MiRNA-196 is up-regulated in glioblastoma but not in anaplastic astrocytoma and has prognostic significance MiRNA-196 in glioblastoma

PURPOSE: MicroRNAs (miRNA) are short non-coding RNAs that can play critical roles in diverse biological processes. They are implicated in tumorigenesis and function as both tumor suppressors and oncogenes. The clinical significance of miRNA expression profiles in malignant gliomas remains unclear. In this study, we examined the expression levels of 365 mature human miRNAs in 12 malignant gliomas, including 8 glioblastomas (GBMs) and 4 anaplastic astrocytomas (AAs), using TaqMan real-time quantitative PCR arrays. A validation study was performed to corroborate a subset of the results, including expression levels of miR-196a, 196b, 21, and 15b, by analyzing 92 malignant gliomas by conventional real time PCR. We modeled the relationship between the expression levels of these miRNAs and the survival rate of 39 GBM patients by Kaplan-Meier method and multivariate analysis. Expression profiles in GBMs and AAs suggested that 16 miRNAs were candidate markers associated with the malignant progression of gliomas. Among them, miR-196a showed the most significant difference ( p =0.0038) and miR-196b also had a high significance ( p= 0.0371). Both miRNAs showed increased expression levels in GBMs relative to both AAs and normal brains in the validation study. Furthermore, patients with high miR-196 expression levels showed significantly poorer survival by the Kaplan-Meier method ( p= 0.0073). Multivariate analysis demonstrated that miR-196 expression levels were an independent predictor of overall survival in all 39 GBM patients ( p= 0.021, HR=2.81). Our results suggest that miR-196 may play a role in the malignant progression of gliomas and may be a prognostic predictor in GBMs. progression of gliomas. We identified several miRNAs showing significantly different expression between AAs and GBMs. Individual Taqman® miRNA real-time PCR assays using 92 malignant glioma tissues were subsequently performed to validate the significances. The up-regulation of miRNA-196 was found to be significantly correlated with the malignant progression of gliomas and poor survival rates. These findings can provide a means to develop a novel targeted molecular therapy for gliomas and can aid in the prediction of patient outcomes.


INTRODUCTION
MicroRNAs (miRNAs) are small non-coding RNA molecules, 19-24 nucleotides in length, which contribute to the regulation of crucial processes, such as cell proliferation, apoptosis, development, and differentiation (1,2). Recent studies have shown that miRNAs are implicated in tumorigenesis and function as tumor suppressor genes or oncogenes (3)(4)(5)). Characterization of the miRNA expression patterns in cancer cells is thought to have substantial value for diagnostic and prognostic determinations as well as for eventual therapeutic interventions (6)(7)(8)(9)(10)(11)(12)(13). Several miRNAs, such as miR-7, 21, 128, 221, and 222, have recently been reported to act as a tumor suppressors or oncogenes in glioblastomas (GBMs), the highest grade glioma (14)(15)(16)(17)(18). Recently, the number of newly identified miRNAs has exploded, however, the biological role of these miRNAs has not yet been elucidated. In addition, the clinical significance of miRNAs in glioma patients is still unclear though several groups have reported the alteration of miRNA expression in glioma (14)(15)(16)(17)(18).
With hundreds of human miRNAs reported thus far and many more awaiting experimental validation, these molecules represent one of the largest classes of gene regulators. As the number of newly discovered miRNAs is still increasing, large-scale screening is necessary to profile the global miRNA expression. MiRNA microarrays, the most commonly used tool for the large scale screening of miRNA expression, do not amplify miRNA, and thus the sensitivity is often compromised in these assays (19). In contrast, the stem-loop reverse transcription-polymerase chain reaction (RT-PCR) can profile miRNA expression with superior sensitivity and specificity. However, RT-PCR is not practical for high-throughput assays. Recently a quantitative RT-PCR-based array method (qPCR-array) became available which enabled high sensitivity and specific quantification of a large number of miRNAs in a single experiment (20,21). In the present study, we applied the qPCR-array method (TaqMan® Human MiRNA Array v1.0) to the analysis of 365 mature human miRNAs for a pilot estimation of the global expression patterns in GBMs and anaplastic astrocytomas (AAs) the second highest grade glioma. Unsupervised clustering analysis successfully segregated the AAs from the majority of the GBMs, indicating that altered miRNAs expression levels are associated with the malignant progression of gliomas. We identified several miRNAs showing significantly different expression between AAs and GBMs. Individual Taqman® miRNA realtime PCR assays using 92 malignant glioma tissues were subsequently performed to validate the significances. The up-regulation of miRNA-196 was found to be significantly correlated with the malignant progression of gliomas and poor survival rates. These findings can provide a means to develop a novel targeted molecular therapy for gliomas and can aid in the prediction of patient outcomes.

Glioma samples and patients
Glioma specimens were obtained from patients during surgery at the Kyushu University Hospital and five affiliated hospitals. A portion of the tumor tissue was saved and made into paraffin sections for histological diagnosis in strict accordance with World Health malignant gliomas as well as three normal brain tissues (for calibration purpose) by conventional real-time PCR. For these 3 normal brains, one of them was purchased from Ambion as total RNA (FirstChoice ® Human Brain Reference RNA), the other two were obtained from 2 patients who underwent treatment in Kyushu University Hospital, one of them was epileptic brain and the other was non-neoplastic portion of the autopsy brain. Finally, we picked up 46 consecutive malignant glioma cases (7 AAs and 39 GBMs), who underwent primary surgery in Kyushu University Hospital from 2002 to 2007, for the further clinical analysis. All of the cases were treated with the same protocol (radiotherapy and chemotherapy (ACNU or Temozolomide)) and well followed up. The present study was approved by the Ethics Committee of the Kyushu University.  (22). Both RNU44 and RNU48 were used as endogenous controls, and non-neoplastic brain tissues were used for calibration.

LOH analysis by microsatellite markers
Tumor and non-tumor DNA were evaluated by a PCR-based LOH assay (23) using 21 microsatellite markers located on chromosomes 1p, 10, and 19q, as shown in the Supplemental Table 2. The allelic status was assessed using the criteria established in a previous study (23).

EGFR gene amplification, EGFR wild-type and vIII expression
For EGFR gene copy number, real-time PCR was performed with the use of the ABI PRISM 7500 Fast Real-time PCR System (Applied Biosystems). All measurements were made in triplicate. LINE1 and Blood DNA were used as endogenous control and calibrator, respectively. A tumor with EGFR amplification was defined when its copy number of EGFR is more than 5. EGFRvIII expression was detected by reverse-transcript-PCR (RT-PCR) as described previously (24). Briefly, total RNA was extracted from frozen tumor samples. Complementary DNA (cDNA) was synthesized and amplified with the use of primers designed specifically to amplify EGFR (1044-bp product) and EGFRvIII (243-bp product).
Wild-type EGFR transcripts were quantified by real-time RT-PCR using the ABI PRISM 7500 Fast Real-time PCR System (Applied Biosystems) with SYBRGreen. Expression of the housekeeping gene HPRT1 was used as endogenous control. The threshold value of overexpression was defined as 2.5-fold of mean expression level of 3 normal brain tissues. The primer sequences were provided in the Supplemental Table S3.

Statistical analysis
Student's t-test was carried out to compare the expression levels of miRNAs in different glioma subtypes. A hierarchical clustering analysis was performed to evaluate the miRNA expression profiles in AA and GBM. The log-rank test was carried out to make univariate assessments of survival rates. Using the Cox proportional hazards regression model, multivariate analysis was performed to identify predictive factors for the survival of GBM patients. Statistical analysis was done using JMP 7.0.

Expression profiles of miRNAs correlated to the malignant progression of gliomas
To test the correlation between miRNAs and the malignant progression of gliomas, we analyzed the expression levels of 365 mature human miRNAs in the total RNA extracted from frozen tissues of four AAs and eight GBMs, using TaqMan real-time quantitative PCR array assays. Experimentally normalized ΔCt values corresponding to the expression levels of 365 miRNAs were used to cluster AAs and GBMs by unsupervised hierarchical clustering. As shown in Fig. 1A, all four AAs and two of the eight GBMs were found to cluster in one main on July 9, 2020. © 2010 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on July 2, 2010; DOI: 10.1158/1078-0432.CCR-10-0207 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research branch, with the other branch containing the remaining GBMs. Thus, AAs were clearly distinguished from the majority of the GBMs. To correlate the crucial miRNA expression levels with glioma progression, Student's t-test was performed to calculate the significant differences between miRNA expression levels in AAs and GBMs. In total, 16 miRNAs (Table1) were found to be differentially expressed in the two histological subtypes, based on the statistical criteria p < 0.05. Using ΔCt values for these 16 miRNAs only, a clustering analysis was conducted again to classify the 12 gliomas (Fig. 1B). This time, the four AAs and eight GBMs were distinctly segregated into 2 clusters, with the exception of one GBM sample, GBM1294. However, GBM1294, which was included in the branch containing the AAs, showed a clear segregation and a relatively long interval from AAs (Fig. 1B). These finding suggest that miRNA expression patterns reflect biological and pathological characteristics of malignant gliomas. Among these 16 miRNAs, miR-196a showed the most significant differential expression (p= 0.0038). Furthermore, this miRNA was expressed approximately 100-fold higher in the GBMs than in the AAs. MiR-196b, which differs by only one base from miR-196a, was also included in the analysis. Recent studies have reported the up-regulation of miR-196a and/or 196b in a variety of tumors, including glioblastoma cell lines (25), prostate (26), or pancreatic cancer (10,27). In addition, miR-21, which was included among the 16 miRNAs listed above, has been previously implicated in glioblastomas (14). MiR-15b, a member of the miR-15/16 family, also showed significantly different expression levels in GBMs and AAs in our results (p= 0.007). We concluded that expression levels of these miRNAs are significantly associated with WHO grade and therefore they could be useful biological markers for the malignant progression of glioma.

Up-regulation of miR-196a and 196b in glioblastomas
To evaluate the correlation between these candidates and malignant progression, we examined the expression of miR-196a, 196b, 15b, and 21 in a panel of 92 malignant glioma tissues, including 74 GBMs and 18 AAs, by individual real-time PCR. As shown in Fig. 2, all of these miRNAs were significantly up-regulated in GBM relative to normal brain tissues (Student's t-test, p<0.001), with the exception of miR-15b when compared with the RNU48 control. The miRNA expression levels of miR-196a, 196b, and 15b were shown to be significantly higher in GBMs than in AAs (Student's t-test, p≤ 0.001). A similar result was observed in miR-21 when RNU48 was used as an endogenous control (Student's t-test, p≤ 0.05). Taken together, our results determined that expression levels of miR-196a and 196b were definitively higher in GBMs compared to either AAs or normal brains, regardless of the endogenous control used, suggesting that they could be relevant to the malignant progression of gliomas.

High levels of miR-196 expression correlated with poor survival in malignant glioma patients
MiRNA expression profiles are reported to have diagnostic and prognostic value in the context of cancer treatment (6)(7)(8)(9)(10)(11)(12)(13). To evaluate the clinical implications of the miRNAs identified in this study, we analyzed the clinical data from 46 malignant glioma patients (7 AAs and 39 GBMs) whose samples were tested in the validation experiment. The correlation between expression levels of miRNAs (determined in the validation experiment) and the overall survival was measured through Kaplan-Meier survival curve analysis with a log-rank comparison using a binomial variable of high or low expression relative to the average expression levels of the miRNAs. No significant correlation was found between the overall survival and the expression levels of the miR-21 (p = 0.5832) and 15b (p = 0.2478). In contrast, highly statistically significant correlation was observed between the overall survival and the expression levels of miR-196a (Fig. 3A, left; p = 0.0052) and 196b (Fig. 3A, middle; p = 0.0149). However, when the analysis was performed on the subset of 39 GBM patients, the correlation was not statistically significant (Fig. 3B, left and middle; p = 0.0519 for miR-196a and p = 0.0619 for miR-196b). MiR-196a and 196b differ by only one nucleic acid and share the majority of predicted target sequences (69.5% for miR-196a and 70.6% for miR-196b, http://miRNA.sanger.uk/cgi-bin/target/v5). Thus, to reasonably evaluate the value of these two miRNAs for prognostic prediction, we divided the GBM patients into two groups: low expression group containing samples in which expression of both miR-196a and 196b were lower than the average, and high expression group, in which expression of either miR-196a or 196b was higher than the average. Based on this grouping, patients with the high expression levels of miR-196 showed a shorter overall survival (p = 0.0003, Fig.3A, right) compared with patients in the low expression group, even among GBM patients (p = 0.0073, Fig. 3B, right). In addition, multivariate analysis using the Cox proportional hazard regression model demonstrated that a high level of miR-196 was an independent and significant (p=0.021, HR=3.37) predictor of short overall survival in GBM patients ( Table 2).

DISCUSSION
Recent studies have implicated miRNAs in a variety of human cancers, and their expression signatures can provide insight into the diagnosis and prognosis of human cancers (6)(7)(8)(9)(10)(11)(12)(13). This study provides the first report of quantitative global miRNA expression profiles, It has been reported that miRNA expression profiles reflect cellular differentiation and distinguish tumors of different developmental origin (4,25). Several recent studies characterized the miRNA expression profiles associated with specific tumor subtypes according to histotype, malignant grade, and clinical features (28)(29)(30)(31)(32). Our study revealed significant differences between the expression levels of 16 miRNAs in the sets of 4 AAs and 8 GBMs. Furthermore, classification of these 12 malignant gliomas via the expression patterns of the 16 miRNAs showed significant correlations with the WHO grade of the tumor. These 16 miRNAs may be involved in tumorigenesis and the progression of malignancy in gliomas, and they may be useful for distinguishing GBMs from AAs.
Interestingly, in our data, two GBMs (GBM1266 and 1294) showed more similar miRNA expression profiles to AAs than to other GBMs ( Figure 1A). Even clustered via the 16 candidate miRNAs, one of them (GBM1294) were still classified into AA group ( Figure 1B).
To exclude the possibility of misdiagnosis, we reviewed the histological sections and confirmed the accuracy of the diagnosis (photomicrographs were shown as Supplementary Figure 1). In addition, it forcefully supported our diagnosis that both of the two tumors showed total chromosome 10 LOH (Supplementary Table 1). However, GBM is a histopathologically and genetically heterogenous tumor, and histopathological diagnosis by rule represents the most malignant part of the tumor. The sampling bias may thus occur even when samples are taken from one same tumor tissue. This is also the common limitation of such studies using frozen sample, however, given that most of the GBM cases fell into the same cluster, we believe our study clearly demonstrated the relevance of miRNAs expression to these histotypes. Nevertheless, it is also possible that there are some subpopulations of GBMs with different miRNA expression patterns even among those meeting the same histopathological criteria of GBM. Further studies on a larger series including investigating the relationship between miRNA expression patterns and clinical courses will be needed.
Among the 16 miRNAs identified, miR-196a was up-regulated by a factor of 100 in GBMs relative to AAs. A similar trend was observed for miR-196b, which differs by only one base from miR-196a. Our data are consistent with a recent study that identified mir-196a and 196b as candidate oncogenes for central nervous system (CNS) tumor cells due to the fact that both genes are expressed at higher levels in CNS tumor-derived cell lines compared with normal brain cells (25). Increased miR-196 expression levels, by factors of 10-100 in some kinds of human cancers, relative to their matched normal tissues, has also been reported in two recent studies (27,33), further supporting the relevance of miR-196 to gliomas. These previous reports have demonstrated that miR-21 is overexpressed in a variety of cancers (34-39). In particular, this miRNA has been suggested to play an important role in preventing apoptosis (14). MiR-21 is also a component in a network of key tumor-suppressive pathways in glioblastoma cells (40). Consistent with these reports, we observed increased expression levels of miR-21 in GBMs relative to normal brain tissues. GBMs were observed to express significantly higher levels of this miRNA in our data. This observation supports a potential role for miR-21 in the malignant progression of gliomas. MiR-15b, a member of miR-15/16 family, was also differentially expressed in GBM and AA tumors. Interestingly, miR-15b and its family members have been reported to be downregulated and function as tumor suppressor genes in several types of cancer (41,42). On the other hand, it has been suggested that miR-15b regulates cell cycle progression and is overexpressed in glioma cells (43), which is consistent with our results.
Several other miRNAs were identified in the screening study, e.g. our result showed that miR-128 expression levels were different by a factor of 0.15 in GBM and AA samples (Table  1). In addition, this miRNA was significantly down-regulated in both AAs and oligodendroglial tumors when compared to normal brain tissues (our unpublished data).
Consistent with our findings, it has been reported that miR-128 is down-regulated in GBMs (16) and functions as a tumor suppressor through the direct repression of the Bmi-1 oncogene (18). Consistently lower expression levels of the miR-302-367 cluster were observed in GBM relative to AA. The human miR-302-367 gene structure has been recently described, characterized, and functionally validated in human stem cells, and it was determined to be a potential stemness regulator in embryonic stem cells (44). This miRNA cluster was demonstrated to reprogram tumor cells into an embryonic stem cell-like state (45). These miRNAs could be significant in glioma progression and may be used as markers to distinguish GBM from AA. These miRNAs may also be involved in the mechanism of malignant transformation in gliomas.
The significance of miRNAs in diagnostic and prognostic determination has been demonstrated in a variety of human cancers, such as chronic lymphocytic leukemia (6), lung cancer (7,8), pancreatic cancer (9, 10), neuroblastomas (11), breast cancers (12), and colon cancers (13). However, investigations into the correlation between miRNA expression patterns and patient survival of GBM, the most frequent and malignant CNS tumor, have not been previously reported. Here, we have provided the first quantitative description of the relationship between deregulated miRNA expression and survival in malignant glioma on July 9, 2020. © 2010 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on July 2, 2010; DOI: 10.1158/1078-0432.CCR-10-0207 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research patients. According to our validation results, miR -21, 196a, 196b, and 15b were found to be expressed at high levels in GBMs, and higher WHO grade gliomas tended to have higher expression levels of these miRNAs. We surveyed the relationship between the expression levels of these miRNAs and the overall survival rate in malignant glioma patients. In contrast with previous studies, which showed evidence that miR-21 overexpression was associated with poor survival and advanced tumor stage (9,13), there was no significant correlation between glioma patient survival and expression levels of miR-21 or miR-15b. In comparison, high miR-196 expression levels were significantly associated with poor survival in WHO grade III and IV glioma patients and even among GBM patients, as determined by Kaplan-Meier analysis. Multivariate analysis demonstrated that high expression level of miR-196 is an independent and significant predictor of poor prognosis in GBM patients.
Our data suggest that a high miR-196 expression level is a valuable marker for pathological diagnosis and prognosis prediction. Although miR-196 up-regulation has been reported in several types of human cancer cells, including glioblastoma cell lines (25,27,33), and its value for prognostic prediction has also been indicated in pancreatic cancer (10), little is known about the function of miR-196 in tumorigenesis. Luthra et al. provided evidence that increased levels of miR-196a stimulated cell proliferation and suppressed apoptosis in other cancer cell lines by regressing annexin A1 (ANXA1), a well-documented mediator of apoptosis and an inhibitor of cell proliferation (33). In contrast, Johnson et al has showed the increased ANXA1 expression in gliomas (46) suggesting that miR-196 may affect glioma cell behavior by another pathway. It should be noted that miR-196 is encoded at 3 paralogous locations in the A, B, and C mammalian HOX clusters and has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. HOX genes are a large family of regulatory genes implicated in the control of developmental processes, and they are involved in the malignant transformation and progression of several types of tumors (47), including gliomas (48). It has been reported that the miR-196 miRNA down-regulates HOXB8, HOXC8, HOXD8, and HOXA7 due to extensive complementarity at sites in the 3' untranslated regions (3' UTRs) of the HOX genes representing each cluster (49). Therefore, it will be important to test the effects of miR-196 knockdown on these HOX genes in glioma cell lines. Furthermore, functional analysis using gain-or loss-of-function strategies should be performed to validate the role of miR-196 in tumorigenesis and the malignant progression of glioblastomas.
In conclusion, we have provided large scale miRNA expression profiles of malignant gliomas. This study identified candidate miRNAs that may correlate with tumorigenesis and the malignant progression of gliomas and may be used to distinguish GBMs and AAs. We also on July 9, 2020. © 2010 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on July 2, 2010; DOI: 10.1158/1078-0432.CCR-10-0207 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research showed that miRNA expression patterns reflect the biological and pathological characteristics of gliomas. Our results suggest that high miR-196 expression levels may play a role in the malignant progression of gliomas and may be a predictor of poor survival in glioblastomas. Fig.1