis of overall survival in archival GBM patients Univariate Analysis Variables Sex Male Female Age (y) ≤60 .60 KPS ,70 ≥70 Tumor size (cm) ,6 ≥6 Extent of resection Not GTR GTR MiR-196a expression Low High

Multivariate Analysis No. of Patients (%)

Median OS (months)

81 (61.36%) 51 (38.64%)

10 10

100 (75.76%) 32 (24.24%)

10 7

26 (19.70%) 106 (80.30%)

9 10

86 (65.15%) 37 (28.03%)

10 9

38 (28.79%) 94 (71.21%)

7 12

P value (log-rank) .924

.021

.031

.717

,.001

,.001 12 7

Variate

HR

Sex Female vs male

1.003

Age (y) .60 vs ≤60

1.637

KPS ≥70 vs ,70

0.563

Tumor size (cm) ≥6 vs ,6

1.277

Extent of resection GTR vs not

0.373

MiR-196a expression High vs low

2.326

P value .989

.078

.033

.333

,.001

.001

Abbreviations: OS, overall survival; HR, hazard ratio; KPS, Karnofsky performance status; GTR, gross total resection.

with cells transfected with scrambled negative control (Fig. 2A). MTT results showed that cells transfected with miR-196a mimics had a significantly increased growth rate as compared with cells transfected with negative control, whereas those transfected with AMO-miR-196a had a significantly decreased proliferation rate (Fig. 2B). To determine the effect of miR-196a on apoptosis, we performed Annexin V and propidium iodide double staining in U87MG or T98G cells. MiR-196a mimic-transfected cells had a significant decrease in Annexin V-positive apoptotic cells as compared with scrambled control-transfected cells, while AMO-miR-196atransfected cells showed a significant increase in Annexin V-positive apoptotic cells (Fig. 2C and D). In addition, after transfection of fluorescent dye labeled FAM-AMO-miR-196a or FAM-NC, we stained U87MG and T98G cells with Hoechst 33258. Our results revealed fluorescent dye concentrated and nuclei condensed and fragmented in FAM-AMO-miR-196a transfected cells, indicating that these cells underwent apoptosis (Fig. 2E). These results demonstrated that ectopic expression of miR-196a promoted cell proliferation and inhibited cell apoptosis in U87MG and T98G cells.

IkBa Is a Direct Target of miR-196a To further explore the regulatory mechanisms of miR-196a in GBM, we analyzed databases miRanda, PicTar, and TargetScan. We found that miR-196a likely regulates the IkBa gene since IkBa may be a target for miR-196a (Fig. 3A). In fact, IkBa has been reported to be a key mediator of cell apoptosis and invasion and is closely associated with survival in GBM patients.14 To determine whether miR-196a could regulate IkBa at mRNA and protein levels, qRT-PCR and Western blot were performed. Our qRT-PCR results showed that the expression of IkBa mRNA in U87MG and T98G cells transfected with miR-196a mimics was downregulated as

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compared with cells transfected with negative control (Fig. 3B). Western blot analysis also revealed that the expression of IkBa protein in U87MG and T98G cells transfected with miR-196a mimics was downregulated as compared with cells transfected with negative control (Fig. 3C). These data demonstrated that miR-196a could regulate IkBa at both mRNA and protein levels. We further examined IkBa protein levels by immunohistochemistry in GBM specimens that were also used to measure miR-196a expression. Our results showed that 87% of GBM specimens exhibited lower protein levels of IkBa as compared with NBTs (Fig. 3D). Further analysis revealed the significant reciprocal association of IkBa protein levels with miR-196a expression in GBM specimens (chi-square test; P , .001; Fig. 3E). To assess whether there was a direct interaction between miR-196a and IkBa, wild-type or mutant 3′ UTR of IkBa gene was constructed into the dual-luciferase reporter plasmid system (Fig. 3A). Subsequently, miR-196a mimics or negative control was cotransfected with the 3′ UTR of IkBa dual-luciferase reporter plasmid into HEK293Tcells. We observed that miR-196a decreased the relative luciferase activity of the reporter containing the wildtype 3′ UTR of IkBa. However, mutations in the predicted binding site of miR-196a in the 3′ UTR of IkBa gene abrogated the aforementioned inhibitory effect of miR-196a, demonstrating that IkBa is a direct target of miR-196a (Fig. 3F).

Inhibition of IkBa Is Essential for miR-196a –induced Proliferation and Suppressed Apoptosis To investigate the inhibitory role of IkBa in the miR-196a-induced proliferation and suppressed apoptosis in GBM cells, we studied the effect of knockdown of IkBa by the specific IkBa siRNA. Fig. 4A shows that IkBa protein expression was significantly

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Fig. 2. Effect of miR-196a on cell proliferation and apoptosis in vitro. (A) miR-196a expression was quantified by qRT– PCR in U87MG and T98G cells 48 h after transfection of miR-196a mimics, AMO-miR-196a, or negative control oligo. (B) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection. (C) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection. (D) Graphical representation of the apoptosis analysis in (C). (E) Hoechst 33258 staining of U87MG and T98G cells 48 hours after transfection of fluorescent dye labeled FAM-miRNA. Arrows indicate apoptotic cells. Original magnification is 400×. Data are presented as mean+SEM for 3 separate experiments performed in duplicate. *P , .05; *P , .001.

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Fig. 3. IkBa is a direct target of miR-196a. (A) The potential interaction between miR-196a and putative binding sites in the IkBa 3′ -UTR. The mutant sequences are equivalent to the wild-type ones, except mutations at the 3′ end of target site are underlined. (B) Relative IkBa mRNA expression was determined by qRT-PCR in U87MG and T98G cells 48 hours after transfection. (C) IkBa protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection. b-actin was used as a loading control. (D) Photomicrographs showing representative results of hematoxylin and eosin staining and immunohistochemical analysis of IkBa protein expression in human GBM specimens and NBTs. Original magnification ×200. (E) A chi-square table showing a significant inverse correlation between miR-196a and IkBa expression (P , .001). (F) Luciferase activities were analyzed in 293 T cells 48 hours after cotransfection of miR-196a mimics and either wild-type or mutant IkBa 3′ -UTR. Data are presented as mean+SEM for 3 separate experiments performed in duplicate. *P , .05; **P , .001.

inhibited, as we expected. Our results further revealed that IkBa siRNA has a similar biological effect on proliferation and apoptosis as miR-196a in GBM cells (Fig. 4B and D). Subsequently, we constructed and transfected pc-IkBa into U87MG or T98G cells. The expression levels of IkBa mRNA and protein in pc-IkBa-transfected cells were significantly increased as compared with pc-emptytransfected cells (Fig. 4E and Supplementary Fig. 1B). After U87MG or T98G cells were cotransfected with both miR-196a mimics and pc-IkBa, we found that proliferation and apoptosis regulated by ectopic expression of miR-196a was rescued by pc-IkBa (Fig. 4F–H). These data suggested that inhibition of IkBa is essential for miR-196a –induced proliferation and suppressed apoptosis. Overexpression of miR-196a can also affect NF-kB regulated expression of several apoptosis-related proteins (Fig. 5A). Western blot analysis indicated that expression levels of NF-kB subunit p65 were markedly increased. As expected, the expression levels of Bax and cleaved caspase-3 proteins were markedly decreased in miR-196a mimics transfected U87MG and T98G

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cells, while Bcl-2 protein levels were significantly increased. However, AMO-miR-196a can reverse the expression of these apoptosis-related proteins (Fig. 5A). Therefore, these results suggested that miR-196a-induced proliferation and suppressed apoptosis in GBM cells could be regulated by inhibition of IkBa via NF-kB-apoptosis signaling.

Inhibition of miR-196a Ameliorates Tumorigenesis of GBM Cells in Vivo To explore whether miR-196a affects tumorigenesis in vivo, U87MG or T98G cells were subcutaneously implanted in the flank of nude mice, respectively. The weight of tumor was substantially lower in animals injected with chemically modified miR-196a inhibitors (AMO-miR-196a), and the mean tumor size at the end of the experiment was markedly smaller in these mice as compared with that in the NC-injected mice (Fig. 5B). The growth curve of tumor xenografts showed that AMO-antagomiR-196a inhibited tumor growth (Fig. 5C). The average tumor weight in the NC

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Fig. 4. Inhibition of IkBa is essential for miR-196a–induced proliferation and inhibited apoptosis. (A) IkBa protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection of specific IkBa siRNA. (B) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection of IkBa siRNA. (C) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection of IkBa siRNA. (D) Graphical representation of the apoptosis analysis in (C). (E) IkBa protein expression was determined by Western blot in U87MG and T98G cells 48 hours after transfection of empty vector + NC oligo, pc-IkBa + NC oligo, or pc-IkBa + miR-196a mimics. (F) Cell viability was determined by MTT assay in U87MG and T98G cells 48 hours after transfection. (G) Cell apoptosis was analyzed by flow cytometry in U87MG and T98G cells 48 hours after transfection. (H) Graphical representation of the apoptosis analysis in (G). Data are presented as mean+SEM for 3 separate experiments performed in duplicate. * P , .05; **P , .001.

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Fig. 5. Inhibition of MiR-196a ameliorates tumorigenesis of GBM cells in vivo. (A) Expression of apoptosis-related proteins was determined by Western blot in U87MG and T98G cells 48 hours after transfection of miR-196a mimics, AMO-miR-196a, or negative control. (B) Photograph showing excised tumors in representative mice in each group implanted with U87MG or T98G cells (n ¼ 8 mice per treatment group) on day 28 after treatment. (C) Graph showing growth inhibition of human tumor xenografts in nude mice treated with AMO-antagomiR-196a. (D) Graph illustrating the excised tumor weight on day 28 after tumor implantation. (E) Relative miR-196a expression was determined by qRT-PCR in xenograft tumor specimens.

group was significantly higher than that in the AMO-antagomiR196a-injected group (Fig. 5D). qRT-PCR analysis was also performed to detect miR-196a expression in tumor xenografts. Our results showed that the expression levels of miR-196a in tumor tissues of AMO-antagomiR-196a-injected mice were significantly lower than those in tumor specimens of NC-injected mice (Fig. 5E). These results indicated that inhibition of miR-196a could ameliorate tumor growth in vivo.

Discussion Although recent reports suggested that miR-196a was upregulated in GBM and correlated with the clinical outcome of GBM patients, its potential regulatory mechanisms in GBM have never been addressed.11 In this study, we presented the first evidence that miR-196a directly interacted with the IkBa 3′ -UTR to suppress IkBa expression and subsequently promote activation of NF-kB, consequently promoting proliferation of and suppressing apoptosis in GBM cells both in vitro and in vivo. Thus, MiR-196a exerted its oncogenic effect in GBM. We further confirmed that miR-196a was upregulated in clinical GBM specimens and that high levels of miR-196a were significantly correlated with shorter overall survival time in a large cohort of GBM patients. Our results from human tumor xenografts in nude mice indicated that inhibition of miR-196a could ameliorate tumor growth in vivo. Thus, our

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findings support the potential role of miR-196a in GBM and may serve as a potential therapeutic target for GBM. Upregulation of miR-196a has been observed in gastric, pancreatic, esophageal, and breast cancers, and strong expression of miR-196a is also associated with a poor prognosis in gastric, pancreatic, and breast cancer patients.9,10,15 – 17 MiR-196a and miR-196b were also correlated with the clinical outcome of GBM patients.11,18,19 Functional analysis revealed that high expression of miR-196a in esophageal, breast, and endometrial cancer cells promoted proliferation and inhibited apoptosis by directly targeting Annexin A1 (ANXA1).20 In gastric cancer, overexpression of miR-196a could contribute to carcinogenesis in vitro and in vivo by suppression of p27kip1.8 However, it has recently been reported that miR-196a has exerted opposite effects in different tumors. For instance, miR-196a is significantly downregulated in melanoma, and overexpression of miR-196a inhibited invasion and metastasis in melanoma and breast cancer cells by suppression of HOX-C8.21,22 In our study, miR-196a could promote proliferation and suppress apoptosis by directly inhibiting IkBa in GBM cells. It is worth noting that single-nucleotide polymorphisms (SNPs) in sequences of miR-196a-2 may contribute to cancer susceptibility and prognosis. These genetic polymorphisms may affect the expression of mature miR-196a. For example, rs11614913 CC or carrying at least one C allele was significantly correlated with high levels of mature miR-196a.23,24 Presence of any variant allele in hsa-miR-196a-2 (rs11614913, CT) was significantly

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associated with a reduced risk for lung, liver, breast, and head and neck cancers.25 – 29 Similarly, SNPs of miR-196a are also closely related to prognosis in glioma.30 These suggest that the presence of mir-196a-2 SNPs may be a reason for abnormal expression of miR-196a in gliomas. IkBa tightly controls NF-kB activation, which binds and tethers NF-kB in the cytoplasm.31 In the classical pathway of NF-kB activation, phosphorylation and degradation of IkBa activates NF-kB complex, a p50-p65 heterodimer that then translocates into the nucleus where it activates a variety of NF-kB target genes31,32 such as apoptosis-related genes Bax and Bcl-2.33,34 Subsequently, Bax and Bcl-2 regulate activation of caspase-3.35,36 In this study, we observed that miR-196a-induced inhibition of IkBa could cause changes of Bax, Bcl-2, and caspase-3. Actually, decreased IkBa expression was frequently observed in various neoplasms, and IkBa is a key regulator of cell adhesion, migration, and cell polarity. For example, increased expression of IkBa predicted improved response and survival in EGFR-mutant lung cancer patients after treatment.37 Recently, Bredel et al revealed that overexpression of IkBa can inhibit GBM cell growth, migration, and colony formation and reduce cell viability.14 Jiang et al. also reported that suppression of IkBa promoted glioma cell invasiveness by upregulation of microRNA-30e*.38 Our results demonstrated that IkBa directly interacted with miR-196a, subsequently inhibiting IkBa expression, which promoted proliferation of and suppressed apoptosis in GBM cells in vitro assays and in a xenotransplantation model. This study provides the mechanistic basis for miR-196a biologic effects on GBM. The present study has several limitations. We only demonstrated the oncogenic effect of miR-196a in GBM. However, we have not applied this study to the anaplastic and low grade of astrocytomas. Recently, Guan et al. demonstrated that the expression levels of miR-196a were higher in GBM as compared with those in anaplastic astrocytoma and that GBM patients with high expression levels of miR-196a had significantly shorter survival periods.11 Thus, it indicated that miR-196a may play a role in the malignant progression of gliomas. In addition, we observed that miR-196a expression was high in a small percentage of GBM specimens in our study, suggesting that some other major factors may contribute to GBM development and prognosis. For example, we previously demonstrated that EGFR amplification and miR-106a and MIB-1 expressions were also significant predictors in GBM patients.39 Moreover, the clinical implications of other important factors, such as mutations of the p53 gene, promotor hypermethylation of O6-methylguanine-DNA methyltransferase, and mutations of IDH genes, have been demonstrated in GBM patients.40,41 In conclusion, we presented the first evidence that miR-196a could promote proliferation of and suppress apoptosis in GBM by inhibition of IkBa both in vitro and in vivo. This study further confirmed that aberrant expression of miR-196a is a common event in GBM. Hence, our results provide new insights into the pathogenesis of GBM and indicate that miR-196a may predict clinical outcome of GBM patients and serve as a new therapeutic target for GBM.

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

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Funding This work was supported by National Natural Science Foundations of China [81302178; 81272788]; Natural Science Foundations of Heilongjiang [QC2013C096]; the Fund of the First Affiliated Hospital of Harbin Medical University [2013B01].

Acknowledgments We thank Dr. Yuandong Dong, Department of Neurosurgery, for data collection.

Conflict of interest statement. None declared.

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