Contribution of MicroRNA-1275 to Claudin11 Protein Suppression via a Polycomb-mediated Silencing Mechanism in Human Glioma Stem-like Cells*

Background: Molecular mechanisms underlying heterogeneity of glioblastoma are poorly understood. Results: Newly identified microRNA-1275, which is controlled by a polycomb-mediated silencing mechanism, regulates expression of oligodendroglial lineage protein, Claudin11, in glioma stem-like cells. Conclusion: MicroRNA-1275 may contribute to the establishment of tissue heterogeneity via epigenetic mechanisms. Significance: We identified a microRNA that is associated with tumor cell differentiation in the oligodendroglial lineage. Glioblastomas show heterogeneous histological features, and tumor cells show distinct phenotypic states that confer different functional attributes and an aggressive character. However, the molecular mechanisms underlying the heterogeneity in this disease are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute to the establishment of tumor heterogeneity. Using a GSC model, we investigated differentially expressed microRNAs (miRNAs) and associated epigenetic mechanisms that regulate the differentiation of GSCs. miRNA profiling using microarray technology showed that 13 and 34 miRNAs were commonly up-regulated and down-regulated in two independent GSC lines during differentiation, respectively. Among this set of miRNAs, quantitative PCR analysis showed that miRNA-1275 (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 with a specific antisense oligonucleotide (anti-miR-1275) in GSCs increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) in the primary microRNA-1275 promoter was closely associated with miR-1275 expression. Treatment with 3-deazaneplanocin A, an inhibitor of H3K27 methyltransferase, attenuated CLDN11 induction by serum stimulation in parallel with sustained miR-1275 expression. Our results have illuminated the epigenetic regulatory pathways of miR-1275 that are closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity seen in the formation of glioblastomas. 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 glioblastomas.

Glioblastoma is the most common and deadly primary brain tumor, which is characterized by intratumoral and intertumoral heterogeneity with histologically different types of cells (1). Such multiple distinct subpopulations of cancer cells within tumors link to the existence of cells that survive surgery and chemotherapy to form recurrent lesions that are resistant to further treatments. Cumulative effort aimed at developing targeted therapies for glioblastomas has been somewhat hampered by complexities arising from tissue heterogeneity (2). Therefore, elucidating the molecular mechanisms underlying tissue heterogeneity of glioblastoma is a fundamental requirement for the development of effective treatments for this dreadful disease.
Evidence from several groups has shown that glioblastoma contain a rare, highly tumorigenic, self-renewing subpopulation of cells, called glioma stem-like cells (GSCs) 2 (3,4). According to recent studies in a breast cancer model, cancer stem-like cells were able to aberrantly differentiate into diverse cell types by signals within the tumor microenvironments; such phenotypic plasticity between stem-like cells and differentiated nonstem-like cells may contribute to the establishment of tumor heterogeneity (5,6). Indeed, the presence of such a cancer stem-like cell subpopulation has been shown in NOD-SCID mouse models to recapitulate the observed heterogeneity of different types of human tumors (7)(8)(9). Thus, GSCs might be a good model for studying tumor cell differentiation and associated tissue heterogeneity. microRNAs (miRNAs) are known to down-regulate the expression of genes by targeting 3Ј-untranslated regions (3Ј-UTRs) of mRNAs via direct mRNA degradation or translational inhibition (10). Studies have shown that miRNAs are potential key regulators of cellular differentiation and proliferation, which have been implicated in many types of cancers (11,12). The roles of miRNAs in glioblastoma have also been studied. miR-9/9* is highly abundant in CD133ϩ cells, and inhibition of miR-9/9* leads to reduced neurosphere formation (13). miR-21 and miR-17-92 cluster were highly expressed in glioblastoma and appear to function in an anti-apoptotic role (14,15). miR-221 and miR-222 were also up-regulated in glioblastoma and shown to be involved in cell cycle regulation via down-regulation of CDKN1B (16). Although the oncogenic or tumor-suppressive roles of these miRNAs in glioblastoma cells have been studied, the regulation of subtype-specific genes by miRNAs in tumor cells, especially in respect to differentiation of GSCs as linked to the establishment of tissue heterogeneity, has not been well studied.
Besides known genetic alterations, aberrant epigenetic alterations have emerged as common hallmarks of many cancers including glioblastoma (17)(18)(19). Epigenetic mechanisms can also regulate the expression of miRNAs (20,21). Emerging data suggest a role for epigenetic controls in regulating tumor cell plasticity, where they can generate multiple distinct cellular subpopulations, thereby contributing to intratumoral heterogeneity (22). Histone H3 lysine 27 trimethylation (H3K27me3), which is catalyzed by polycomb repressor complex 2 (PRC2) and recognized by PRC1, is regarded as an easily reversible modification (23,24). Recent data imply that PRC could act not only to maintain stemness and determine the proper lineage in pluripotent stem cells, but also to guide their further developmental processes by proper regulation of subtype-specific genes in progenitor cells (25).
Here, we investigated the molecular effects of the newly identified miR-1275 in GSC differentiation, whose expression is regulated via a PRC2-H3K27me3-dependent epigenetic mechanism in response to environmental cues, and assessed the relationship between stem-like cell differentiation and tissue heterogeneity, especially the oligodendroglial component in glioblastomas. Our results suggest that this developmental microRNA is regulated via an epigenetic pathway that contributes to the phenotypic diversity of glioblastoma tissues, which may in turn provide a better understanding of the heterogeneity of glioblastoma in the context of human neurodevelopment.

EXPERIMENTAL PROCEDURES
Cell Cultures-Glioblastoma tissue samples were obtained from patients undergoing surgical treatment at Nagoya University Hospital, Japan, after they provided written informed consent. The procedures used for derivation of GSCs (1228-GSC, 316-GSC, and 222-GSC) were described previously (26,27). Briefly, dissociated tumor cells were cultured in neurobasal medium comprising Neurobasal media, with N2 and B27 supplements (Invitrogen), along with human recombinant basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (20 ng/ml each; R&D Systems, Minneapolis, MN). Serum-induced brain tumor cells (S-BTCs) were established by culturing GSCs in DMEM (Invitrogen) containing 10% fetal bovine serum (FBS). T98, MDA231, MCF7, SKBR3, and PC3 cell lines were grown in DMEM with 10% FBS. A human neural stem cell (NSC) line was generated from the human fetal telencephalon as described previously (28). This human NSC line is capable of self-renewal and can differentiate into cells of neuronal and glial lineages, both in vivo and in vitro. NSCs were cultured in DMEM containing 0.1 mM 2-mercaptoethanol. After 2 days, NSCs were transferred to DMEM containing 0.5 M retinoic acid (Sigma-Aldrich) and incubated for two additional days to induce differentiation. For the inhibition of PRC2 activity, GSCs were plated in dishes for 24 h before 3-deazaneplanocin A (DZNep) treatment (5 M). GSCs were treated with DZNep or PBS (control) for 2-7 days. For the generation of stable knockdown of EZH2 in GSC (EZH2-KD GSC), retroviral vectors were used as reported previously (29).
miRNA Microarray-Human miRNA Microarray V3 kits (G4470C; Agilent Technologies, Santa Clara, California) were used according to the manufacturer's protocols. This microarray system contains probes for all 866 human and 89 human viral miRNAs reported from the Sanger database v12.0 (miR-Base). Each miRNA species is printed 20 times with replicate probes on the array. Total RNA was isolated from GSCs (1228, 316) and corresponding S-BTCs (continuous serum exposure for 21 days) with TRIzol reagent (Invitrogen). One hundred ng of total RNA was labeled with pCp-Cy3 (Agilent Technologies) and 15 units of T4 RNA ligase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 16°C for 2 h. Labeled samples were purified with Micro Bio-Spin 6 columns (Bio-Rad) and hybridized to microarrays at 55°C with rotation at 20 rpm for 20 h. Microarrays were scanned by an Agilent Scanner (Agilent Scan Control 7.0 software) and analyzed using Agilent Feature Extraction 10.7 software for miRNA microarray profiling. Raw data were normalized and analyzed with the use of GeneSpring software (version 11.5; Silicon Genetics, Redwood City, CA). The data of our microarray are available in the ArrayExpress database with accession code: GSE36201. To analyze the target genes of differentially expressed miRNAs, we performed computational analysis using two miRNA target databases, namely TargetScan and the microRNA Search software from the Memorial Sloan-Kettering Cancer Center.
Manipulation of miR-1275 in Cell Lines-To examine the effects of miR-1275 (accession number, NR_031681), cells were transfected with the precursor molecules mimicking miR-1275 (pre-miR-1275 precursor, final concentration of 30 or 100 nM, Applied Biosystems) or negative control miRNA (pre-miR negative control 1, 30 or 100 nM, Applied Biosystems) according to the manufacturer's instructions. Inhibition of miR-1275 was performed using an anti-miR-1275 inhibitor (anti-miR-1275 inhibitor, 30 or 100 nM, Applied Biosystems) or a negative control anti-RNA (anti-miR inhibitor negative control 1, 30 or 100 nM, Applied Biosystems) according to the manufacturer's instructions.
Statistics-The statistical significance of the differences observed was determined by paired Student's t test or one-way analysis of variance (StadView software version 5.0; Abacus Concepts, Berkeley, CA). All reported p values were two-sided, with p Ͻ 0.05 considered statistically significant.

RESULTS
miRNA Expression Profiling during GSC Differentiation and Target Prediction-Glioblastoma spheroid cultures, which are enriched with GSCs, converted into differentiated adherent cells in response to serum within 48 h and were termed S-BTCs along with contributing to an attenuation of tumorigenicity in NOD-SCID mice as was reported previously (4,26). To investigate the miRNAs involved in GSC differentiation, we examined miRNA expression before and after exposure to differ entiation-promoting serum condition in two GSC models (316-and 1228-GSCs) using miRNA expression microarrays. Among the 866 human miRNAs on the microarray, 32 and 43 miRNAs were up-regulated, and 128 and 56 miRNAs were down-regulated with a greater than 2-fold difference before and after serum exposure in 1228-GSCs and 316-GSCs, respectively (supplemental Tables S1 and S2). Among these differentially expressed miRNAs, 13 and 34 miRNAs were commonly upregulated and down-regulated in both 1228-GSCs and 316-GSCs, respectively (Fig. 1A).
We computationally analyzed the list of target genes of these differentially expressed miRNAs in two GSC lines using two independent publicly available database-related software programs, TargetScan and the microRNA Search software, because each program uses its own unique algorithms to measure complimentarily. Among the list of miRNAs and their targets, we focused on the miRNAs for which relevant targets are closely associated with neural cell differentiation. A profound relationship was found between miR-1275 and CLDN11 (also known as oligodendrocyte-specific protein, OSP), a tight junction protein that is a major and essential component of central nervous system myelin and has been shown to increase in expression when oligodendrocyte progenitors develop into mature oligodendrocytes (32). Predicted target genes of miR-1275 that were commonly identified within two databases are listed in supplemental Table 3. miR-1275 appeared to have a strong target bias for the 3Ј-UTR mRNA of CLDN11; it has two predictive seed sequences in this 3Ј-UTR (Fig. 1B). Consistent with the microarray analysis, quantitative RT-PCR showed that the expression of miR-1275 was down-regulated during GSC differentiation, together with up-regulation of CLDN11 in three independent GSC lines (1228-, 316-, and 222-GSCs; Fig.  1, C and D). Expression of the other significant miR-1275 targets, such as GATA2B, CEBPG, PAX5, and NEUROD2 that are listed in the database and were validated by qPCR or Western blot analysis, did not appear to be significantly concordant with miR-1275 expression in our model, although PAX5 and NEUROD2 are associated with neural differentiation (33, 34) (supplemental Fig. 1, A and B). We also analyzed the expression levels of miR-205 in GSCs, which has been reported as a poten-tial regulator of CLDN11 in human embryonic stem cell differentiation (35), and found that miR-205 expression was at an extremely low level, and there was no evidence of dynamic alteration in response to serum in GSCs (supplemental Fig. 1C). Intriguingly, an inverse correlation between miR-1275 and CLDN11 expression was also found in the NSCs. miR-1275 expression was down-regulated during the differentiation of NSCs induced by retinoic acid, together with CLDN11 up-regulation (supplemental Fig. 1D).
Interaction between miR-1275 and Its Binding Sites in the CLDN11 3Ј-UTR-Because miRNAs negatively regulate mRNA expression by repressing translation or directly cleaving the targeted mRNA via imperfect binding to their 3Ј-UTR, we experimentally assessed the interaction between miR-1275 and the 3Ј-UTR of the CLDN11 mRNA. First, we examined the levels of miR-1275 expression in five cancer cell lines, namely glioma cell line (T98), three breast cancer cell lines (MDA-MB-231, MCF7, and SKBR3), and a prostate cancer cell line (PC3), and in NSC by quantitative RT-PCR analysis. Low level of miR-1275 expression was observed in T98, whereas all the breast cancer cell lines and the prostate cancer cell line, as well as NSCs, showed moderate to high levels of miR-1275 expression ( Fig. 2A).
To investigate the potential relationship between CLDN11 3Ј-UTR and miR-1275, the CLDN11 3Ј-UTR, which contained two predictive miR-1275 target sites, was cloned just downstream of the firefly luciferase coding sequence (pmir-CLDN11-3Ј-UTR) and transfected into the MDA231, MCF7, SKBR3, PC3, and T98 cell lines. Significant repression of luciferase activities was observed in the MDA-MB-231, MCF7, SKBR3, and PC3 cell lines, which expressed a high level of miR-1275, as compared with the control luciferase activity (Fig. 2B). On the other hand, no obvious changes of luciferase activity were observed in the T98 line, which expressed a substantially lower level of miR-1275.
Next, to further examine the direct interaction of miR-1275 with the potential target sites in the CLDN11 3Ј-UTR, we cotransfected the pmir-CLDN11-3Ј-UTR together with expression plasmids for miR-1275 into T98 cells. Significant repression of luciferase activities was observed under the miR-1275 expression condition (Fig. 2C). However, although it is statistically significant, the effect of robust miR-1275 up-regulation (more than 600 times higher than untransfected cells) in T98 cells transfected with pre-miR-1275 resulted in relatively small changes (decreases of around 30 -40%) in CLDN113Ј-UTR luciferase activity. In contrast, CLDN11 3Ј-UTR luciferase activity was prominently down-regulated in MDA231, MCF7, SKBR3, and PC3 cells as compared with T98 cells, although the difference in miR-1275 expression between these two groups was less than 10 times (Fig. 2, A and B). These data suggest the existence of another mechanism, such as other types of microR-NAs in addition to miR-1275, that is involved in the regulation of CLDN11 expression in T98 cells.
Conversely, repression of luciferase activities in PC3 was impaired by down-regulation of miR-1275 with a specific miR-1275 inhibitor (anti-miR-1275, 100 nM) (supplemental Fig. 2A). In addition, pmir-CLDN11-3Ј-UTR constructs with one or two mutated miR-1275 binding sites showed stepwise increase in luciferase activity in PC3 cells (supplemental Fig. 2B). Taken together, these results indicated that miR-1275 directly regulates CLDN11 expression by targeting the two binding sites in the 3Ј-UTR of CLDN11 mRNA.
miR-1275 Inhibits CLDN11 Protein Expression in GSC Cell Lines-To further assess the direct relationship between miR-1275 and CLDN11 expression in GSCs, which showed a high level of miR-1275 and a low level of CLDN11 mRNA expression, we examined the effects of anti-miR-1275 on CLDN11 expression in GSC cell lines. GSC lines were treated with anti-miR-1275, a negative control miRNA inhibitor (anti-N), or without any antisense molecules (mock control). In response to anti-miR-1275, mRNA expression of CLDN11 was up-regulated along with the down-regulation of miR-1275 in three GSC lines (Fig. 3A). Consistently, both CLDN11 protein level and the number of CLDN11-positive cells were significantly increased in GSC spheroids after treatment with anti-miR-1275 in the three GSC lines (Fig. 3B).
We also found that GSCs treated with anti-miR-1275 showed significant growth suppression as compared with those treated with anti-N control, suggesting that inhibition of this miRNA could induce CLDN11 expression with rapid inhibition of GSC proliferation (Fig. 3C). Further, we confirmed that inhibition of CLDN11 by siRNA suppressed cell proliferation of GSCs (Fig. 3D).
miR-1275 Is Regulated by PRC2-mediated H3K27me3-Epigenetic control of developmental genes has emerged as a key mechanism for differentiation of NSCs in response to differentiation-inducing cues. Notably, a PRC2-H3K27me3-mediated gene silencing mechanism plays an essential part in the repression of key development-associated genes (36). To investigate whether this epigenetic mechanism is involved in the regulation of miR-1275, we examined the association between epigenetic status of the promoter region of pri-miR-1275 and miR-1275 expression in GSC lines. The transcriptional start site of pri-miR-1275 was determined by the DBTSS database (Fig. 4A) and confirmed by RT-PCR (supplemental Fig. 3) (37). Concordant with miR-1275 down-regulation during GSC differentiation into S-BTC, ChIP-PCR analysis showed that enrichment of H3K27me3 repressive mark and YY1 protein, together with loss of H3K9 acetylation (H3K9Ac) active mark in S-BTCs (Fig.  4A). Compellingly, treatment of GSCs with a potent PRC2 inhibitor, DZNep, showed sustained miR-1275 expression along with a small effect on CLDN11 expression in the presence of serum (Fig. 4B, supplemental Fig. 4A). To clarify whether miR-1275 is regulated by an epigenetic pathway, especially PRC2-mediated mechanism, we further examined the effect of EZH2, a catalytic component of PRC2, inhibition on expression of miR-1275 and CLDN11 using an shRNA system (EZH2-KD). EZH2-KD impaired serum-induced miR-1275 repression, resulting in disruption of CLDN11 induction (supplemental Fig. 4B). These data suggested that induction of miR-1275 by differentiation-promoting serum conditions was responsible for increased expression of CLDN11 via the PRC2-H3K27me3 epigenetic regulatory pathway.
Immunohistochemical Analysis of CLDN11 in Clinical Samples-To assess the impact of our findings in respect to the clinical features of glioblastoma, we investigated the expression pattern of CLDN11 in glioblastoma (n ϭ 3) and normal brain (n ϭ 3) tissues. CLDN11 was highly expressed in mature oligodendrocytes in normal brain (Fig. 5, A and B) (38). By contrast, in all three glioblastoma specimens, a major part of the tumor tissue was CLDN11-negative. Only one specimen showed a certain area composed of CLDN11-positive tumor cells (Fig. 5, C-E). To clarify whether these CLDN11-positive cells correspond to the oligodendroglial lineage, which sometimes present in a small fraction of glioblastomas (39,40), we investigated the expression pattern of CLDN11 in comparison with major neural cell lineage markers, such as GFAP (an astrocyte lineagespecific marker), microtubule-associated protein 2 (MAP2, a neuron lineage-specific marker), and oligodendrocyte lineage transcription factor 2 (OLIG2, a oligodendrocyte lineage-specific marker). CLDN11-positive cells and GFAP-positive cells existed on a mutually exclusive basis in glioblastoma specimens. No MAP2-positive cells were found in the glioblastoma tissues examined. Notably, in small fractions of glioblastomas, CLDN11-positive cells were also positive for the anti-OLIG2 antibody, suggesting that CLDN11-positive cells in the glioblastoma tissues correspond to the oligodendroglial lineage and that regulation of miR-1275 may contribute, at least in part, to form the tissue heterogeneity corresponding to the oligodendroglial lineage cells in glioblastomas (Fig. 5, F-I).

DISCUSSION
Glioblastoma displays heterogeneous histological features, with subsets of tumor cells often showing distinct phenotypic states that differ in functional attributes. This phenotypic heterogeneity might be explained, at least in part, via a cancer stem-like cell model (5). In this study, using a GSC-based differentiation model (4,26,27), we investigated the expression of miRNAs and associated epigenetic mechanisms that regulate the differentiation of GSCs. In response to serum-induced differentiation conditions, acquisition of H3K27me3 modification and enrichment of YY1 protein binding, which has been implicated to act as a modular scaffold protein to recruit PRC2 to the target genes (41,42), together with loss of H3K9Ac in the pri-miR-1275 promoter region, led to the silencing of miR-1275 and derepression of its target, CLDN11, in GSCs. These findings support a model for the establishment of tissue heterogeneity where the tumor environment affects the expression of a subset of genes including miRNAs via plastic epigenetic mechanisms, resulting in distinct types of cell differentiation. We also examined DNA methylation status of a CpG island within the CLDN11 promoter and found that no DNA methylation changes were detected in all three GSC lines, suggesting that this highly stable epigenetic machinery was not directly involved during GSC differentiation (supplemental Fig. 5). The significant difference between PRC2-H3K27me3 and DNA methylation centers on the stability of repression; indeed, H3K27me3-mediated gene silencing can change dynamically during differentiation, as opposed to DNA methylation within CpG islands, which is highly stable and irreversible without artificially altering key factors in cells (43).
The most prevalent form of glioma is referred to as astrocytoma. However, glioblastoma also contains an oligodendrocyte component in some cases. This oligodendroglial component has been detected in ϳ20% of glioblastomas; moreover, the presence of oligodendroglial components has been reported to be predictive of longer survival of patients in certain studies (39,40). Our immunohistochemical examination revealed that CLDN11-positive tumor cells also showed OLIG2 positivity in a specific region of tumors, with this area being in concordance with an oligodendroglial component; this suggests that CLDN11 expression via down-regulation of miR-1275 contrib- utes to the formation of oligodendroglial component in glioblastoma.
We noted that GSC with ectopic expression of miR-1275 could convert into S-BTC with GFAP and Tuj1 positives under differentiation-promoting serum conditions (supplemental Fig. 6). This suggests that miR-1275 particularly affected the expression of CLDN11, which is an important protein for oligodendroglial lineage differentiation but is not a master regulator of the other differentiation-associated genes in GSCs.
Although miR-205 has been reported as a potential regulator of CLDN11 in human embryonic stem cell differentiation, this miRNA did not appear to be involved in GSC differentiation into oligodendroglial lineage in our model (35). Another miRNA, miR-219, has been shown to be necessary and sufficient to promote oligodendrocyte differentiation (44). However, expression of this miRNA was also at an undetectable level in our microarray analysis of the three GSC cell types (Array-Express, accession codes: GSE36201). In addition to GSCs, we also found that miR-1275 may affect the expression of CLDN11 during NSC differentiation mediated by retinoic acid exposure. Because miRNAs are potential key regulators of differentiation and miRNA-deficient mice with Dicer deletions showed signif-icant impairment of oligodendrocyte differentiation and production of myelin genes (44,45), miR-1275 might also be one of the key miRNAs for myelination and oligodendroglial lineage differentiation.
Recent studies indicate that miRNAs are essential regulators of GSCs. miR-9/9* is highly abundant in CD133ϩ cells, and inhibition of miR-9/9* leads to reduced neurosphere formation (13). Members of the miR-17-92 cluster are down-regulated upon GSC differentiation. Transfection of inhibitors of the miR-17-92 cluster induced increased apoptosis and decreased cell proliferation (15). Consistent with these previous studies, down-regulation of miR-9/9* and miR-17, miR-19b, and miR-20a, components of the miR-17-92 cluster, were detected during GSC differentiation in our microarray analysis. In addition to these findings, we found that suppression of miR-1275 in GSCs induced oligodendroglial lineage-associated protein (CLDN11) along with rapid inhibition of GSC proliferation.
The loss of tumor-suppressive miRNAs enhances the expression of target oncogenes, whereas increased expression of oncogenic miRNAs (known as oncomirs) can repress tumorsuppressor genes in cancers (46). Members of the Claudin gene family are expressed in both a tissue stage-specific and a devel- opmental stage-specific manner. CLDN11 expression is highly regulated during development and may play an important role in growth and differentiation of oligodendrocytes. In cancer cells, CLDN11 appears to have a tumor-suppressor function in gastric cancer, bladder cancer, and meningioma (47)(48)(49). Indeed, inhibition of CLDN11 by siRNA in GSCs induced cell proliferation (Fig. 3D). Given these tumor-suppressor functions of CLDN11, miR-1275 may also have a context-dependent effect; in cancer cells (including our GSCs), it works as an oncomir, inhibition of which may also lead the suppression of tumor cell growth. This is also explained by the ideas that developmentally important genes also have crucial roles during tumor progression in a lineage-specific context (50).
In summary, our data provide evidence that repression of miR-1275 via PRC2-H3K27me3 results in CLDN11 up-regulation, which may be required for the tumor cell differentiation and contribute to the establishment of tissue heterogeneity in glioblastoma. In addition, miR-1275 targets CLDN11, which represses the proliferation of tumor cells, as was found in the current study. These results have illuminated the epigenetic regulatory pathways for tumor cell differentiation by miR-1275 in glioblastomas, inhibition of which may be a potential target for cancer therapy as a new clinical application.