REVIEW ARTICLE

Journal of

New Advances of microRNAs in Glioma Stem Cells, With Special Emphasis on Aberrant Methylation of microRNAs BING ZHAO,1,2* ER-BAO BIAN,1,2 JIA LI,1,2

AND

Cellular Physiology

JUN LI3

1

Department of Neurosurgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, China

2

Cerebral Vascular Disease Research Center, Anhui Medical University, Hefei, China

3

School of Pharmacy, Anhui Medical University, Hefei, China

Malignant brain tumors are thought to be originate from a small population of cells that display stem cell properties, including the capacity of self-renewal, multipotent differentiation, initiation of tumor tissues. Cancer stem cells (CSCs) have been identified in gliomas in which they are named as glioma stem cells (GSCs). GSCs, sharing some characteristics with normal neural stem cells (NSCs), contribute to the cellular origin for primary gliomas and the recurrence of malignant gliomas after current conventional therapy. Recently, increasing evidences have showed that miRNAs play a central role in GSCs. In this review we focus on the role of GSCs in gliomas and in the abnomal expression of miRNAs in GSCs. Furthermore, we also discuss epigenetic dysregulation of tumor-suppressor miRNAs by promoter DNA methylation is involved in the regulation of GSCs biology. Recent advances in understanding dysregulated expression of miRNAs and methylation of tumor-suppressor miRNAs in GSCs and their possible use as new therapeutic targets of gliomas. J. Cell. Physiol. 229: 1141–1147, 2014. ß 2013 Wiley Periodicals, Inc.

Gliomas are the most common histological type of malignant brain tumor and they account for over 30% of all primary brain and central nervous system tumors (Dolecek et al., 2012). Gliomas are classified by the 2007 World Health Organization into four grades depending on their histological features (Gladson et al., 2010). Glioblastoma multiforme(GBM), known as Grade IV gliomas, are the most malignant and aggressive primary tumors of the brain and account for over a half of all gliomas (Kohler et al., 2011). GBMs are characterized by highly infiltrating nature, extensive invasion into the surrounding normal brain tissue, and associated with high recurrence rates secondary to inherent resistance to standard radiachemotherapies, with a 5-year survival of patients of no more than 10% (Kang and Kang, 2007; Stupp et al., 2009). GBMs are often found at an advanced stage, and therefore there is little understanding as to the early cellular origins of GBMs. Recent research on the cell of origin for these tumors suggests that GBMs originate from CSCs, termed glioma stem cells (GSCs), as opposed to differentiated glial cells, as previously believed (Dai et al., 2001; Bachoo et al., 2002; Persano et al., 2013). GSCs are highly infiltrative and possess stem-like characteristics similar with normal neural stem cells (NSCs), including the expression of neural stem cell markers, the capacity for self-renewal and long-term proliferation, the formation of neurospheres and the ability to differentiate into multiple nervous system lineages (Galli et al., 2004; Singh et al., 2004; Yuan et al., 2004). Recently, several groups have identified GSCs in samples from patients with gliomas, and a small number of GSCs with resistances against conventional chemotherapy and radiotherapy are sufficient to cause recurrent tumors (Singh et al., 2003; Lee et al., 2006; Panagiotakos and Tabar, 2007; Kong et al., 2013). Therefore, targeting GSCs may offer a new avenue of therapeutic intervention for gliomas. Recently, several studies have demonstrated that microRNAs (miRNAs) have been implicated in gliomas initiation, progression, metastasis, and, importantly, in the formation and maintenance of GSCs (Huang et al., 2010; Zhang ß 2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .

et al., 2012; Wang et al., 2013). Dysregulation of miRNAs in GSCs have been observed and several miRNAs have been elucidated to regulate GSC functions, which demonstrate their close association with gliomas. Such regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for gliomas by virtue of their ability to regulate gliomas initiation, progression and metastasis. Herein we review the current understanding and recent advance of miRNAs involved in the control of GSCs functions and

Abbreviations: GBM, glioblastoma multiforme; GSCs, glioma stem cells; NSCs, normal neural stem cells; miRNAs, microRNAs; CSC, cancer stem cell; CSCs, cancer stem cells; pri-miRNAs, primary transcripts; DGCR8, DiGeorge syndrome critical region gene 8; RISC, RNA-induced silencing complex; UTR, untranslated region; CSMD1, CUB and SUSHI multiple domain; TSGs, tumor suppressor genes; HXK2, hexokinase 2; EGFR, epithelial growth factor receptor; PDGFR, platelet-derived growth factor receptor-a; H3K27me3, histone H3 lysine 27 trimethylation; SAM, S-adenosylL-methionine; 5-azadC, 5-aza-20 -deoxycytidine; CTGF, connective tissue growth factor; OSCC, oral squamous cell carcinoma. Bing Zhao and Er-Bao Bian contributed equally to this work. Conflicts of interest: none. Contract grant sponsor: National Science Foundation of China; Contract grant number: 81072066. *Correspondence to: Bing Zhao, Department of Neurosurgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province, China 230601; Cerebral Vascular Disease Research Center, Anhui Medical University, 678 Fu Rong Road, Hefei, Anhui Province, China 230601. E-mail: [email protected] Manuscript Received: 7 December 2013 Manuscript Accepted: 12 December 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 20 December 2013. DOI: 10.1002/jcp.24540

1141

1142

ZHAO ET AL.

methylation of miRNAs in GSCs, which will broaden our understanding of the regulatory mechanisms of GSCs and may contribute significantly to the gliomas therapy. Cancer Stem Cells and Glioma Stem Cells

The cancer stem cell (CSC) hypothesis has provided an alternative framework for understanding cancer heterogeneity, tumorigenesis and cancer progression (Xu et al., 2012). Indeed, the first evidence for the existence of a small subpopulation of cancer stem cells (CSCs) was reported in acute myeloid leukemia (Lapidot et al., 1994). Soon after, researchers suspected that such insights might also apply to solid tumors and found the existence of CSCs in a number of solid organ malignancies such as breast, pancreas, head and neck, colon, and brain (Bonnet and Dick, 1997; Esposito et al., 2002; Al-Hajj et al., 2003; Galli et al., 2004; Prince et al., 2007; Ricci-Vitiani et al., 2007). CSCs are identified as only a distinct population of tumor cells with stem cell characteristics, such as asymmetric cell division, unlimited growth, and multipotency, that later differentiate into rapidly proliferating progenitor-like and more differentiated cells, defining the histopathology of the tumor (Clarke et al., 2006). Recently several studies have reported that CSCs play central roles tumor initiation, proliferation, progression, invasion, metastasis, recurrence, and resistance to therapies (Croker and Allan, 2008). Ignatova et al. (2002) were the first to describe that stem-like cells, isolated clonogenic, neurosphereforming precursors from surgery specimens of human GBM, exist in human cortical glial tumors. More recently, a number of studies have demonstrated that GSCs, glioma tumor initiating cells and brain tumor stem-like cells, shared characteristics with normal stem cells, especially adult somatic stem cells, including the capacities for self-renewal, differentiation and maintained proliferation (Marsden et al., 2009; Tabatabai and Weller, 2011). GSCs expressed neuronal markers on differentiation, with several key determinants of NSCs fate, that can be often used for their identification. NSCs markers such as CD133, nestin, Oct4, SOX2, Bmi1, Musashi, CD44, CD15, or ABC transporter proteins have been widely used for the identification and isolation of GSCs (Ignatova et al., 2002; Hemmati et al., 2003; Yao et al., 2011). Some of these markers may not be fully specific for GSCs, but in all cases, they has been used as a marker for enrichment of GSC population from GBM tumors or xenografts, which are more tumorigenic than the rest of the tumor cells (Beier et al., 2007). Overall, controversy persists in relation to the functional significance of GSC based on their frequency, propagation rate, and correlation between tumorigenicity and differently defined stem cell markers (Binello and Germano, 2011). However, accumulated evidences have support the pivotal role of GSCs in gliomas. Currently, a significant number of studies have been devoted to unraveling the mechanisms of action behind GSCs, generating numbers of potential targets. Overview of microRNA

miRNAs represent a class of small non-coding, single-stranded RNA molecules containing 19–25 nucleotides (nt) (Bartel, 2004). miRNAs are capable of regulating gene expression through the sequence-selective targeting of mRNAs, resulting in translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences (Krol et al., 2010; Kozomara and Griffiths-Jones, 2011) (Fig. 1). Over 1,500 miRNAs have been identified in animal genomes and each has the potential to target a number of different mRNAs. In addition, each mRNA can conceivably be controlled by several miRNAs (Wilson and Huys, 2013). Thus, miRNAs consist of a complex regulatory JOURNAL OF CELLULAR PHYSIOLOGY

network that regulates every cellular process, including cell proliferation, apoptosis, differentiation, development, and tumorigenesis (Macfarlane and Murphy, 2010; Lopez-Serra and Esteller, 2012). miRNAs are generated by multiple steps, starting in the nucleus and continuing in the cytoplasm. At first, miRNAs are transcribed into long primary transcripts (primiRNAs) that is typically transcribed from the genome by RNAPolymerase II (RNApolII) and is subsequently capped and polyadenylated (Kim, 2005). The pri-miRNA is further processed by the RNA polymerase III Drosha and its cofactor Pasha which comprise the DiGeorge syndrome critical region gene 8 (DGCR8) complex to generate 70–100-nucleotide long pre-miRNAs that have a hairpin structure with a 30 -overhang (Cho, 2007; Bartel, 2009). Pre-miRNAs are then exported out of the nucleus by expotin-5 into the cytosol where pre-miRNA undergo final cleavage into 22 nt, double-stranded, mature miRNA: miRNA star duplex by the RNase III nuclease Dicer. Finally, the mature single-stranded miRNA, or guide strand, is preferentially incorporated into an enzyme complex named RNA-induced silencing complex (RISC), which composed of Dicer, TRBP, and a protein of the Argonaute superfamily (Hansen and Obrietan, 2013). In contrast, the other strand which is called the passenger strand is usually degraded. Within this complex, miRNAs bind to targets by complementary base pairing within the 30 untranslated region (UTR) of mRNA (Bartel, 2004). Dysregulated microRNA in Glioma Stem Cells

There are rapidly accumulating evidences implicating important roles of miRNAs in the regulation of pathological processes of gliomas. In addition, recent work in literatures demonstrated that the dysregulation of miRNAs has been involved in the regulation of GSCs biology (Fig. 2). Furthermore, of these dysregulated miRNAs, some were found to be involved in the regulation of including proliferation, cell cycle, apoptosis, invasion, migration, self-renewal, differentiation of GSC. Recent studies found that upregulated expression of miRNAs, including miR-10a, miR-10b, miR-9, miR-17, and miR106, was identified as oncogenic miRNAs, leading to enhanced invasion, migration, and self-renewal and reduced apoptosis and differentiation of GSC. Lang et al. (2012) demonstrated that the upregulation of miR-10a and miR-10b expression in GSCs repressed the expression of CUB and SUSHI multiple domain (CSMD1), a tumor suppressor gene, through the predicted targeting sites in CSMD1 30 UTR. Guessous et al. also demonstrated that the upregulated expression of miR-10b was observed in GSCs. Inhibition of miR-10b with a specific antagomir inhibited the proliferation and reduced invasion and migration of GSCs while overexpression of miR-10b induced cell migration and invasion. In addition, miR-10b inhibition significantly decreased the in vivo growth of stem cell-derived orthotopic GBM xenografts (Guessous et al., 2013). miR-9, miR-17, and miR-106b were highly abundant in CD133 positive cells. Inhibition of miR-9 or miR-17 resulted in reduced neurosphere formation and stimulated cell differentiation by targeting Calmodulin-binding transcription activator 1 (CAMTA1). Enforced expression of CAMTA1, a putative transcription factor, led to reduced neurosphere formation and tumor growth in nude mice, suggesting that CAMTA1 can function as tumor suppressor. In addition, CAMTA1 expression was correlated with patient survival (Schraivogel et al., 2011). These findings suggest that the upregulated expression of miRNAs, serving as key regulators of oncogenes, targets tumor suppressor genes (TSGs), which is associated with GSCs biology. However, numbers of downregulated expressions of miRNAs were found in GSCs. For example, Zhao et al. showed

miRNA IN GLIOMA STEM CELLS

Fig. 1.

Overview of miRNA of biogenesis and funciton.

that miR-143 was significantly down-regulated in glioma tissues and GSCs, while miR-143 over-expression inhibited glycolysis by directly targeting hexokinase 2(HXK2), and promoted differentiation of GSCs. Moreover, miR-143 inhibited proliferation of GSCs under hypoxic conditions and decreased tumor formation capacity of GSCs in vivo (Zhao et al., 2013b). More importantly, the low expression of miR-143 in GBM was correlated with poor patient prognosis (Lee et al., 2013a). These results suggest that miR-143 may be a better prognostic factor and potential target of treatment of gliomas. In addition, miR-125b was downregulated in human U251 glioma stem cells. Inhibition of miR-125b-induced U251 glioma stem cell proliferation was due to cell cycle arrest at the G1/S transition and involved the cell cycle regulated proteins CDK6 and CDC25A, whereas miR-125b overexpression decreased CDK6 and CDC25A expression (Shi et al., 2010). Wu et al. reported that miR-125b is significantly down-regulated in CD133 positive GSCs, whereas enforced expression of miR125b inhibits the proliferation of CD133 positive GSCs and reduces the expression of stem marker. Furthermore, two binding sites for miR-125b were identified in the 30 UTR of E2F2 and overexpression of miR-125b in CD133 positive GSCs repressed the endogenous level of E2F2 protein (Wu et al., 2012). Shi et al. reported that miR-125b had no effects on the invasion of primary glioblastoma CD133-negative cells but that the upregulation or downregulation of miR-125b could inhibit and promote the invasion of corresponding CD133 positive cells, respectively. Further research into the underJOURNAL OF CELLULAR PHYSIOLOGY

lying mechanism demonstrated that the effects of miR-125b on the invasion of glioblastoma CD133 positive cells were related to the change of the expression of MMPs (MMP-2 and MMP-9) and corresponding inhibitors (RECK and TIMP3) (Shi et al., 2012). Zhao et al. report that miR-153 expression was down-regulated in GBM tissues relative to normal brain tissues, and in CD133 positive cells relative to CD133 negative cells. Transient transfection of miR-153 into GSCs can inhibit their stemness properties, such as impairing self-renewal ability and inducing differentiation. Meanwhile, miR-153 can also repress GSCs growth and induce apoptosis (Zhao et al., 2013a). miR-124 is down-regulated in glioma tissues, human glioma cell lines and GSCs (Silber et al., 2008; Lang et al., 2012; Xia et al., 2012; Wei et al., 2013). The delivered miR-124 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs (Lee et al., 2013b). Three oncogenes, NRAS, PIM3, and SNAI2, as downstream targets of miR-124, which has been shown to regulate stem cell functions and is often up-regulated in glioma (Lang et al., 2012; Xia et al., 2012). overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133 positive cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo (Xia et al., 2012). Silber et al. showed that

1143

1144

ZHAO ET AL.

Fig. 2.

Overview of dysregulation miRNAs regulation of GSCs by targeting, directly or indirectly, tumor suppressor genes or oncogenes.

transfection of miR-124 induced G1 cell cycle arrest in glioma cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins. Transfection of miR-124 also induced morphological changes and differentiation of CD133 positive cells (Silber et al., 2008). These findings suggest that miRNAs play an important role in CD133 positive cells from gliomas. A recent study demonstrated that miR-34a expression was down-regulated in GBM tissues as compared with normal brain and in mutant p53 gliomas as compared with wild-type p53 gliomas. Enforced expression of miR-34a in glioma cell lines strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion (Li et al., 2009b). miR-34a expression also inhibited various malignancy endpoints in GSCs and induces GSCs differentiation (Guessous et al., 2010). In addition, miR-34a expression inhibited c-Met reporter activities in glioma cells and Notch-1 and Notch-2 30 -untranslated region reporter activities in glioma cells and stem cells. Overexpression of c-Met or Notch-1/Notch-2 transcripts lacking the 30 -untranslated region sequences partially reversed the effects of miR-34a on cell cycle arrest and cell death in glioma cells and stem cells, respectively. Also, transient expression of miR-34a in glioblastoma cells strongly inhibited in vivo glioma xenograft growth (Li et al., 2009b). Mei et al. demonstrated that overexpression of miR-146a inhibited the glioma development of a human GBM cell line in an orthotopic xenograft model. Such an inhibitory function of miR-146a on gliomas was largely through downregulation of Notch1, which plays a key role in neural stem cell maintenance and is a direct target of miR-146a. Accordingly, miR-146a modulated neural JOURNAL OF CELLULAR PHYSIOLOGY

stem cell proliferation and differentiation and reduced migration of GSCs. Conversely, knockdown of miR-146a upregulates Notch1 and promotes tumorigenesis of malignant astrocytes (Mei et al., 2011). Chen et al. showed that miR-107 was down-regulated in GSCs, whereas over-expression of miR-107 suppressed proliferation and down-regulated Notch2 protein and stem cell marker (CD133 and Nestin) expression in GSCs. Furthermore, enhanced miR-107 expression significantly inhibited GSCs invasion and reduced MMP-12 expression. miR-107 also suppressed GSCs xenograft growth in vivo (Chen et al., 2013). Tu et al. demonstrated that restoring the expression of miR-218, a miRNA commonly downregulated in glioma, dramatically reduced the migration, invasion, and proliferation of glioma cells. Expression of the stem cellpromoting oncogene Bmi1, known as a functional downstream target of miR-218, was decreased after overexpression of miR218 in glioma cells. MiR-218 also blocked the self-renewal of GSCs, suggesting that miR-218 regulated stem cell growth by targeting the GSCs marker Bmi1 (Tu et al., 2013). These results suggest that miRNAs are involved in regulation of gliomas though targeting the key molecular of GSCs. miR-128 was strongly down-regulated in GBM and decreased expression of miR-128 correlates with aggressive human glioma subtypes. miR-128 repressed GSCs growth and mediated differentiation by targeting oncogenic receptor tyrosine kinases epithelial growth factor receptor (EGFR) and platelet-derived growth factor receptor-a (PDGFR) (Godlewski et al., 2008). In addition, miR-128 negatively regulated the expression of Bmi-1, a neural stem cell and glioma maintenance factor, directly through its predicted miR-128

1145

miRNA IN GLIOMA STEM CELLS

binding site. Overexpression of miR-128 caused a decrease in histone H3 lysine 27 trimethylation (H3K27me3) and Akt phosphorylation, and up-regulation of p21 levels, consistent with Bmi-1 down-regulation (Godlewski et al., 2008). In addition to Bmi-1, a component of PRC1, miR-128 directly targeted mRNA of SUZ12, a key component of PRC2, This blocked the partially redundant functions of PRC1/PRC2, thereby significantly reducing PRC activity and its associated histone modifications. Moreover, knockdown of miR-128 expression in nonmalignant mouse and human NSCs led to elevated expression of PRC components and increased clonogenicity (Peruzzi et al., 2013). Epigenetic analysis revealed that gain of H3K27me3 mediated by PRC2 in the primary miR1275 promoter was closely associated with miR-1275 expression (Katsushima et al., 2012). miR-1275 was consistently down-regulated during GSC differentiation, along with the up-regulation of its target, CLDN11, an important protein during oligodendroglial lineage differentiation. Inhibition of miR-1275 in GSCs increased the expression of CLDN11, together with significant growth suppression. Treatment with 3-deazaneplanocin A, an inhibitor of H3K27 methyltransferase, attenuated CLDN11 induction by serum stimulation in parallel with sustained miR-1275 expression. Given that inhibition of miR-1275 induces expression of oligodendroglial lineage proteins and suppresses tumor cell proliferation, this may be a potential therapeutic target for GBM. These findings suggest that epigenetic modification controlling of miRNAs expression is involved in the pathology of gliomas.

receptor, apoptosis, and angiogenesis (Widschwendter and Jones, 2002). Methyltransferase inhibitors such as 5-aza-20 deoxycytidine (5-azadC) is incorporated into RNA and, to a lesser extent, into DNA strands. The incorporated 5-azadC disrupts the interaction between DNA and DNMTs through nitrogen instead of carbon, in the 5-position of the modified pyrimidine, which finally promotes its proteosomal degradation (Santi et al., 1984; Stresemann and Lyko, 2008). Therefore, 5-azadC can be utilized to reverse the effects of methylation, including the reduction of mutations at methylated CpG sites, reactivation of genes suppressed by hypermethylation (Bender et al., 1998; Li et al., 2009a). Downregulation of miRNAs expression in gliomas may be regulated by diverse mechanisms, including DNA methylation, histone modification, or post-transcriptional process (Burgess et al., 2008; Deng et al., 2008; Lee and Vasudevan, 2013). Of these, DNA methylation is related to repression of miRNAs possessing promoter-associated CpG islands. In addition, the expressions and functions of these tumor-suppressor miRNAs can be reversed and restored by DNA hypomethylation treatment (Liu et al., 2013). Therefore, a better understanding of DNA methylation of tumor-suppressor miRNAs is pivotal for the treatment of gliomas. Recently, increasing studies have described the role of DNA methylation of tumor-suppressor miRNAs, including miR-211, miR-204, miR-23b, miR-145, and miR-137, in GSCs (Table 1).

Overview of DNA Methylation

miR-211, a miRNA predicted to target MMP-9, suppression in GBM involved aberrant methylation-mediated epigenetic silencing of the miR-211 promoter. Additionally, shRNA specific for MMP-9 promoted miR-211 expression via demethylation of miR-211 promoter-associated CpG islands. miR-211 overexpression led to the activation of the intrinsic mitochondrial/Caspase-9/3-mediated apoptotic pathway in both glioma cells and CSCs (Asuthkar et al., 2012). Ying et al. demonstrated that miR-204 was markedly downregulated in glioma and NSCs, miR-204 simultaneously suppressed selfrenewal, stem cell-associated phenotype, and migration of glioma cells by targeting the stemness-governing transcriptional factor SOX4 and the migration-promoting receptor EphB2. Restoring miR-204 expression in glioma cells suppressed tumorigenesis and invasiveness in vivo and increased overall host survival. Further evaluation revealed that the miR204 promoter was hypermethylated and that attenuating promoter methylation was sufficient to upregulate miR-204 in glioma cells (Ying et al., 2013). miR-23b was frequently methylated in GSCs but not in parallel U87 cells. Meanwhile, miR-23b expression was also markedly reduced in GSCs compared with matching U87 cells. However, treatment with 5-azadC increased the expression of miR-23b in GSCs. In addition, ectopic expression of miR-23b in GSCs induced cell cycle arrest and proliferation inhibition (Geng et al., 2012). These results suggest that miRNAs hypermethylation in GSCs is a central event in gliomas. Lee et al. showed that expression of miR-145 was downregulated in astrocytic tumors compared to normal brain

DNA methylation occurs by the addition of a methyl group from S-adenosyl-L-methionine (SAM) substrates to the cytosine catalyzed by DNA (cytosine-5)-methyltransferases, resulting in 5-methylcytosine (Yu et al., 2011). Currently, three DNA methyltransferases have functional enzymatic activity in mammals, namely, DNMT1, DNMT3A, and DNMT3B (Bestor, 2000; Rottach et al., 2009). DNMT3A and DNMT3B fall in the group of de novo methyltransferases enzymes that are able to methylate previously unmethylated CpG sequences, while the most abundant DNMT1, which has a higher affinity for hemimethylated DNA, is responsible for propagation and maintenance of established DNA methylation patterns (Okano et al., 1999; Baylin and Herman, 2000). The aberrant DNA methylation, for example hypomethylation and hypermethylation, is co-operatively regulated by these enzymes. Hypomethylation associated with early stage genetic instability and up-regulation of gene expression can occur at normally methylated DNA sequences as repeated sequences, as well as both encoding regions and introns of genes (Kisseljova and Kisseljov, 2005; Wilson et al., 2007; Esteller, 2008). Another form of aberrant DNA methylation, CpG islands hypermethylation in the promoter region of specific genes, is found in numbers of sites in carcinogenesis. Promoter hypermethylation is related to gene transcriptional repression, and can affect carcinogenesis particularly when the affected genes are tumor suppressor genes or other genes involved in the cell cycle, DNA mismatch repair, cell-to-cell interaction, steroid

Aberrant Methylation of miRNA in Glioma Stem Cells

TABLE 1. Methylation of miRNAs in GSCs miRNAs

Gene expression

Targets

Biological process

References

miR-211 miR-204 miR-23b miR-145 miR-137

Downregulation Downregulation Downregulation Downregulation Downregulation

MMP9 SOX4, EphB2 — CTGF, SCP-1, SOX2 RTVP-1

Apoptotic Migration, invasiveness self-renewal Cell cycle, proliferation Migration, invasion, adhesion Differentiation, self-renewal

Asuthkar et al., 2012 Ying et al., 2013 Geng et al., 2012 Lee et al., 2013a; Lee et al., 2013b Bier et al., 2013

JOURNAL OF CELLULAR PHYSIOLOGY

1146

ZHAO ET AL.

specimens and in glioma cells and GSCs compared to normal astrocytes and NSCs. Moreover, the low expression of miR145 in GBM was correlated with poor patient prognosis. Overexpression of miR-145 significantly decreased the migration and invasion of glioma cells, and the expression of connective tissue growth factor (CTGF), as a novel target of miR-145 (Lee et al., 2013a). Speranza et al. reported that intracranial injection of glioblastoma neurospheres overexpressing miR-145 delays significantly tumor development: deriving tumors showed a significant down-regulation of NEDD9. Moreover, a significant inhibition of invasion in silencing experiments with GB-NS shNEDD9, and an upregulation of miR-145 in shNEDD9 (Speranza et al., 2012). Furthermore, the delivered miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs (Lee et al., 2013b). Most importantly, miR-145 had a tumor-suppressive function in glioblastoma in that it reduced proliferation, adhesion, and invasion of glioblastoma cells, apparently by suppressing the activity of oncogenic proteins Sox9 and ADD3. Reduced levels of miR-145 resulted in neoplastic transformation and malignant progression in glioma due to unregulated activity of these proteins. The CpG islands hypermethylation of miR-145 promoter was observed in glioma cells. 5-azadC, a potent inhibitor of DNA methyltransferases, treatment reversed the repression of miR-145 in glioma cells (Rani et al., 2013). These findings suggest that the expression of miR-145 mediated by DNA methylation in GSCs may provide molecular mechanisms for gliomas. Epigenetic inactivation of the tumor-suppressor miR-137 was reported in oral squamous cell carcinoma, colorectal carcinogenesis, squamous cell carcinoma, gastric cancer, and gliomas (Balaguer et al., 2010; Chen et al., 2011; Langevin et al., 2011; Bier et al., 2013). The expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and NSCs. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2, and Shh. Transfection of cells with miR-137 decreased the expression of RTVP-1, a novel target of miR-137 in GSCs, and the luciferase activity of RTVP-1 30 -UTR reporter plasmid. The promoter hypermethylation of miR-137 was observed in the GBM specimens (Bier et al., 2013). In primary tumors of oral squamous cell carcinoma (OSCC) with paired normal oral mucosa, down-regulation of miRNA expression through tumor-specific hypermethylation was more frequently observed for miR-137. The expression of miR-137 was restored by treatment with 5-azadC in OSCC cells lacking miR-137 expression (Kozaki et al., 2008). These results suggest that the loss of miR-137 due to hypermethylation may be involved in regulation of GSCs. Conclusion and Future Perspectives

In this review, we summarize our current view of the biology of miRNAs and the role of miRNAs in GSCs. miRNAs are frequently de-regulated by genetic and this dysregulated expression involves in gliomagenesis by functioning as TSGs or interfering with the expression and function of TSGs or oncogenes. These findings suggest the potential use of tumorsuppressor miRNA mimics as a cancer therapy in tumors lacking certain critical tumor-suppressor miRNAs. Recently, Tsuruta et al. (2011) reported that dsRNA mimicking miR-152 administered with atelocollagen to SCID mice could suppress the in vivo growth of an endometrial cancer cell line, which result in us to consider the possibility of miRNA replacement JOURNAL OF CELLULAR PHYSIOLOGY

therapy for cancer. The discovery and understanding of the biology of miRNAs has unveiled a new level of gene regulation that certainly offers new promise for the treatment of gliomas. These miRNAs, which are embedded in a CpG island and keep epigenetically silenced by promoter hypermethylation based on DNA methylation can re-express when treated with epigenetic drugs (Karpf and Jones, 2002). For instance, pharmacological grade DNA methyltransferase inhibitors such as 5-azadC have been approved for the treatment of myelodysplastic syndrome (Kuendgen and Lubbert, 2008). Therefore, these findings on methylation of tumor-suppressor miRNAs may provide a foundation for the use of epigenetic drugs in the treatment of gliomas. We foresee that the analysis of the methylation of miRNAs may lead to novel hypotheses about pathogenesis and may identify targets of research and hopefully even therapies. Literature Cited Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. 2003. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988. Asuthkar S, Velpula KK, Chetty C, Gorantla B, Rao JS. 2012. Epigenetic regulation of miRNA211 by MMP-9 governs glioma cell apoptosis, chemosensitivity and radiosensitivity. Oncotarget 3:1439–1454. Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, Tang Y, DeFrances J, Stover E, Weissleder R, Rowitch DH, Louis DN, DePinho RA. 2002. Epidermal growth factor receptor and Ink4a/Arf: Convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 1:269–277. Balaguer F, Link A, Lozano JJ, Cuatrecasas M, Nagasaka T, Boland CR, Goel A. 2010. Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis. Cancer Res 70:6609–6618. Bartel DP. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116:281–297. Bartel DP. 2009. MicroRNAs: Target recognition and regulatory functions. Cell 136:215– 233. Baylin SB, Herman JG. 2000. DNA hypermethylation in tumorigenesis: Epigenetics joins genetics. Trends Genet 16:168–174. Beier D, Hau P, Proescholdt M, Lohmeier A, Wischhusen J, Oefner PJ, Aigner L, Brawanski A, Bogdahn U, Beier CP. 2007. CD133(þ) and CD133() glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67:4010– 4015. Bender CM, Zingg JM, Jones PA. 1998. DNA methylation as a target for drug design. Pharm Res 15:175–187. Bestor TH. 2000. The DNA methyltransferases of mammals. Hum Mol Genet 9:2395–2402. Bier A, Giladi N, Kronfeld N, Lee HK, Cazacu S, Finniss S, Xiang C, Poisson L, de Carvalho AC, Slavin S, Jacoby E, Yalon M, Toren A, Mikkelsen T, Brodie C. 2013. MicroRNA-137 is downregulated in glioblastoma and inhibits the stemness of glioma stem cells by targeting RTVP-1. Oncotarget 4:665–676. Binello E, Germano IM. 2011. Targeting glioma stem cells: A novel framework for brain tumors. Cancer Sci 102:1958–1966. Bonnet D, Dick JE. 1997. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737. Burgess R, Jenkins R, Zhang Z. 2008. Epigenetic changes in gliomas. Cancer Biol Ther 7:1326– 1334. Chen L, Chen XR, Chen FF, Liu Y, Li P, Zhang R, Yan K, Yi YJ, Xu ZM, Jiang XD. 2013. MicroRNA-107 inhibits U87 glioma stem cells growth and invasion. Cell Mol Neurobiol 33:651–657. Chen Q, Chen X, Zhang M, Fan Q, Luo S, Cao X. 2011. miR-137 is frequently downregulated in gastric cancer and is a negative regulator of Cdc42. Digest Dis Sci 56:2009– 2016. Cho WC. 2007. OncomiRs: The discovery and progress of microRNAs in cancers. Mol Cancer 6:60. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM. 2006. Cancer stem cells—Perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344. Croker AK, Allan AL. 2008. Cancer stem cells: Implications for the progression and treatment of metastatic disease. J Cell Mol Med 12:374–390. Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC. 2001. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev 15:1913– 1925. Deng S, Calin GA, Croce CM, Coukos G, Zhang L. 2008. Mechanisms of microRNA deregulation in human cancer. Cell Cycle 7:2643–2646. Dolecek TA, Propp JM, Stroup NE, Kruchko C. 2012. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neuro Oncol 14:v1–49. Esposito I, Kleeff J, Bischoff SC, Fischer L, Collecchi P, Iorio M, Bevilacqua G, Buchler MW, Friess H. 2002. The stem cell factor-c-kit system and mast cells in human pancreatic cancer. Lab Invest 82:1481–1492. Esteller M. 2008. Epigenetics in cancer. N Engl J Med 358:1148–1159. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A. 2004. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021. Geng J, Luo H, Pu Y, Zhou Z, Wu X, Xu W, Yang Z. 2012. Methylation mediated silencing of miR-23b expression and its role in glioma stem cells. Neurosci Lett 528:185–189. Gladson CL, Prayson RA, Liu WM. 2010. The pathobiology of glioma tumors. Annu Rev Pathol 5:33–50. Godlewski J, Nowicki MO, Bronisz A, Williams S, Otsuki A, Nuovo G, Raychaudhury A, Newton HB, Chiocca EA, Lawler S. 2008. Targeting of the Bmi-1 oncogene/stem cell

miRNA IN GLIOMA STEM CELLS

renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res 68:9125–9130. Guessous F, Alvarado-Velez M, Marcinkiewicz L, Zhang Y, Kim J, Heister S, Kefas B, Godlewski J, Schiff D, Purow B, Abounader R. 2013. Oncogenic effects of miR-10b in glioblastoma stem cells. J Neurooncol 112:153–163. Guessous F, Zhang Y, Kofman A, Catania A, Li Y, Schiff D, Purow B, Abounader R. 2010. microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle 9:1031–1036. Hansen KF, Obrietan K. 2013. microRNA as therapeutic targets for treatment of depression. Neuropsychiatr Dis Treat 9:1011–1021. Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI. 2003. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci USA 100:15178–15183. Huang Z, Cheng L, Guryanova OA, Wu Q, Bao S. 2010. Cancer stem cells in glioblastoma— Molecular signaling and therapeutic targeting. Protein Cell 1:638–655. Ignatova TN, Kukekov VG, Laywell ED, Suslov ON, Vrionis FD, Steindler DA. 2002. Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro. Glia 39:193–206. Kang MK, Kang SK. 2007. Tumorigenesis of chemotherapeutic drug-resistant cancer stemlike cells in brain glioma. Stem Cells Dev 16:837–847. Karpf AR, Jones DA. 2002. Reactivating the expression of methylation silenced genes in human cancer. Oncogene 21:5496–5503. Katsushima K, Shinjo K, Natsume A, Ohka F, Fujii M, Osada H, Sekido Y, Kondo Y. 2012. Contribution of microRNA-1275 to Claudin11 protein suppression via a polycombmediated silencing mechanism in human glioma stem-like cells. J Biol Chem 287:27396– 27406. Kim VN. 2005. MicroRNA biogenesis: Coordinated cropping and dicing. Nat Rev Mol Cell Biol 6:376–385. Kisseljova NP, Kisseljov FL. 2005. DNA demethylation and carcinogenesis. Biochemistry 70:743–752. Kohler BA, Ward E, McCarthy BJ, Schymura MJ, Ries LA, Eheman C, Jemal A, Anderson RN, Ajani UA, Edwards BK. 2011. Annual report to the nation on the status of cancer, 1975– 2007, featuring tumors of the brain and other nervous system. J Natl Cancer Inst 103:714– 736. Kong BH, Park NR, Shim JK, Kim BK, Shin HJ, Lee JH, Huh YM, Lee SJ, Kim SH, Kim EH, Park EK, Chang JH, Kim DS, Kim SH, Hong YK, Kang SG, Lang FF. 2013. Isolation of glioma cancer stem cells in relation to histological grades in glioma specimens. Childs Nerv Syst 29:217–229. Kozaki K, Imoto I, Mogi S, Omura K, Inazawa J. 2008. Exploration of tumor-suppressive microRNAs silenced by DNA hypermethylation in oral cancer. Cancer Res 68:2094– 2105. Kozomara A, Griffiths-Jones S. 2011. miRBase: Integrating microRNA annotation and deepsequencing data. Nucleic Acids Res 39:D152–D157. Krol J, Loedige I, Filipowicz W. 2010. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597–610. Kuendgen A, Lubbert M. 2008. Current status of epigenetic treatment in myelodysplastic syndromes. Ann Hematol 87:601–611. Lang MF, Yang S, Zhao C, Sun G, Murai K, Wu X, Wang J, Gao H, Brown CE, Liu X, Zhou J, Peng L, Rossi JJ, Shi Y. 2012. Genome-wide profiling identified a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. PloS ONE 7:e36248. Langevin SM, Stone RA, Bunker CH, Lyons-Weiler MA, LaFramboise WA, Kelly L, Seethala RR, Grandis JR, Sobol RW, Taioli E. 2011. MicroRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck. Cancer 117:1454–1462. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. 1994. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645–648. Lee HK, Bier A, Cazacu S, Finniss S, Xiang C, Twito H, Poisson LM, Mikkelsen T, Slavin S, Jacoby E, Yalon M, Toren A, Rempel SA, Brodie C. 2013a. MicroRNA-145 is downregulated in glial tumors and regulates glioma cell migration by targeting connective tissue growth factor. PloS ONE 8:e54652. Lee HK, Finniss S, Cazacu S, Bucris E, Ziv-Av A, Xiang C, Bobbitt K, Rempel SA, Hasselbach L, Mikkelsen T, Slavin S, Brodie C. 2013b. Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal. Oncotarget 4:346–361. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W, Park JK, Fine HA. 2006. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391–403. Lee S, Vasudevan S. 2013. Post-transcriptional stimulation of gene expression by microRNAs. Adv Exp Med Biol 768:97–126. Li Q, Bartlett DL, Gorry MC, O’Malley ME, Guo ZS. 2009a. Three epigenetic drugs upregulate homeobox gene Rhox5 in cancer cells through overlapping and distinct molecular mechanisms. Mol Pharmacol 76:1072–1081. Li Y, Guessous F, Zhang Y, Dipierro C, Kefas B, Johnson E, Marcinkiewicz L, Jiang J, Yang Y, Schmittgen TD, Lopes B, Schiff D, Purow B, Abounader R. 2009b. MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res 69:7569–7576. Liu SV, Fabbri M, Gitlitz BJ, Laird-Offringa IA. 2013. Epigenetic therapy in lung cancer. Front Oncol 3:135. Lopez-Serra P, Esteller M. 2012. DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer. Oncogene 31:1609–1622. Macfarlane LA, Murphy PR. 2010. MicroRNA: Biogenesis, function and role in cancer. Curr Genomics 11:537–561. Marsden CG, Wright MJ, Pochampally R, Rowan BG. 2009. Breast tumor-initiating cells isolated from patient core biopsies for study of hormone action. Methods Mol Biol 590:363–375. Mei J, Bachoo R, Zhang CL. 2011. MicroRNA-146a inhibits glioma development by targeting Notch1. Mol Cell Biol 31:3584–3592. Okano M, Bell DW, Haber DA, Li E. 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257. Panagiotakos G, Tabar V. 2007. Brain tumor stem cells. Curr Neurol Neurosci Rep 7:215– 220. Persano L, Rampazzo E, Basso G, Viola G. 2013. Glioblastoma cancer stem cells: Role of the microenvironment and therapeutic targeting. Biochem Pharmacol 85:612–622.

JOURNAL OF CELLULAR PHYSIOLOGY

Peruzzi P, Bronisz A, Nowicki MO, Wang Y, Ogawa D, Price R, Nakano I, Kwon CH, Hayes J, Lawler SE, Ostrowski MC, Chiocca EA, Godlewski J. 2013. MicroRNA-128 coordinately targets Polycomb Repressor Complexes in glioma stem cells. Neuro Oncol 15:1212– 1224. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE. 2007. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 104:973– 978. Rani SB, Rathod SS, Karthik S, Kaur N, Muzumdar D, Shiras AS. 2013. MiR-145 functions as a tumor-suppressive RNA by targeting Sox9 and adducin 3 in human glioma cells. Neuro Oncol 15:1302–1316. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. 2007. Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115. Rottach A, Leonhardt H, Spada F. 2009. DNA methylation-mediated epigenetic control. J Cell Biochem 108:43–51. Santi DV, Norment A, Garrett CE. 1984. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci USA 81:6993– 6997. Schraivogel D, Weinmann L, Beier D, Tabatabai G, Eichner A, Zhu JY, Anton M, Sixt M, Weller M, Beier CP, Meister G. 2011. CAMTA1 is a novel tumour suppressor regulated by miR-9/9 in glioblastoma stem cells. EMBO J 30:4309–4322. Shi L, Wan Y, Sun G, Gu X, Qian C, Yan W, Zhang S, Pan T, Wang Z, You Y. 2012. Functional differences of miR-125b on the invasion of primary glioblastoma CD133-negative cells and CD133-positive cells. Neuromol Med 14:303–316. Shi L, Zhang J, Pan T, Zhou J, Gong W, Liu N, Fu Z, You Y. 2010. MiR-125b is critical for the suppression of human U251 glioma stem cell proliferation. Brain Res 1312:120–126. Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M, Vandenberg SR, Ginzinger DG, James CD, Costello JF, Bergers G, Weiss WA, Alvarez-Buylla A, Hodgson JG. 2008. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med 6:14. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. 2003. Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. 2004. Identification of human brain tumour initiating cells. Nature 432: 396–401. Speranza MC, Frattini V, Pisati F, Kapetis D, Porrati P, Eoli M, Pellegatta S, Finocchiaro G. 2012. NEDD9, a novel target of miR-145, increases the invasiveness of glioblastoma. Oncotarget 3:723–734. Stresemann C, Lyko F. 2008. Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. Int J Cancer 123:8–13. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO, European Organisation for Research, Treatment of Cancer Brain Tumour, Radiation Oncology Group, National Cancer Institute of Canada Clinical Trials Group. 2009. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10:459–466. Tabatabai G, Weller M. 2011. Glioblastoma stem cells. Cell Tissue Res 343:459–465. Tsuruta T, Kozaki K, Uesugi A, Furuta M, Hirasawa A, Imoto I, Susumu N, Aoki D, Inazawa J. 2011. miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer. Cancer Res 71:6450–6462. Tu Y, Gao X, Li G, Fu H, Cui D, Liu H, Jin W, Zhang Y. 2013. MicroRNA-218 inhibits glioma invasion, migration, proliferation, and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1. Cancer Res 73:6046–6055. Wang W, Dai LX, Zhang S, Yang Y, Yan N, Fan P, Dai L, Tian HW, Cheng L, Zhang XM, Li C, Zhang JF, Xu F, Shi G, Chen XL, Du T, Li YM, Wei YQ, Deng HX. 2013. Regulation of epidermal growth factor receptor signaling by plasmid-based microRNA-7 inhibits human malignant gliomas growth and metastasis in vivo. Neoplasma 60:274–283. Wei J, Wang F, Kong LY, Xu S, Doucette T, Ferguson SD, Yang Y, McEnery K, Jethwa K, Gjyshi O, Qiao W, Levine NB, Lang FF, Rao G, Fuller GN, Calin GA, Heimberger AB. 2013. miR-124 inhibits STAT3 signaling to enhance T cell-mediated immune clearance of glioma. Cancer Res 73:3913–3926. Widschwendter M, Jones PA. 2002. DNA methylation and breast carcinogenesis. Oncogene 21:5462–5482. Wilson AS, Power BE, Molloy PL. 2007. DNA hypomethylation and human diseases. Biochim Biophys Acta 1775:138–162. Wilson JA, Huys A. 2013. miR-122 Promotion of the hepatitis C virus life cycle: Sound in the silence. Wiley Interdiscip Rev RNA 4:665–676. Wu N, Xiao L, Zhao X, Zhao J, Wang J, Wang F, Cao S, Lin X. 2012. miR-125b regulates the proliferation of glioblastoma stem cells by targeting E2F2. FEBS Lett 586:3831– 3839. Xia H, Cheung WK, Ng SS, Jiang X, Jiang S, Sze J, Leung GK, Lu G, Chan DT, Bian XW, Kung HF, Poon WS, Lin MC. 2012. Loss of brain-enriched miR-124 microRNA enhances stemlike traits and invasiveness of glioma cells. J Biol Chem 287:9962–9971. Xu Q, Yuan X, Yu JS. 2012. Glioma stem cell research for the development of immunotherapy. Adv Exp Med Biol 746:216–225. Yao XH, Liu Y, Chen K, Gong W, Liu MY, Bian XW, Wang JM. 2011. Chemoattractant receptors as pharmacological targets for elimination of glioma stem-like cells. Int Immunopharmacol 11:1961–1966. Ying Z, Li Y, Wu J, Zhu X, Yang Y, Tian H, Li W, Hu B, Cheng SY, Li M. 2013. Loss of miR-204 expression enhances glioma migration and stem cell-like phenotype. Cancer Res 73:990– 999. Yu NK, Baek SH, Kaang BK. 2011. DNA methylation-mediated control of learning and memory. Mol Brain 4:5. Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, Farkas DL, Black KL, Yu JS. 2004. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23:9392– 9400. Zhang Y, Dutta A, Abounader R. 2012. The role of microRNAs in glioma initiation and progression. Front Biosci 17:700–712. Zhao S, Deng Y, Liu Y, Chen X, Yang G, Mu Y, Zhang D, Kang J, Wu Z. 2013a. MicroRNA-153 is tumor suppressive in glioblastoma stem cells. Mol Biol Rep 40:2789–2798. Zhao S, Liu H, Liu Y, Wu J, Wang C, Hou X, Chen X, Yang G, Zhao L, Che H, Bi Y, Wang H, Peng F, Ai J. 2013b. miR-143 inhibits glycolysis and depletes stemness of glioblastoma stem-like cells. Cancer Lett 333:253–260.

1147