Therapeutic potential of targeting micro RNA ‐10b in established intracranial glioblastoma: first steps toward the clinic
2016; Springer Nature; Volume: 8; Issue: 3 Linguagem: Inglês
10.15252/emmm.201505495
ISSN1757-4684
AutoresNadiya M. Teplyuk, Erik J. Uhlmann, Galina Gabriely, Natalia Volfovsky, Yang Wang, Jian Teng, Priya Karmali, Eric G. Marcusson, Merlene Peter, Athul Mohan, Yevgenya Kraytsberg, Ron Cialic, E. Antonio Chiocca, Jakub Godlewski, Bakhos A. Tannous, Anna M. Krichevsky,
Tópico(s)RNA Interference and Gene Delivery
ResumoResearch Article10 February 2016Open Access Source DataTransparent process Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic Nadiya M Teplyuk Nadiya M Teplyuk Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Erik J Uhlmann Erik J Uhlmann Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Galina Gabriely Galina Gabriely Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Natalia Volfovsky Natalia Volfovsky Simons Foundation, New York, NY, USA Search for more papers by this author Yang Wang Yang Wang Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jian Teng Jian Teng Department of Neurology, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Priya Karmali Priya Karmali Regulus Therapeutics Inc., San Diego, CA, USA Search for more papers by this author Eric Marcusson Eric Marcusson Regulus Therapeutics Inc., San Diego, CA, USA Search for more papers by this author Merlene Peter Merlene Peter Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Athul Mohan Athul Mohan Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yevgenya Kraytsberg Yevgenya Kraytsberg Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ron Cialic Ron Cialic Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author E Antonio Chiocca E Antonio Chiocca Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jakub Godlewski Jakub Godlewski Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Bakhos Tannous Bakhos Tannous Department of Neurology, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Anna M Krichevsky Corresponding Author Anna M Krichevsky Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Nadiya M Teplyuk Nadiya M Teplyuk Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Erik J Uhlmann Erik J Uhlmann Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Galina Gabriely Galina Gabriely Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Natalia Volfovsky Natalia Volfovsky Simons Foundation, New York, NY, USA Search for more papers by this author Yang Wang Yang Wang Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jian Teng Jian Teng Department of Neurology, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Priya Karmali Priya Karmali Regulus Therapeutics Inc., San Diego, CA, USA Search for more papers by this author Eric Marcusson Eric Marcusson Regulus Therapeutics Inc., San Diego, CA, USA Search for more papers by this author Merlene Peter Merlene Peter Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Athul Mohan Athul Mohan Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Yevgenya Kraytsberg Yevgenya Kraytsberg Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Ron Cialic Ron Cialic Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author E Antonio Chiocca E Antonio Chiocca Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jakub Godlewski Jakub Godlewski Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Bakhos Tannous Bakhos Tannous Department of Neurology, Massachusetts General Hospital, Boston, MA, USA Search for more papers by this author Anna M Krichevsky Corresponding Author Anna M Krichevsky Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Nadiya M Teplyuk1, Erik J Uhlmann1, Galina Gabriely1, Natalia Volfovsky2, Yang Wang1, Jian Teng3, Priya Karmali4, Eric Marcusson4, Merlene Peter1, Athul Mohan1, Yevgenya Kraytsberg1, Ron Cialic1, E Antonio Chiocca5, Jakub Godlewski5, Bakhos Tannous3 and Anna M Krichevsky 1 1Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA 2Simons Foundation, New York, NY, USA 3Department of Neurology, Massachusetts General Hospital, Boston, MA, USA 4Regulus Therapeutics Inc., San Diego, CA, USA 5Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA *Corresponding author. Tel: +1 617 525 5195; E-mail: [email protected] EMBO Mol Med (2016)8:268-287https://doi.org/10.15252/emmm.201505495 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR-10b regulates cell cycle and alternative splicing, often through the non-canonical targeting via 5′UTRs of its target genes, including MBNL1-3, SART3, and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR-10b ASO led to targets' derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes. Synopsis Oncogenic miR-10b highly expressed in glioblastoma and absent in normal brain tissues promotes the growth of heterogeneous glioma cell types. Teplyuk et al. describe common genes regulated by miR-10b in glioma stem cells and demonstrate the efficacy of miR-10b inhibition in established GBM models. mRNA splicing and processing factors are regulated by miR-10b through the non-canonical 5′UTR binding in glioma-derived neurospheres. Growth of orthotopic glioblastoma (GBM) xenografts and allografts in mouse models is attenuated by local and systemic delivery of miR+NA inhibitors. No systemic toxicity of miR-10b inhibitors' administration by local or systemic routes was observed. Introduction Glioblastoma (GBM), also known as grade IV astrocytoma, is the most aggressive primary brain cancer. Despite intensive treatment that includes surgery, chemotherapy, and radiotherapy, the median survival of GBM patients is around 14 months. Only a few drugs are proven to possess some therapeutic efficacy and administered for GBM patients. However, significant toxicity of those treatments and a very high rate of disease recurrence turn further care strategies to palliative. Glioblastoma is a highly heterogeneous disease that is generally classified into four subtypes, mesenchymal, classical, proneural, and neural, based on genetic alterations, gene expression patterns, and putative cellular origin (Verhaak et al, 2010). Numerous mutations, expression, and epigenetic alterations occur in different GBM subtypes with frequencies ranging between 3 and 50% (Cancer Genome Atlas Research, 2008). Considering the highly heterogenic molecular landscape of GBM, identification of common regulators of tumor growth and progression across GBM is very important. High expression of microRNA-10b (miR-10b) represents a rare unifying event for gliomas, as it occurs in at least 90% of all GBM cases across all disease subtypes, as well as in low-grade gliomas (Gabriely et al, 2011b). Importantly, miR-10b inhibition compromises proliferation and survival of glioma cells without affecting normal neural cells cultured in vitro, and initial evidence also suggests the suppressive effects on tumor growth in vivo (Gabriely et al, 2011b; Dong et al, 2012; Lin et al, 2012; Guessous et al, 2013; Teplyuk et al, 2015). Therefore, considering the lack of miR-10b expression in the normal brain, targeting this molecule in GBM might represent a unique opportunity for specific and non-toxic therapy. Substantial preclinical studies in vivo are required to evaluate the potency and efficacy of miR-10b therapeutic targeting for GBM treatments. miR-10b is a powerful oncogenic miRNA promoting growth and metastasis and indicative of poor prognosis in various types of cancer (Ma et al, 2007, 2010; Nakata et al, 2011; Li et al, 2012; Nishida et al, 2012; Liu et al, 2013b; Mussnich et al, 2013; Nakayama et al, 2013; Sun et al, 2013; Wang et al, 2013; Ouyang et al, 2014). Depending on the cancer type and genetic context, miR-10b acts through pleiotropic mechanisms, including control of cell proliferation, survival, migration, invasion, and epithelial-to-mesenchymal transition, and directly targeting different genes in various cells and tissues. Although several miR-10b targets have been identified in GBM and other tumors, their regulation appears highly cell- and context- specific (Ma et al, 2010; Gabriely et al, 2011a; Han et al, 2014; Teplyuk et al, 2015). For GBM, a pathology originating from genetically and epigenetically diverse backgrounds, the common mechanisms underlying miR-10b functions in survival of tumor cells are unknown. Here, we investigated the role of miR-10b in heterogeneous GBM-initiating stem-like cells (GSC) in vitro, as well as in orthotopic GBM xenograft mouse models in vivo. GSC is a subpopulation of tumor cells with tissue stem cell properties, capable of self-renewal and providing an origin to the rest of the tumor. GSC are highly resistant to conventional chemo- and radiation therapies and associated with the disease recurrence (Bao et al, 2006; Liu et al, 2006). Therefore, development of efficient GSC targeting strategies is critically important. Patient-derived GSC growing in neurosphere cultures are highly tumorigenic; injected to athymic mice, they form aggressive and invasive tumors, more reminiscent of human GBM than glioma cell lines derived xenografts (Galli et al, 2004; Singh et al, 2004). As other glioma cells, GSC express high levels of miR-10b, and their growth is suppressed by miR-10b inhibition (Gabriely et al, 2011b; Guessous et al, 2013); however, the mechanisms underlying this growth regulation have not been identified yet. Here, we utilized GSC to investigate the mechanisms governing miR-10b-mediated survival of cancer cells in vitro and miR-10b potency as therapeutic target in vivo. Using the whole-genome expression profiling of three distinct GSC cultures, we demonstrated that cell cycle and mRNA processing/alternative splicing are the major cellular mechanisms commonly affected by miR-10b in GSC. Inhibition of miR-10b caused derepression of multiple mRNA targets, in most cases by non-canonical non-seed binding to their 5′UTRs. Several splicing factors were validated as direct miR-10b targets in GSC, including MBNL2 and MBNL3. Altogether, miR-10b inhibition led to a global shift in splicing patterns of GSC. To assess the therapeutic efficacy of miR-10b inhibition for GBM, we studied the effects of anti-miR-10b treatment on GSC-derived established intracranial GBM xenograft in athymic mice and complemented this study with experiments on syngeneic GL261 glioma model in immunocompetent mice. In a series of in vivo experiments, we demonstrated that anti-miR-10b ASO, administered during the exponential phase of tumor growth, significantly reduced progression of established intracranial GBM. Three delivery routes for the miR-10b ASO inhibitor, including direct intratumoral injections, continuous osmotic delivery, and systemic intravenous (i.v.) injections, proved efficient in inhibiting the growth of orthotopic GBM. This study, therefore, provides a preclinical rationale for clinical evaluation of the miR-10b targeting therapies against GBM. Results GSC as a model to study miR-10b function To characterize GSC as model to study miR-10b oncogenic function, we have determined miR-10b expression in three genetically distinct patient-derived GBM neurosphere cultures, GBM4, GBM6, and GBM8 (also referred as MGG4, MGG6, and MGG8; Wakimoto et al, 2009). The expression of stem cell markers, pluripotency, and tumorigenic properties of these GSC were characterized earlier (Wakimoto et al, 2009). The three types of GSC carry diverse genetic alterations and distinct phenotypic features and potentially represent mesenchymal and proneural subtypes (Wakimoto et al, 2012). We found that miR-10b was expressed in all three GSC cultures at the levels comparable to those observed in GBM cell lines, such as A172, U87, and LN229 (Fig EV1), in agreement with our observation that miR-10b is similarly expressed across various GBM subtypes in The Cancer Genome Atlas (TCGA) (Appendix Fig S1). In contrast, miR-10b expression was detected neither in primary and early passage cultures of normal human neural stem cells (NSC), nor in human astrocytes (Fig EV1). Click here to expand this figure. Figure EV1. miR-10b expression in GBM stem-like cells (GSC), relative to human GBM cell lines, normal human brain tissues, and brain cellsThe relative expression levels of miR-10b have been assessed by qRT–PCR analysis and normalized to levels of snRNA U6. NSCs—primary normal embryonic neural stem cells of the indicated passages. Source data are available online for this figure. Download figure Download PowerPoint Consistent with previous data (Guessous et al, 2013), inhibition of miR-10b had a strong effect on viability of GSC (Fig 1A). GSC transfected with miR-10b ASO formed neurospheres similar to control cultures, suggesting that their neurosphere-forming capacity was unaltered. However, at a later time point (day four post-transfection) the neurospheres started to exhibit markers of apoptosis followed by the massive cell death and sphere disaggregation. This process resulted in significant reduction in both number and size of the GSC neurospheres (Fig 1B). As it was previously shown for glioma cell lines (Gabriely et al, 2011b), miR-10b inhibition led to the cleavage of caspase 3 and caspase 7 in GSC, indicative of the induction of apoptotic cell death (Fig 1C). A significant increase in the number of apoptotic cells occurred in miR-10b depleted cultures by day five post-transfection, as also indicated by the double staining with 7-aminoactinomycin D (or propidium iodide) and Annexin V (Figs 1D and EV2). Therefore, miR-10b inhibition strongly reduced the viability of heterogeneous GSC, similarly to its effects on other subpopulations of glioma cells. At the same time, inhibition of miR-10b in differentiating GSC cultures attenuated expression of stem cell markers Nestin and OCT4 and elevated astrocytic marker GFAP (Appendix Fig S2), indicating that in differentiating conditions, miR-10b helps to maintain stem cell properties of GSC. Figure 1. MiR-10b inhibition reduces viability and enhances apoptosis of GSCGSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled "miR-10b-i") or non-targeting control oligonucleotide, or treated with Lipofectamine 2000 only (Mock). Cell viability was monitored at day 5 after transfection as described in 4. The number and size of GSC colonies were monitored at day 5 after transfection. miR-10b inhibition induces cleavage of caspases 3 and 7 in GSC, as determined by Western blot analysis at day 5 after transfection with the inhibitor. The signals were quantified using ImageJ and normalized to beta-actin. Flow cytometry analysis of Annexin V and 7-AAD staining of GSC GBM8 at day 5 after miR-10b inhibition. Data information: (A, B) Statistical significance of the difference was determined by Student's t-test, with P-values < 0.001 indicated by three asterisks. Numbers of replicates and exact P-values are included in Appendix Table S4. Source data are available online for this figure. Source Data for Figure 1 [emmm201505495-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Annexin V and propidium iodide staining of GSC at day 5 after miR-10b inhibitionGSC neurospheres were dissociated to single cell suspension and transfected with either miR-10b inhibitor (labeled "miR-10b-i") or non-targeting control oligonucleotide. Cells were stained with Annexin V and propidium iodide at day 5 after transfections. Cells were collected on microscopic slides by Cytospin, and fluorescence images were taken immediately after staining. Source data are available online for this figure. Download figure Download PowerPoint MiR-10b affects cell cycle and spicing machinery in GSC through non-conventional gene targeting To identify the common targets regulated by miR-10b in heterogeneous GSC, we transfected GBM4, GBM6, and GBM8 cultures with the miR-10b ASO or the corresponding control oligonucleotide and conducted genomewide microarrays expression profiling at 24 h post-transfection. This time frame allowed identification of direct mRNA targets and molecular pathways modulated, prior to the massive changes in gene expression associated with apoptosis. The 1,429 probe sets corresponding to transcripts and splice variants of 956 genes exhibited significantly altered expression (more than 1.2-fold, P < 0.05) in all three types of GSC after miR-10b inhibition. Five hundred and twenty-two probe sets have shown up-regulation, and 907 probe sets were down-regulated by the anti-miR-10b treatment. Functional annotation using Ingenuity Pathway Analysis highlighted cell cycle as one of the major functions affected by miR-10b inhibition. A total of 119 genes related to bioterm "cell cycle" were affected by miR-10b in GBM4, GBM6, and GBM8 cells (Figs 2A and EV3). This was in line with previously reported effects of miR-10b on glioma cell cycle progression (Gabriely et al, 2011b; Teplyuk et al, 2015). More detailed analysis of the expression dataset by Gene Set Enrichment Analysis (GSEA) indicated that genes related to bioterms "RNA processing" and "RNA splicing" were the most strongly enriched among the miR-10b-regulated genes in all three types of GSC (Table EV1). Expression of multiple splicing factors and components of splicosome complex were significantly affected by anti-miR-10b ASO (Fig 2B). Furthermore, alternative splice variants of numerous transcripts exhibited differential regulation based on the microarrays data (Fig EV4) and further validation for a subset of genes by isoform-specific qRT–PCR analysis (Appendix Fig S3), indicating that global splicing pattern of GSC shifted upon miR-10b inhibition. Importantly, mRNA processing and splicing-related bioterms exhibited a strong correlation with miR-10b expression in GBM TCGA dataset as well (Table EV2), substantiating the idea of miR-10b-regulated splicing in human GBM tumors in situ. Figure 2. miR-10b regulates cell cycle- and splicing-related genes in GSCThree types of GSC (GBM4, GBM6, and GBM8) were transfected with miR-10b ASO, and gene expression was analyzed 24 h later by the Affymetrix microarrays. The heatmaps' colors intensity demonstrates altered expression of the genes (up- or down-regulated relative to the mock-treated samples) with the fold change > 1.2 and P < 0.05 in at least two out of the three GSC cultures. The genes associated with "cell cycle" bioterm have been selected using Ingenuity Pathway Analysis. The treatment with the miR-10b inhibitor is indicated as "miR-10b-i". The genes associated with "RNA splicing" bioterm have been selected using Gene Ontology (GO). The treatment with the miR-10b inhibitor is indicated as "miR-10b-i". Arrows depict the genes selected as candidate direct targets for further study. miR-10b-binding motifs are enriched in 5′UTRs of the genes up-regulated by miR-10b ASO. The graph shows the probability that enrichment of the miR-10b motifs in mRNAs up-regulated vs. unchanged (P < 0.05) by anti-miR-10b does not occur by chance. The miR-10b octamer motifs' composition of the 5′UTRs was compared between transcripts up- and down-regulated on the microarrays. The relative frequencies of various miR-10b-binding motifs are shown, indicating that mostly miR-10b 3′-end-binding motifs are enriched in the up-regulated mRNAs. Source data are available online for this figure. Source Data for Figure 2 [emmm201505495-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Regulation of cell cycle-related genes by miR-10b in GSCChange in the expression of genes related to "cell cycle" bioterm upon miR-10b inhibition in GSC, as determined by the whole-genome expression profiling. The cell cycle-associated genes were selected using the Ingenuity Pathway Analysis. "miR-10b-i" indicates the treatment with miR-10b inhibitor. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Regulation of splice isoforms by miR-10b inhibition in GSC, as determined by the whole-genome expression profilingEach row of the heatmap demonstrates a pair of probe sets corresponding to different splice isoforms of the same gene, and regulated inversely (with fold change > 1.2 on a left side, and < 0.8 on a right side, and P < 0.05 in at least two out of three GSC cultures). Affymetrix prob set IDs are indicated in parenthesis. Download figure Download PowerPoint We next utilized the array analysis to identify common direct mRNA targets regulated by miR-10b in GSC. The classical mechanism of miRNA regulation involves the binding of 5′ miRNA "seeds" (7–8 nt motifs) to the complementary sequences within 3′UTRs of corresponding mRNA targets (Lewis et al, 2005), which accounts for the majority of miRNA–target interactions (Helwak et al, 2013). However, our computational analysis revealed no enrichment of the miR-10b seeds in the 3′UTRs of genes derepressed by miR-10b inhibition in GSC (data not shown). Emerging data suggest that miRNAs may also function via non-canonical (non-seed-mediated and/or non-3′UTR-based) mechanisms (Lytle et al, 2007; Chi et al, 2012). To explore this possibility, we scanned 5′UTRs, 3′UTRs, and complete coding sequences (CDS) of the derepressed/up-regulated genes for potential 6- to 9-nucleotide-long miR-10b complementary motifs. We found that 5′UTRs but not 3′UTRs of mRNAs up-regulated by anti-miR-10b treatment were significantly enriched in such motifs (Fig 2C). Of note, the majority of miR-10b octamers that were strongly overrepresented in the population of up-regulated transcripts (relative to the population of down-regulated or unaltered transcripts) corresponded to the miR-10b 3′ end and not to the conventional 5′ seed (Fig 2D). These results suggest that miR-10b most frequently binds to the 5′UTRs of its targets and functions via a non-conventional non-seed-mediated mechanism. Regulation of splicing factors by miR-10b through non-canonical binding within 5′UTR We have further selected eight splicing factors as potential direct miR-10b targets using the following criteria: (i) Their genes were derepressed in at least two out of three GSC types by miR-10b inhibitor; (ii) expression of those genes is reduced in GBM versus the normal brain in The Cancer Genome Atlas (TCGA) or Oncomine datasets (Fig 3, Appendix Table S1), (Bredel et al, 2005; Liang et al, 2005; Lee et al, 2006; Sun et al, 2006; Murat et al, 2008); and (iii) corresponding mRNAs possess a putative miR-10b binding site in either 3′UTR or 5′UTR (Fig 4A). Derepression of these genes by miR-10b inhibition was validated by qRT–PCR analysis in GBM4, GBM6, GBM8, and BT74 GSC cultures, as well as additional glioma cell lines LN215 and U251 (Fig 4B). Notably, in all cases we observed a weak/moderate but statistically significant effect on these mRNAs, typical for miRNA regulation. Additional experiments on 5′UTR targets MBNL1-3, SART3, and RSRC1 demonstrated that transfections of miR-10b mimic down-regulated expression of these proteins in GSC (Fig 4C). Figure 3. Expression analysis of splicing factor mRNAs in various GBM datasets The genes encoding splicing factors down-regulated by miR-10b are expressed at lower levels in various GBM datasets relative to their expression in normal brain tissues. In contrast, many splicing factors up-regulated by miR-10b are overexpressed in the GBM datasets. Data information: (A, B) Six high-content GBM microarray datasets from the Oncomine resource (https://www.oncomine.org/resource/login.html), including TCGA_BrainGBM (2), Bredel Brain2 (31), Lee Brain (32), Liang Brain (33), Murat Brain (34), and Sun Brain (35), that collectively contain information for 858 GBM and 52 control samples, have been utilized for the analysis. The data is presented as log2 fold change between GBM and normal brain tissues. Source data are available online for this figure. Source Data for Figure 3 [emmm201505495-sup-0007-SDataFig3.xls] Download figure Download PowerPoint Figure 4. miR-10b regulates splicing factors through the non-canonical binding within 5′UTRs Putative miR-10b binding sites within 5′UTRs of candidate splicing factors mRNAs. qRT–PCR analysis validates that mRNA of MBNL1-3, SART3, RSRC1, and other splicing factors are derepressed by miR-10b ASO in different GSC and GBM cell lines. mRNA expression levels were normalized to GAPDH expression. Regulation of representative splicing-related proteins by miR-10b mimic in GSC, as demonstrated by Western blot analysis. The signals were quantified using ImageJ and normalized to beta-actin. The ratios between miR-10b mimic expressing and control samples are indicated. miR-10b mimic regulates 5′UTR luciferase reporter containing a single miR-10b complementary site. miR-10b mimic regulates 5′UTR luciferase reporters of some splicing factors genes bearing wild-type (WT) but not mutated (Mut) miR-10b binding sites. Data information: (B, D, and E) Statistical significance of the differences was determined by Student's t-test, **P < 0.01 and ***P < 0.001. Numbers of replicates and exact P-values are included in Appendix Table S4. Source data are available online for this figure. Source Data for Figure 4 [emmm201505495-sup-0008-SDataFig4.pdf] Download figure Download PowerPoint To validate the direct binding and regulation, 5′UTRs or 3′UTR fragments of these genes were cloned into 5′UTR LightSwitch reporter or 3′UTR PsiCheck2 reporter vectors, respectively, depending on the location of the best predicted miR-10b binding sites. We have also constructed a 5′UTR reporter of miR-10b activity by inserting a single miR-10b complementary site into the 5′UTR of luciferase gene within the
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