Cancer Epidemiology 38 (2014) 152–156

Decreased expression of microRNA-200b is an independentunfavorable prognostic factor for glioma patients

Donghai Men 1, Yuansheng Liang 1, Liyi Chen *

Department of Neurosurgery, The Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China

A R T I C L E I N F O

Article history:

Received 18 September 2013

Received in revised form 7 January 2014

Accepted 20 January 2014

Available online 18 February 2014

Keywords:

MicroRNA-200b

Glioma

Real-time quantitative RT-PCR assay

Prognosis

A B S T R A C T

Background and aim: As a member of the microRNA (miR)-200 family, miR-200b has been recognized as

one of the fundamental regulators of epithelial–mesenchymal transition, chemosensitivity, cell

proliferation, and cell cycle. Especially in glioma, miR-200b targets the CREB1 gene and suppresses

the tumor cell growth in vitro. However, its involvement in human glioma has not yet been determined.

The aim of this study was to investigate the clinical significance of miR-200b expression in this disease.

Methods: miR-200b expression in 266 pairs of human gliomas and matched nonneoplastic brain tissues

was measured by real-time quantitative RT-PCR assay.

Results: Compared with nonneoplastic brain tissues, the expression level of miR-200b was significantly

decreased in glioma tissues (tumor vs. normal: 2.87 � 2.05 vs. 8.78 � 2.50, P < 0.001). Of 266 patients with

gliomas, 166 (62.41%) were in low miR-200b expression group. In addition, we found that the glioma tissues

from high-grade tumors (grade III and IV) had much lower miR-200b expression than glioma tissues from

low grade tumors (grade I and II). Moreover, the expression level of miR-200b was positively correlated with

Karnofsky performance status (KPS) scores of glioma tissues. The results of a 60-month follow-up in 266

glioma patients further demonstrated that lower miR-200b expression was correlated with worse

progression-free survival and overall survival in the patients with grade III and IV gliomas. Both univariate

and multivariate analyses revealed that miR-200b was an independent prognostic indicator for glioma.

Conclusion: These findings prove that the decreased expression of miR-200b may be associated with

malignant tumor progression and poor prognosis in patients with gliomas, suggesting the potential role

of miR-200b in glioma management. miR-200b may be a novel and valuable signature for predicting the

clinical outcome of patients with gliomas.

� 2014 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1. Introduction

Gliomas account for the most common human primary tumorsof the central nervous system and have a tendency to invade thesurrounding brain tissue [1]. These tumors are characterized by arapid infiltrative growth pattern making complete surgicalresection impossible [2]. Gliomas are divided into four histopath-ologic grades based on the degree of malignancy according toWorld Health Organization (WHO) classification: low-gradeastrocytomas (WHO grade I–II), anaplastic astrocytomas (WHOgrade III), and glioblastoma (GBM, WHO grade IV) [3]. Despite therecent advances in tumor diagnosis and treatment, includingsurgery, radiotherapy and chemotherapy, the median survival time

* Corresponding author. Tel.: +86 759 2369961; fax: +86 759 2369961.

E-mail addresses: 475080010@qq.com (D. Men), lys845@163.com (Y. Liang),

lysghq@163.com (L. Chen).1 These authors offer the equal contributions to this study.

1877-7821/$ – see front matter � 2014 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.canep.2014.01.003

is only one year and few patients survive for two years. The 5-yearsurvival rate of low-grade glioma is 30–70%, while the mediansurvival time of the most aggressive type GBM, which grows andinfiltrates rapidly, is from 9 to 12 months [4]. Several clinicopath-ologic features have been used as prognostic factors for gliomas,such as histopathologic grades and Karnofsky performance status(KPS) score. Unfortunately, these factors may not estimateprognosis in glioma patients exactly because of patients’ hetero-geneous [5]. Accumulating evidence reveals that a number ofbiological and molecular factors are involved in the development,progression, metastasis, and drug resistance of gliomas. However,few molecular signatures have been validated and widely acceptedas prognostic indicators in clinical practice. Since more preciseprognostic predictors and more effective therapies for gliomas arerequired, it is extremely necessary to identify novel molecularsignatures that can predict the clinical outcome and response totreatment of this disease with reliable clinical significance.

MicroRNAs (miRNAs) are a new class of single-stranded non-coding small RNAs with about 17–27 nucleotides in length [6].

Table 1Association between miR-200b expression and different clinicopathological

features of human gliomas.

Clinicopathological features No. of cases miR-200b expression P

High (n, %) Low (n, %)

Age

<49 110 41 (37.27) 69 (62.73) NS

�49 156 59 (37.82) 97 (62.18)

Gender

Male 138 45 (32.61) 93 (67.39) NS

Female 128 55 (42.97) 73 (57.03)

WHO grade

I–II 98 94 (95.92) 4 (4.08) <0.001

III–IV 168 6 (3.57) 163 (96.43)

KPS score

<80 148 30 (20.27) 118 (79.73) 0.02

�80 118 70 (59.32) 48 (40.68)

Extent of resection

�98% 200 76 (38.00) 124 (62.00) NS

<98% 66 24 (36.36) 42 (63.64)

Tumor size

�5 cm 166 56 (33.73) 110 (66.37) NS

<5 cm 100 44 (44.00) 56 (56.00)

Note: ‘NS’ refers to the difference without statistical significance.

D. Men et al. / Cancer Epidemiology 38 (2014) 152–156 153

Functionally, miRNAs can regulate gene expression by post-transcriptional silencing of target mRNAs by binding to the 30-untranslated region (30-UTR) of target transcripts [7]. Each miRNAcan affect a number of mRNAs, and depending upon its targets,plays different roles in biological or pathological processes.Emerging evidence has strongly suggested that a large numberof miRNAs may function as oncogenes and/or tumor suppressors invarious cellular processes during cancer initiation and progression,including cell proliferation, stem cell renewal, and differentiation[8]. Especially, there have been a number of miRNAs, the abnormalexpression of which has been demonstrated to be a common andimportant feature in human gliomas. For example, the down-regulation of miR-34a [9], miR-203 [10], miR-383 [11], miR-145[12], miR-326 [13], and miR-375 [14], and the upregulation of miR-650 [15], miR-372 [16], miR-17 [17], miR-221/222 [18] and miR-355 [19] all predict poor prognosis in patients with gliomas. Basedon these findings, the altered expression of oncogenic and tumor-suppressing miRNAs may be widely associated with tumorigenesisof human gliomas.

The miR-200 family consists of miR-200a, miR-200b, miR-200c,miR-141 and miR-429, which are clustered and expressed as twoseparate polycistronic pri-miRNA transcripts with the miR-200b-200a-429 cluster at chromosomal location 1p36 and miR-200c-141 cluster at chromosomal location 12p13 [20]. Functionally,members in this family can regulate the transcription factors Zeb1and Ets-1 as well as Suz12, a subunit of the polycomb repressorcomplexes [21]. Recent studies have demonstrated that the miR-200 family plays a tumor suppressive role in a wide range ofhuman cancers, such as gastric cancer, pancreatic cancer,colorectal cancer, breast cancer and endometrial carcinoma [22–26]. Interestingly, Siebzehnrubl et al. [27] revealed that the Zeb1-miR-200 feedback loop could interconnect the processes ofinvasion, chemoresistance and tumorigenesis in GBM throughthe downstream effectors ROBO1, c-MYB and MGMT. As a memberof the miR-200 family, miR-200b has been recognized as one of thefundamental regulators of epithelial–mesenchymal transition(EMT), chemosensitivity, cell proliferation, and cell cycle [28].Recent studies have indicated that the down-regulation of miR-200b and the accompanied up-regulation of the EMT inducer Zeb1and Zeb2 are highly correlated with the mesenchymal and drug-resistant phenotypes of cancer cells [29]. Nevertheless, over-expression of miR-200b is also observed in several types of tumors.Especially in glioma, miR-200b targets the CREB1 gene andsuppresses the tumor cell growth in vitro [30]. However, itsinvolvement in human glioma has not yet been determined. Theaim of this study was to investigate the clinical significance of miR-200b expression in this disease.

2. Materials and methods

2.1. Patients and tissue samples

This study was approved by the Research Ethics Committee ofthe Affiliated Hospital of Guangdong Medical College. Writteninformed consent was obtained from all of the patients. Allspecimens were handled and made anonymous according to theethical and legal standards.

A total of 266 pairs of human gliomas and matchednonneoplastic brain tissues obtained from 266 patients withprimary gliomas were collected from Department of Neurosurgery,the Affiliated Hospital of Guangdong Medical College between1998 and 2008. The patients were 151 males and 115 females, witha median age of 49 years (range, 26–82 years). All patientsunderwent MRI a few days before and within 72 h after surgery.The extent of tumor resection was determined using thepostoperative MRI scans. Surgical resection was defined as

macroscopic total resection, partial resection or biopsy. Accordingto WHO criteria, 98 of the 266 gliomas were classified as low-grade[46 pilocytic astrocytomas (WHO I) and 52 diffuse astrocytomas(WHO II)], and 168 were classified as high-grade gliomas [68anaplasia astrocytomas (WHO III), and 100 primary glioblastomas(WHO IV)]. None of the patients had received chemotherapy orradiotherapy prior to surgery. The clinicopathologic features of allthe patients were summarized in Table 1. Each nonneoplastic braintissue was acquired a minimum of 4 cm from the tumor tissues.Resected tissue samples were immediately cut and snap-frozen inliquid nitrogen before stored at �80 8C until RNA extraction.

Clinical follow-up was available for all patients (median, 18months; range, 1–138 months). At the last follow-up, 106 patientswere still alive, whereas 160 had died. The period between theinitial operation and death was defined as the overall survival time.The period between the initial operation and tumor recurrence ordeath was defined as progression-free survival time. Patients, whodied of diseases not directly related to their gliomas or due tounexpected events, were excluded from this study.

2.2. Real-time quantitative RT-PCR for miRNA

miR-200b expression in 266 pairs of human gliomas andmatched nonneoplastic brain tissues was measured by real-timequantitative RT-PCR assay. Total RNA was extracted from tissuesamples using TRIzol (Invitrogen) according to the manufacturer’sprotocol. The stem-loop reverse-transcription (RT) primers weredesigned as follows: miR-200b, 50-GTC GTA TCC AGT GCA GGG TCCGAG GTA TTC GCA CTG GAT ACG ACT CAT CAT-30, and U6, 50-CGCTTC ACG AAT TTG CGT GTC A-30. RT reactions contained 10 ngof total RNAs, 50 nmol/l stem-loop RT primer, 1� RT buffer,0.25 mmol/l each of deoxynucleotide triphosphate (dNTP), 3.33 U/ml MultiScribe reverse transcriptase, and 0.25 U/ml RNase Inhibi-tor. The 7.5-ml reaction volumes were incubated in Bio-Rad i-Cycler (Bio-Rad Laboratories, Hercules, CA, USA) in a 96-well platefor 30 min at 16 8C, 30 min at 42 8C, 5 min at 85 8C, and then held at4 8C. Real-time PCR was performed using an Applied Biosystems7500 real-time PCR system. The 10-ml PCR included 0.6 ml of RTproducts, 1� TaqMan Universal PCR master mix, and 1 ml ofprimers and probe mix of the TaqMan MicroRNA Assays. The PCRprimers were designed as follows: miR-200b sense, 50-GCG GCTAAT ACT GCC TGG TAA-30, and reverse, 50-GTG CAG GGT CCG AGG

D. Men et al. / Cancer Epidemiology 38 (2014) 152–156154

T-30; U6 sense, 50-CGC TTC GGC AGC ACA TAT ACT A-30, and reverse,50-CGC TTC ACG AAT TTG CGT GTC A-30. Relative quantification oftarget miRNA expression was evaluated using the comparativecycle threshold (CT) method. The raw data were presented as therelative quantity of target miRNA, normalized with respect toRNU6B. Each sample was examined in triplicate. Mean normalizedgene expression � standard deviation (SD) was calculated fromindependent experiments.

2.3. Statistical analysis

All computations were carried out using the software of SPSSversion 13.0 for Windows (SPSS Inc., IL, USA). Data were expressedas mean � SD. The Chi-square test was used to assess miR-200bexpression with respect to clinicopathological parameters. Thesurvival curves of the patients were determined using the Kaplan–Meier method and Cox regression, and the log-rank test was used forstatistical evaluations. Differences were considered statisticallysignificant when P was less than 0.05.

3. Results

3.1. Decreased expression of miR-200b in human glioma tissues

The expression levels of miR-200b in all glioma tissues withWHO grade I–IV and nonneoplastic brain tissues were shown inFig. 1. Compared with nonneoplastic brain tissues, the expressionlevel of miR-200b was significantly decreased in glioma tissues(tumor vs. normal: 2.87 � 2.05 vs. 8.78 � 2.50, P < 0.001). Theresults showed that the highest expression of miR-200b was achievedin nonneoplastic brain tissues, while grade IV glioma tissue displayedthe lowest expression level. Interestingly, the expression levels ofmiR-200b in glioma tissues with four WHO grades were allsignificantly lower than that in nonneoplastic brain tissues (WHO

Fig. 1. miR-200b expression in 266 pairs of glioma tissues and matched

nonneoplastic brain tissues were respectively detected by real-time quantitative

RT-PCR assay. Compared with nonneoplastic brain tissues (N), the expression level

of miR-200b was significantly decreased in glioma tissues (G) (tumor vs. normal:

2.87 � 2.05 vs. 8.78 � 2.50, P < 0.001). The results showed that the highest

expression of miR-200b was achieved in nonneoplastic brain tissues, while grade

IV glioma tissue (GIV) displayed the lowest expression level. Interestingly, the

expression levels of miR-200b in glioma tissues with four WHO grades were all

significantly lower than that in nonneoplastic brain tissues [WHO grade I tumor (GI)

vs. normal (N): 5.99 � 1.38 vs. 8.78 � 2.50, P < 0.001; WHO grade II tumor (GII) vs.

normal (N): 3.80 � 1.41 vs. 8.78 � 2.50, P < 0.001; WHO grade III tumor (GIII) vs.

normal (N): 2.85 � 0.72 vs. 8.78 � 2.50, P < 0.001; WHO grade IV tumor (GIV) vs.

normal (N): 0.94 � 0.61 vs. 8.78 � 2.50; P < 0.001].

grade I tumor vs. normal: 5.99 � 1.38 vs. 8.78 � 2.50, P < 0.001; WHOgrade II tumor vs. normal: 3.80 � 1.41 vs. 8.78 � 2.50, P < 0.001;WHO grade III tumor vs. normal: 2.85 � 0.72 vs. 8.78 � 2.50,P < 0.001; WHO grade IV tumor vs. normal: 0.94 � 0.61 vs.8.78 � 2.50; P < 0.001). Moreover, the median value of the expressionlevels of miR-200b in all 266 patients with gliomas (median = 2.56)was used as the cutoff point to divide patients into two groups: lowand high miR-200b expression groups, respectively. Of 266 patientswith gliomas, 166 (62.41%) were in low miR-200b expression group.

3.2. Association of miR-200b expression with clinicopathological

parameters of human gliomas

Table 1 summarizes the association between miR-200bexpression and clinicopathological parameters in gliomas. Asignificant relationship was observed between miR-200b expres-sion and the histopathologic grade. We found that the gliomatissues from high-grade tumors (grade III and IV) had much lowermiR-200b expression than glioma tissues from low grade tumors(grade I and II) (P < 0.001). Moreover, the expression level of miR-200b was positively correlated with KPS scores of glioma tissues(P = 0.02). No statistically significant association of miR-200bexpression with age at diagnosis, gender of patients, extent ofresection, and tumor size was found (both P > 0.05, Table 1).

3.3. Association of high miR-200b expression with poor prognosis in

patients with gliomas

To determine the prognostic value of miR-200b expression inhuman glioma, clinical follow-up was available for all patients. Theresults of Kaplan–Meier survival analysis in the glioma patientswith pathological grades I–IV are shown in Fig. 2. In grade I and IIglioma patients, miR-200b expression did not significantlycorrelate with shorter progression-free survival and overallsurvival (both P > 0.05, Fig. 2A and B), while the expression levelsof miR-200b displayed prognostic significance for progression-freesurvival and overall survival of glioma patients with grade III andIV. The median values of progression-free survival and overallsurvival of the patients with low miR-200b expression were 9.61months (95% CI, 6.9–12.8 months) and 13.89 months (95% CI,11.06–17.83 months), respectively, which were significantlyshorter than those in the patients with high miR-200b expressionwho had a median progression-free survival value of 14.66 months(95% CI, 9.82–20.92 months) and a median overall survival value of20.19 months (95% CI, 13.68–26.29 months) (P = 0.023 and 0.012,respectively; Fig. 2C and D).

Moreover, Cox regression analysis was performed to determinethe association of miR-200b expression with progression-freesurvival and overall survival of glioma patients with grade III andIV. As shown in Table 2, both univariate and multivariate analysesrevealed that miR-200b was an independent prognostic indicatorfor glioma.

4. Discussion

Despite tremendous efforts in developing multimodal treat-ments, the clinical outcome of glioma patients, especially for high-grade gliomas, remains unfavorable. The survival time variesconsiderably between patients. Heterogeneity in both cytologyand gene expression makes it difficult to coordinate effectivetherapeutic strategies which work for every patient. Thus, it is ofgreat significance to realize the underlining molecular mechan-isms and to identify powerful prognostic indicator for humangliomas. In the current study, we examined the miR-200bexpression in glioma tissues at various WHO grades. We foundthat the tissues from high-grade glioma (grade III and IV) had much

Fig. 2. Kaplan–Meier curves for progression-free survival (A and C) and overall survival (B and D) in patients with gliomas divided according to miR-200b expression. In grade I

and II glioma patients, miR-200b expression did not significantly correlate with shorter progression-free survival and overall survival (both P > 0.05, A and B), while the

expression levels of miR-200b displayed prognostic significance for progression-free survival and overall survival of glioma patients with grade III and IV (P = 0.023 and 0.012,

respectively; C and D).

D. Men et al. / Cancer Epidemiology 38 (2014) 152–156 155

lower miR-200b expression level than low-grade glioma (grade I–II) and nonneoplastic brain tissues. The results of a 60-monthfollow-up in 266 glioma patients further demonstrated that lowermiR-200b expression was correlated with worse progression-freesurvival and overall survival in the patients with grade III and IVgliomas. Therefore, we present evidence here for miR-200b as apotential prognostic indicator for human gliomas.

The distinct miRNA expression patterns in different cancer typeshave been identified by profiling approaches. The functionaldiscovery of miRNAs may enable to offer insight into regulationof gene expression and complexity of carcinogenesis. Interestingly,

Table 2Univariate and multivariate analysis of prognostic parameters in glioma patients with

Variables Progression-free survival

Univariate log-rank test Cox multivariable an

Hazard ratio P Hazard ratio

Age at diagnosis (years)

<49 vs. �49 0.56 NS 0.71

Gender

Male vs. female 0.68 NS 0.89

WHO grade

I–II vs. III–IV 4.32 0.011 6.93

KPS

<80 vs. �80 1.67 NS 1.90

Extent of surgery

�98% vs. <98% 1.46 NS 1.62

Tumor size

�5 cm vs. <5 cm 1.88 NS 2.09

miR-200b expression

High vs. low 2.88 0.023 4.62

the influence of miRNAs may be dependent on cancer type. SomemiRNAs act as oncogenes, by contributing to the transformedphenotype when expressed at high levels in cancers. Theseoncogenic miRNAs may function by suppressing tumor suppressorgenes. In contrast, some miRNAs act as tumor suppressors byallowing the expression of oncogenes, and are weakly expressed orabsent in tumors [31]. EMT manifests through downregulation of E-cadherin and successive loss of cell–cell adhesion, leading to amesenchymal phenotype. It occurs during embryogenesis tosupport tissue remodeling and has been proposed as the fundamen-tal step in the metastasis of epithelial cancers, contributing to

grade III–IV by Cox regression analysis.

Overall survival

alysis Univariate log-rank test Cox multivariable analysis

P Hazard ratio P Hazard ratio P

NS 0.42 NS 0.69 NS

NS 0.55 NS 0.76 NS

0.009 2.98 0.020 4.36 0.012

NS 1.11 NS 1.29 NS

NS 1.09 NS 1.33 NS

NS 1.65 NS 1.87 NS

0.012 2.11 0.038 2.90 0.022

D. Men et al. / Cancer Epidemiology 38 (2014) 152–156156

increased cell motility and invasion [32]. This process mayaccelerate invasiveness, dissemination, and metastasis of epithelialtumor cells in several malignancies [33]. As a member of the miR-200 family, miR-200b is involved in the inhibition of EMT bymaintaining the epithelial phenotype through directly targeting theE-cadherin transcriptional repressors ZEB1 and ZEB2, therebyinducing upregulation of E-cadherin [34]. It is thereby not surprisingthat miR-200b has been demonstrated to be down-regulated orupregulated in various advanced cancers. For example, Manavalanet al. [35] indicated that the reduced miR-200b expression maycontribute to endocrine resistance in breast cancer cells; Feng et al.[36] reported that down-regulation of miR-200b could lead to E2F3overexpression and in turn contribute to chemoresistance of lungadenocarcinoma cells to docetaxel. Tang et al. [37] revealed thatmiR-200b was downregulated in the gastric cancer specimens andcell lines, and the expression levels of miR-200b may be significantlycorrelated with the clinical stage, T stage, lymph node metastasisand survival of patients; Kurashige et al. [38] also identified ZEB2 as atarget gene for miR-200b and demonstrated that miR-200b mayregulate ZEB2 expression and thus control metastasis in gastriccancer. Here, our data showed the decreased expression of miR-200bin human glioma tissues compared with nonneoplastic brain tissues,which was similar with previous findings on other cancers [35–38]and the report of Peng et al. [30] on human gliomas. We also foundthat the extent of miR-200b downregulation depended onpathological grades of gliomas. The tissues with advanced patho-logical grades and low KPS scores may express lower miR-200b. Inour survival analysis, the aberrant expression miR-200b did notsignificantly correlate with prognosis of glioma patients with lowpathological grades (I–II), which may be due to the fact that low-grade tumors have a lower risk of recurrence and death as opposedto high-grade tumors, thus concealing the prognostic effect of miR-200b. Thus, it is more meaningful and accurate to assess theprognostic value of miR-200b in the patients with high-gradegliomas (WHO grade III and IV gliomas). More importantly, when thepatients with grade III and IV gliomas were stratified by miR-200bexpression, the patients with low miR-200b expression showedsignificantly shorter progression-free survival and overall survivalthan those with high miR-200b expression, implying that miR-200bmay be an independent indicator for predicting survival of gliomapatients.

In conclusion, these findings prove that the decreased expres-sion of miR-200b may be associated with malignant tumorprogression and poor prognosis in patients with gliomas,suggesting the potential role of miR-200b in glioma management.miR-200b may be a novel and valuable signature for predicting theclinical outcome of patients with gliomas.

Conflict of interest

None.

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