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• W1680693606 abstract "Using in silico analysis of The Cancer Genome Atlas (TCGA), we identified microRNAs associated with glioblastoma (GBM) survival, and predicted their functions in glioma growth and progression. Inhibition of two “risky” miRNAs, miR-148a and miR-31, in orthotopic xenograft GBM mouse models suppressed tumor growth and thereby prolonged animal survival. Intracranial tumors treated with uncomplexed miR-148a and miR-31 antagomirs exhibited reduced proliferation, stem cell depletion, and normalized tumor vasculature. Growth-promoting functions of these two miRNAs were, in part, mediated by the common target, the factor inhibiting hypoxia-inducible factor 1 (FIH1), and the downstream pathways involving hypoxia-inducible factor HIF1α and Notch signaling. Therefore, miR-31 and miR-148a regulate glioma growth by maintaining tumor stem cells and their niche, and providing the tumor a way to activate angiogenesis even in a normoxic environment. This is the first study that demonstrates intratumoral uptake and growth-inhibiting effects of uncomplexed antagomirs in orthotopic glioma. Using in silico analysis of The Cancer Genome Atlas (TCGA), we identified microRNAs associated with glioblastoma (GBM) survival, and predicted their functions in glioma growth and progression. Inhibition of two “risky” miRNAs, miR-148a and miR-31, in orthotopic xenograft GBM mouse models suppressed tumor growth and thereby prolonged animal survival. Intracranial tumors treated with uncomplexed miR-148a and miR-31 antagomirs exhibited reduced proliferation, stem cell depletion, and normalized tumor vasculature. Growth-promoting functions of these two miRNAs were, in part, mediated by the common target, the factor inhibiting hypoxia-inducible factor 1 (FIH1), and the downstream pathways involving hypoxia-inducible factor HIF1α and Notch signaling. Therefore, miR-31 and miR-148a regulate glioma growth by maintaining tumor stem cells and their niche, and providing the tumor a way to activate angiogenesis even in a normoxic environment. This is the first study that demonstrates intratumoral uptake and growth-inhibiting effects of uncomplexed antagomirs in orthotopic glioma. Since the discovery of the first oncogenic miRNAs involved in cancer,1Chan JA Krichevsky AM Kosik KS MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells.Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2228) Google Scholar,2He L Thomson JM Hemann MT Hernando-Monge E Mu D Goodson S et al.A microRNA polycistron as a potential human oncogene.Nature. 2005; 435: 828-833Crossref PubMed Scopus (3137) Google Scholar,3Hayashita Y Osada H Tatematsu Y Yamada H Yanagisawa K Tomida S et al.A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation.Cancer Res. 2005; 65: 9628-9632Crossref PubMed Scopus (1377) Google Scholar critical functions of miRNA in dysregulated gene expression underlying human malignancies have been well established.4Iorio MV Croce CM microRNA involvement in human cancer.Carcinogenesis. 2012; 33: 1126-1133Crossref PubMed Scopus (487) Google Scholar The sequence-specific targeting of growth-promoting miRNAs emerges as a new and promising therapeutic strategy in oncology, following the recent success of the first miRNA-based human clinical trials.5Janssen HL Reesink HW Lawitz EJ Zeuzem S Rodriguez-Torres M Patel K et al.Treatment of HCV infection by targeting microRNA.N Engl J Med. 2013; 368: 1685-1694Crossref PubMed Scopus (1729) Google Scholar Others and we have previously identified two onco-miRNAs, miR-21 and miR-10b, as strongly elevated in glioblastoma, the most common malignant brain tumors in adults.1Chan JA Krichevsky AM Kosik KS MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells.Cancer Res. 2005; 65: 6029-6033Crossref PubMed Scopus (2228) Google Scholar,6Sasayama T Nishihara M Kondoh T Hosoda K Kohmura E MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC.Int J Cancer. 2009; 125: 1407-1413Crossref PubMed Scopus (241) Google Scholar,7Gabriely G Yi M Narayan RS Niers JM Wurdinger T Imitola J et al.Human glioma growth is controlled by microRNA-10b.Cancer Res. 2011; 71: 3563-3572Crossref PubMed Scopus (235) Google Scholar Each of these miRNAs downregulates a number of tumor suppressor genes, and their knockdown in glioma cells results in activation of proapoptotic genes, and suppresses cell cycle and invasiveness.7Gabriely G Yi M Narayan RS Niers JM Wurdinger T Imitola J et al.Human glioma growth is controlled by microRNA-10b.Cancer Res. 2011; 71: 3563-3572Crossref PubMed Scopus (235) Google Scholar,8Gabriely G Wurdinger T Kesari S Esau CC Burchard J Linsley PS et al.MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.Mol Cell Biol. 2008; 28: 5369-5380Crossref PubMed Scopus (767) Google Scholar,9Krichevsky AM Gabriely G miR-21: a small multi-faceted RNA.J Cell Mol Med. 2009; 13: 39-53Crossref PubMed Scopus (826) Google Scholar Identification of miR-296, a regulator of glioma-induced angiogenesis,10Würdinger T Tannous BA Saydam O Skog J Grau S Soutschek J et al.miR-296 regulates growth factor receptor overexpression in angiogenic endothelial cells.Cancer Cell. 2008; 14: 382-393Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar further supported the diverse roles of miRNAs in various aspects of gliomagenesis. More recently, miRNAs have been implicated as important regulators of glioblastoma stem cell (GSC) maintenance, epigenetic pathways, metabolism, and invasiveness.11Godlewski J Krichevsky AM Johnson MD Chiocca EA Bronisz A Belonging to a network-microRNAs, extracellular vesicles, and the glioblastoma microenvironment.Neuro Oncol. 2015; 17: 652-662Crossref PubMed Scopus (72) Google Scholar Of note, however, is that none of these miRNAs is significantly associated with survival, suggesting that their targeting may be insufficient as a therapeutic strategy for gliomas. Dysregulated miRNAs are considered to be essential players in carcinogenesis, and thus are potential therapeutic targets. Most previous screens, therefore, were primarily based on differential expression analyses. The Cancer Genome Atlas (TCGA) now enables us to apply a principally different in silico approach for identifying tumor-promoting miRNAs, and predicting the cellular pathways that they may regulate. In this work, we identified two risky miRNAs, miR-148a and miR-31, strongly associated with shortened GBM patient survival and with many biological functions promoting glioma growth. Using orthotopic xenograft models of GBM, we demonstrate that delivery of specific antagomirs to established GBM and the resulting suppression of these miRNAs reduce tumor growth and extend survival. We identify FIH1 as the common direct target of miR-148a and miR-31 that mediates their effects on HIF1α and Notch pathways and, thereby, regulates stem cells and angiogenesis in GBM. To discover critical miRNA regulators of gliomagenesis, we first identified those associated with glioma patient survival. Previous attempts to associate miRNA expression with GBM survival have been made based on the first release of the GBM TCGA data.12Srinivasan S Patric IR Somasundaram K A ten-microRNA expression signature predicts survival in glioblastoma.PLoS One. 2011; 6: e17438Crossref PubMed Scopus (164) Google Scholar,13Kim TM Huang W Park R Park PJ Johnson MD A developmental taxonomy of glioblastoma defined and maintained by MicroRNAs.Cancer Res. 2011; 71: 3387-3399Crossref PubMed Scopus (164) Google Scholar We considered the 5-year survival of an expanded, more recent and divergent TCGA dataset consisting of 240 filtered GBM patients and 98 filtered low-grade glioma (LGG) patients. For each cancer type, we used an iterative approach to classify patients into low and high expression groups for each miRNA. We evaluated the relative survival of the low and high expression groups for each miRNA via the log-rank test, then used Cox proportional hazards regression to classify miRNAs as risky or protective, stratifying on patient age at diagnosis in both analyses. From 534 miRNAs considered in GBM, 25 showed association with survival (Figure 1a; adjusted log-rank P value < 0.03). Sixty-three of 378 miRNAs considered exhibited association with survival in LGG (Supplementary Table S2). Notably, only several common risky (including miR-221/222, miR-135b, and miR-148a) and one protective (miR-9) miRNA have been identified for both GBM and LGG (Supplementary Tables S1 and S2 and Figure S1). We next asked whether miRNAs significantly associated with survival may represent critical regulators of gene expression that actively modulate tumor growth or host response. Our previous work indicated that integrated miRNA and mRNA analysis might suggest potential biological functions for a miRNA regulator.7Gabriely G Yi M Narayan RS Niers JM Wurdinger T Imitola J et al.Human glioma growth is controlled by microRNA-10b.Cancer Res. 2011; 71: 3563-3572Crossref PubMed Scopus (235) Google Scholar We correlated miRNA expression for a subset of survival-associated miRNAs with mRNA expression of ~18,000 genes, using the GBM TCGA database. The lists of mRNAs correlating with selected miRNAs were further analyzed for pathway enrichment patterns using Gene Ontology and GSEA databases. The regulatory functions of several survival-associated miRNAs have been previously reported, allowing us to validate the predictive power of our approach. For example, the risky miR-221/222 family that correlates in GBM TCGA with proliferation, anti-apoptosis, and cell migration-related bioterms (Supplementary Figure S2) has been characterized as an oncogenic cluster that confers therapy resistance and promotes glioma cell invasion and survival.14le Sage C Nagel R Egan DA Schrier M Mesman E Mangiola A et al.Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation.EMBO J. 2007; 26: 3699-3708Crossref PubMed Scopus (698) Google Scholar,15Medina R Zaidi SK Liu CG Stein JL van Wijnen AJ Croce CM et al.MicroRNAs 221 and 222 bypass quiescence and compromise cell survival.Cancer Res. 2008; 68: 2773-2780Crossref PubMed Scopus (264) Google Scholar,16Quintavalle C Garofalo M Zanca C Romano G Iaboni M del Basso De Caro M et al.miR-221/222 overexpession in human glioblastoma increases invasiveness by targeting the protein phosphate PTPμ.Oncogene. 2012; 31: 858-868Crossref PubMed Scopus (154) Google Scholar,17Zhang C Zhang J Hao J Shi Z Wang Y Han L et al.High level of miR-221/222 confers increased cell invasion and poor prognosis in glioma.J Transl Med. 2012; 10: 119Crossref PubMed Scopus (106) Google Scholar A protective miR-9 that correlates with neuroglial differentiation related bioterms (Supplementary Figure S2) is suppressed by mutant EGFR signaling and known to play a tumor-suppressor role in GBM.18Gomez GG Volinia S Croce CM Zanca C Li M Emnett R et al.Suppression of microRNA-9 by mutant EGFR signaling upregulates FOXP1 to enhance glioblastoma tumorigenicity.Cancer Res. 2014; 74: 1429-1439Crossref PubMed Scopus (54) Google Scholar From the list of the top-ranked high-risk miRNAs (Figure 1a), we selected two molecules, miR-148a and miR-31 (Figure 1b), both practically unexplored in the GBM context. While each demonstrated highly diverse levels in GBM, their expression distributions were vastly different. MiR-148a was elevated in the majority of GBM specimens, whereas miR-31 was often downregulated relative to the normal brain tissues (Figure 1c). Nevertheless, they demonstrated overlapping correlation patterns with mRNA bioterms (Figure 1d). Specifically, both miRNAs correlated in TCGA with endothelial cell growth, wound healing, response to external stimulus, and H-Ras oncogene bioterms. In addition, miR-31 expression correlated with proliferation and immune response-related genes, and anticorrelated with glial differentiation, whereas miR-148a correlated with antiapoptosis, extracellular matrix, and response to hypoxia genes and anticorrelated with neuronal differentiation. This bioinformatics analysis suggested complex regulatory functions for these miRNAs, associated not only with gene expression in tumor cells per se, but also in cells of the angiogenic niche and tumor microenvironment. We therefore hypothesized that, in gliomas expressing high levels of miR-148a or miR-31, inhibition of these miRNAs may reduce tumor growth and prolong survival. We also hypothesized that it might cause more pronounced effects in orthotopic GBM models in vivo than in cellular models lacking micro-environmental cues. As the first step toward investigating the roles of miR-148a and miR-31 in gliomagenesis, we tested expression of miR-148a and miR-31 in a panel of glioma cell lines and low passage patient-derived GBM neurospheres. Similar to the high expression range observed in tumor specimens, cultured glioma cells also exhibited widely diverse expression (Figure 2a,b). Both miRNAs were also expressed in primary human brain microvascular endothelial cells, HBECs (Figure 2a,b). To further investigate the functions of miR-148a and miR-31 in glioma, we selected a panel of cell lines and tumor neurospheres that express high or moderate levels of both miRNAs, and utilized highly specific and potent antisense 2’-O-MOE-PO miRNA inhibitors (ASO).19Esau CC Inhibition of microRNA with antisense oligonucleotides.Methods. 2008; 44: 55-60Crossref PubMed Scopus (214) Google Scholar Liposome-based transfection of such antagomirs to LN229, LN382 and U251 glioma cells resulted in a 90% knockdown efficiency (Figure 2c). Suppressing miR-31 or miR-148a in GBM lines, tumor neurospheres, or human primary endothelial cells has not resulted in significant phenotypic changes, nor has it altered cell viability or metabolic activity up to 120 hours post-transfection (Figure 2d). Similarly, there was no effect on cell cycle progression or migration in a wound-healing assay (data not shown). In contrast, we have observed, a prominent effect on cell invasion in some, although not all, glioma lines tested (Figure 2e). Overexpression of miR-148a and miR-31 with corresponding synthetic mimics also has not altered the growth of glioma cell lines (Supplementary Figure S3). In order to better understand the role of these two miRNAs in tumor growth, we employed two orthotopic GBM xenograft models. LN229 and MGG75 gliomas, expressing relatively high levels of both miR-148a and miR-31, have been selected. The cells, transduced with mCherry and Luciferase constructs, were injected into the deep white matter in nude mice, the tumor growth was monitored using the whole body bioimaging, and body weight and survival tracked. LN229 cells formed fast-growing tumors with relatively defined boundary, some infiltrating cells, and rarely detectable small hemorrhage areas or necrotic centers (Figure 3a, left panel). The second, slower-growing model is based on low-passage MGG75 cultures, grown as tumor neurospheres in serum-free media, and thereby better retaining initial genetic and tumorigenic properties.20Wakimoto H Mohapatra G Kanai R Curry Jr, WT Yip S Nitta M et al.Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells.Neuro Oncol. 2012; 14: 132-144Crossref PubMed Scopus (167) Google Scholar The MGG75 tumors were highly diffusive and invasive, with hemorrhagic rim and necrotic center (Figure 3a, right panels). The miR-148a or miR-31 were suppressed in the established tumors by stabilized 2’-α-flouro MOE-PS ASO.21Eckstein F Phosphorothioates, essential components of therapeutic oligonucleotides.Nucleic Acid Ther. 2014; 24: 374-387Crossref PubMed Scopus (341) Google Scholar To ensure efficient and continuous local delivery of either miR-31 or miR-148a inhibitors to the intracranial tumors, and avoid repeated neurosurgeries, we used osmotic pumps (Figure 3b,c). We first examined the effects of knocking down miR-31 or miR-148a in the intracranial LN229. Since this is a very aggressive model, the uncomplexed ASOs were administered to the intracranial tumors starting from day 7 after cell implantation (15 μg daily), when the tumors reached a relative luciferase signal of about ~106 photons/sec and exhibited exponential growth. The osmotic delivery continued over four weeks. To verify the delivery of the antagomirs, LN229 brain tumors were excised, sectioned, and immunostained with an antibody that recognizes the phosphorothioate backbone of the oligonucleotides. The tumor cells showed strong immunoreactivity, indicating the uptake of ASOs by at least 95% of the cells (Figure 4a and Supplementary Figure S5). In addition, the qRT-PCR analysis of RNA extracted from the intracranial LN229 tumors demonstrated that miR-148a and miR-31 become almost undetectable after the corresponding treatments and, thus, validated the high efficacy of osmotic delivery (Figure 4a, right panels).Figure 4Silencing miR-31 or miR-148a in intracranial LN229 tumors reduces tumor burden and extents animal survival. (a) The inhibitors are taken-up by tumor cells, resulting in reduced miR-148a/miR-31 levels. LN229 tumors are immunostained with the antibody detecting the intracellular ASOs (anti-6653, left panel, green). miR-31 or miR-148a expression is markedly diminished in the tumor, as detected by qRT-PCR. Results are normalized by the uniformly expressed miR-99a. Data are average from four to five animals. *P < 0.001. Scale bar = 50 μm. (b) Silencing miR-31 or miR-148a markedly reduces tumor burden. The tumor growth is monitored by luciferase imaging in vivo, and is expressed in photon flux per second. There are eight to nine animals per group at the treatment initiation, and each dot represents an animal/tumor. *P = 0.026; NS is not significant. The insert illustrates tumor imaging in representative animals, 28 days after treatment initiation. (c) Silencing miR-31 or miR-148a helps maintain the body weight in mice bearing intracranial tumors. N = 8–9 animals per group. (d) Silencing miR-31 or miR-148a significantly extends animal survival, analyzed by Kaplan-Meier plot. N = 8–9 animals per group. *P = 0.0279 and **P = 0.0483, by log-rank test. (e) Silencing miR-31 or miR-148a significantly increases tumor cell apoptosis. *P = 0.0062 and **P = 0.0024. Scale bar = 150 μm. Each dot represents one analyzed image. (f) Silencing miR-148a reduces tumor cell proliferation, as indicated by Ki67 proliferative marker. *P = 0.0025 and NS = not significant. Scale bar = 150 μm. (g) Silencing miR-31 or miR-148a normalizes tumor vasculature. Blocking miR-31 or miR-148a leads to the reduction of CD31 positive areas (left panels) and decreases tumor blood vessel diameter (right panel). Tumor vessels look smaller and less torturous. *P < 0.001. Scale bar = 150 μm. (h) Silencing miR-31 or miR-148a diminishes tumor cell self-renewal, as indicated by CD133 staining. Upper panels show the CD133-positive tumor cells. *P < 0.001. Scale bar = 25 μm. (i) Silencing miR-31 or miR-148a reduces tumor invasion. Two representative tumors are shown for each treatment group. The distance between infiltrating tumor cells and tumor mass (M) was measured in three tumors per group, three sites per tumor. Blocking either miRNA reduces the distance between infiltrative tumor cells and tumor mass. *P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Intratumoral inhibition of miR-31 or miR-148a led to pronounced beneficial effects for the animals bearing LN229 tumors (n = 8–9 per group). It markedly reduced the rate of tumor growth (Figure 4b). At least 50% of mice treated with the inhibitors of either miR-31 or miR-148a maintained healthy body weight, whereas the control group treated with a nontargeting oligonucleotide of the same chemistry lost their weight dramatically over the course of the study (Figure 4c). Correspondingly, the inhibition of either miR-31 or miR-148a led to a significant increase in median survival (log rank-test, control versus anti-miR-31, P = 0.0279; control versus anti-miR-148a, P = 0.0483) (Figure 4d). These results support the idea that high levels of either miR-31 or miR-148a contribute to glioma growth and shortened survival in GBM. To explore the mechanism underlying the therapeutic effects of the miRNA inhibitors, at the end point of the study tumors were excised and analyzed (n = 4–5 tumors per group). Inhibition of either miR-148a or miR-31 had significant effects on tumor histopathology. First, it increased the number of apoptotic cells detected by TUNEL (Figure 4e). The number of cells labeled with Ki67, a marker of cell proliferation, was decreased by anti-miR-148a, but not by anti-miR-31 (Figure 4f). As “endothelial cells” bioterm was associated with both miR-148a and miR-31 in GBM TCGA (Figure 1d), and tumor angiogenesis is a critical factor modulating gliomagenesis, we further examined tumor angiogenesis in the LN229 tumors. There was a profound morphological difference between the CD31-stained large abnormal vessels characteristic for the control tumors, and normalized vessels resembling brain vasculature, observed in anti-miR-148a and anti-miR-31 groups. Total CD31-positive areas and vessel diameter distribution were dramatically altered (Figure 4g). Furthermore, blocking either miRNA also reduced the number of GSCs, as indicated by CD133 staining. In case of anti-miR-148a, CD133 positivity dropped almost by 60% (Figure 4h). Finally, we took advantage of the limited invasiveness of LN229 tumors to examine the migratory properties of the control and miRNA targeted groups. Quantitative analysis of cells migrating away from the tumor mass revealed that inhibition of miR-148a or miR-31 resulted in a 50–60% decrease in travel distance (Figure 4i), indicating that tumor cell invasiveness is reduced. Therefore, a number of key characteristics of GBM, including self-renewal and invasiveness of tumor cells, as well as tumor vascularization, have been modulated in the LN229 model by the specific miR-148a and miR-31 inhibitors. The key characteristics of human high-grade glioma include its highly infiltrative nature and the presence of a GSC population that might be responsible for tumor formation, expansion, resilience, and recurrence.22Heddleston JM Hitomi M Venere M Flavahan WA Yang K Kim Y et al.Glioma stem cell maintenance: the role of the microenvironment.Curr Pharm Des. 2011; 17: 2386-2401Crossref PubMed Scopus (62) Google Scholar,23Piccirillo SG Reynolds BA Zanetti N Lamorte G Binda E Broggi G et al.Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.Nature. 2006; 444: 761-765Crossref PubMed Scopus (979) Google Scholar To validate our conclusions in an animal model that more closely recapitulates human pathology, we employed the MGG75 tumor neurosphere model that forms highly invasive and infiltrative intracranial GBM (Figure 3a). In cultures at least 90–95% of the MGG75 neurospheres were positive for neural stem cell markers nestin and SOX2 (data not shown). Small spheres were intracranially implanted and the experiments performed similarly to those described above for LN229, with the exception of treatment being initiated at 100 days postinjection, due to slower tumor engraftment and progression. As previously detected in the LN229 model, continuous inhibition of miR-31 or miR-148a also produced marked health beneficial effects in animals bearing MGG75 intracranial tumors (Figure 5). First, it suppressed tumor growth rate (Figure 5a). Second, while 90% of the animals treated with the control ASO lost at least 20% body weight and showed high morbidity or mortality, only 10 and 40% of anti-miR-31 and anti-miR-148a animals, respectively, showed some weight loss (Figure 5b). Consequently, animal survival was extended significantly (Figure 5c). To provide further insights into the delayed tumor growth and pathology, we stained and quantified the tumor sections for cell proliferation marker Ki67, the stem cell markers SOX2 and nestin, and tumor angiogenesis marker CD31. There was a significant reduction of Ki67, SOX2, and CD31 immunolabeling in the brain tumor sections of animals treated with anti-miR-31 or anti-miR-148a (Figure 5d,e,g). Though the reduction of nestin signal did not reach statistical significance, a decreasing trend was also observed (Figure 5f).Figure 5Silencing miR-31 or miR-148a in intracranial MGG75 tumors reduces tumor burden and extents animal survival. (a) Continuous delivery of miRNA inhibitors over a period of 8 weeks significantly reduces MGG75 growth rate, as monitored by luciferase imaging. N = 8–9 mice per group. *P < 0.05 in all time points examined. (b) Silencing miR-31 or miR-148a helps maintain the body weight in mice bearing MGG75 tumors. N = 10–11 mice per group. (c) Silencing miR-31 or miR-148a significantly extends survival of MGG75-bearing mice, as analyzed by Kaplan-Meier plot. N = 10–11 mice per group. *P < 0.0001 and **P = 0.0251, by log-rank test. (d) Silencing miR-31 or miR-148a reduces tumor cell proliferation, as indicated by Ki67 proliferative marker. *P <0.0001. Scale bar = 150 μm. Each dot represents an analyzed image. (e) Silencing miR-31 or miR-148a markedly reduces percentage of SOX2-positive cells. *P < 0.0001. Scale bar = 150 μm. (f) Silencing miR-31 or miR-148a slightly reduces percentage of nestin-positive cells. NS, not significant. Scale bar = 150 μm. (g) Silencing miR-31 or miR-148a normalizes tumor vasculature. Blocking miR-31 or miR-148a reduces the percentage of abnormal blood vessels, and decreases tumor blood vessel diameter (data not shown). Tumor vessels look smaller and less torturous. *P < 0.01. Scale bar = 150 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Human TCGA datasets and experiments with mouse orthotopic glioma xenografts support the idea that suppressing miR-31 and miR-148a activity could slow down glioma growth rate and extend survival. Remarkably, the in vivo consequences of inhibiting either miR-148a or miR-31 were surprisingly similar. The slower growth of tumors with suppressed miR-148a or miR-31 can be likely attributed to the reduced tumor cell proliferation (Ki67), number of GSCs (CD133/SOX2/NESTIN), and partly normalized tumor blood vessels. This led us to hypothesize that miR-31 and miR-148a may regulate common targets and downstream pathways in the GBM microenvironment. We, therefore, sought to identify common targets for miR-31 and miR-148a that could be involved in the self-renewal and angiongenesis-related phenotypes, observed in our experiments. Computational algorithms predicted numerous putative targets for miR-31 and miR-148a, and some of them have important functions in angiogenesis and stem cell maintenance (Table 1 and Supplementary Table S3). We found that one of the targets potentially shared by miR-148a and miR-31, hypoxia inducing factor 1α inhibitor (HIF1AN) also known as the factor inhibiting hypoxia (FIH1; predicted by three out of five algorithms for miR-31, and by two algorithms for miR-148a), is indeed regulated by each of these miRNAs in glioma cells (Figure 6a). Other tested putative targets (PTEN, EGLN3, ACVR1, DICER1, AKAP7, BACH2, CUL5, NARG1, NFAT5, SLC24A3, and SULF1) were not regulated in glioma cells (data not shown). FIH1 is an asparaginyl hydroxylase that negatively regulates HIF1α stability and transcriptional activity in normoxic conditions; it is destabilized by hypoxia, which leads to HIF1α transcription and thereby upregulation of VEGF and angiogenesis.24Lisy K Peet DJ Turn me on: regulating HIF transcriptional activity.Cell Death Differ. 2008; 15: 642-649Crossref PubMed Scopus (172) Google Scholar,25Majmundar AJ Wong WJ Simon MC Hypoxia-inducible factors and the response to hypoxic stress.Mol Cell. 2010; 40: 294-309Abstract Full Text Full Text PDF PubMed Scopus (1633) Google Scholar FIH1 also interacts with and hydroxylates Notch intracellular domain (NICD), and thereby represses NICD transcription that is critical for both angiogenesis and maintaining neural stem cell properties.26Gustafsson MV Zheng X Pereira T Gradin K Jin S Lundkvist J et al.Hypoxia requires notch signaling to maintain the undifferentiated cell state.Dev Cell. 2005; 9: 617-628Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar,27Zheng X Linke S Dias JM Zheng X Gradin K Wallis TP et al.Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways.Proc Natl Acad Sci USA. 2008; 105: 3368-3373Crossref PubMed Scopus (199) Google Scholar Furthermore, HIF1α and Notch have been shown to interact in GBM, and HIF1α requires Notch pathway for the maintenance of GSCs.28Qiang L Wu T Zhang HW Lu N Hu R Wang YJ et al.HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway.Cell Death Differ. 2012; 19: 284-294Crossref PubMed Scopus (216) Google Scholar FIH1 therefore may represent a very attractive common target for miR-31 and -148a implicated in GBM.Table 1Common computationally predicted targets for both miR-148a and miR-31 that are associated with angiogenesis, cellular response to hypoxia, and stem cell (SC) maintenance biotermsGene nameGO termsNumber of Programs predicting miR-148aNumber of Programs predicting miR-31Pearson Correlation miR-148aP value; miR-148aPearson Correlation miR-31P value miR-31ACVR1Angiogenesis410.1410.0210.224<0.001BACH1Hypoxia11−0.0250.6880.0030.95" @default.
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• W1680693606 date "2015-07-01" @default.
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• W1680693606 title "The Cancer Genome Atlas Analysis Predicts MicroRNA for Targeting Cancer Growth and Vascularization in Glioblastoma" @default.
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