Identification of intrinsic subtype-specific prognostic microRNAs in primary glioblastoma

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant type of glioma. Integrated classification based on mRNA expression microarrays and whole–genome methylation subdivides GBM into five subtypes: Classical, Mesenchymal, Neural, Proneural-CpG island methylator phenotype (G-CIMP) and Proneural-non G-CIMP. Biomarkers that can be used to predict prognosis in each subtype have not been systematically investigated. METHODS: In the present study, we used Cox regression and risk-score analysis to construct respective prognostic microRNA (miRNA) signatures in the five intrinsic subtypes of primary glioblastoma in The Cancer Genome Atlas (TCGA) dataset. RESULTS: Patients who had high-risk scores had poor overall survival compared with patients who had low-risk scores. The prognostic miRNA signature for the Mesenchymal subtype (four risky miRNAs: miR-373, miR-296, miR-191, miR-602; one protective miRNA: miR-223) was further validated in an independent cohort containing 41 samples. CONCLUSION: We report novel diagnostic tools for deeper prognostic sub-stratification in GBM intrinsic subtypes based upon miRNA expression profiles and believe that such signature could lead to more individualized therapies to improve survival rates and provide a potential platform for future studies on gene treatment for GBM

PKM2 promotes glucose metabolism and cell growth in gliomas through a mechanism involving a let-7a/c-Myc/hnRNPA1 feedback loop

AbstrAct Tumor cells metabolize more glucose to lactate in aerobic or hypoxic conditions than non-tumor cells. Pyruvate kinase isoenzyme type M2 (PKM2) is crucial for tumor cell aerobic glycolysis. We established a role for let-7a/c-Myc/hnRNPA1/PKM2 signaling in glioma cell glucose metabolism. PKM2 depletion via siRNA inhibits cell proliferation and aerobic glycolysis in glioma cells. C-Myc promotes up-regulation of hnRNPA1 expression, hnRNPA1 binding to PKM pre-mRNA, and the subsequent formation of PKM2. This pathway is downregulated by the microRNA let-7a, which functionally targets c-Myc, whereas hnRNPA1 blocks the biogenesis of let-7a to counteract its ability to downregulate the c-Myc/hnRNPA1/PKM2 signaling pathway. The down-regulation of c-Myc/ hnRNPA1/PKM2 by let-7a is verified using a glioma xenograft model. These results suggest that let-7a, c-Myc and hnRNPA1 from a feedback loop, thereby regulating PKM2 expression to modulate glucose metabolism of glioma cells. These findings elucidate a new pathway mediating aerobic glycolysis in gliomas and provide an attractive potential target for therapeutic intervention

Evaluation of biological functions of H19 in U87 and U251 cells.

<p>(A) Transfection efficiency of si-H19 in U87 and U251 cells was indicated by PCR. (B–C) si-H19 inhibited the invasiveness of U87 and U251 cells. Cells were examined for cell invasion in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet. The invasiveness of U87 and 251 cells was attenuated with the decreased expression of H19. (D–E) Wound healing assay of glioma cells transfected with either control or the si-H19, respectively. The wound healing was photographed at different time points and wounded gaps were analyzed by measuring the distance of migrating cells for 3 different areas for each wound. *P<0.05, **P<0.01.</p

H19 expression in glioma tissues.

<p>(A) H19 levels were analyzed in glioma tissues of the CGGA glioma datasets (61 cases of grade II, 33 cases of grade III and 64 cases of grade IV). (B) The expression of H19 was analyzed in glioma tissues of the GSE16011 glioma datasets. (C–D) H19 expression with two probes was analyzed in glioma tissues of the Rembrant glioma datasets.</p

Effect of H19/miR-675 signaling on glioma cell biology.

<p>(A) CDH13 expression changes following transfection with si-H19 and miR-675 identified by Western blots. (B–C) Effect of si-H19 and miR-675 on glioma cell invasion, after differential treatment in the <i>in vitro</i> transwell invasion assay. (D–E) Cell migration change was analyzed by scratch wound assay. *P<0.05, **P<0.01.</p

Long Non-Coding RNA H19 Promotes Glioma Cell Invasion by Deriving miR-675

<div><p><i>H19</i> RNA has been characterized as an oncogenic long non-coding RNA (lncRNA) in breast and colon cancer. However, the role and function of lncRNA H19 in glioma development remain unclear. In this study, we identified that H19/miR-675 signaling was critical for glioma progression. By analyzing glioma gene expression data sets, we found increased H19 in high grade gliomas. H19 depletion via siRNA inhibited invasion in glioma cells. Further, we found H19 positively correlated with its derivate miR-675 expression and reduction of H19 inhibited miR-675 expression. Bioinformatics and luciferase reporter assays showed that miR-675 modulated Cadherin 13 expression by directly targeting the binding site within the 3′ UTR. Finally, introduction of miR-675 abrogated H19 knockdown-induced cell invasion inhibition in glioma cells. To our knowledge, it is first time to demonstrate that H19 regulates glioma development by deriving miR-675 and provide important clues for understanding the key roles of lncRNA-miRNA functional network in glioma.</p></div

H19 regulates the expression of miR-675 which is associated with cell invasion.

<p>(A) MiR-675 expression in 158 glioma tissues of the CGGA glioma datasets. (B) The data shows that miR-675 positively correlated with H19, in 158 glioma samples. (C–D) MiR-675 positively correlated with H19 in HGG of 158 glioma samples. (E) si-H19 decreases miR-675 expression compared with NC in U87 and U251 cells. (F–G) The effects of miR-675 inhibitor on the invasion of U87 and U251 cells were assessed by transwell invasion assay. (H–I) The results of in vitro scratch wound assay showed that knockdown miR-675 inhibit the migration of glioma cells. *P<0.05, **P<0.01.</p

CDH13 is a directly target of miR-675.

<p>(A) Correlations of miR-675 with CDH13 in glioma tissues. (B) Putative binding sites of miR-675 within theCDH13 3′UTR, as predicted by miRanda and Pictar alghorithms. (C) CDH13 protein levels were measured in U87 and U251 cells at 48 h post-transfection. (D) MiR-675 down-regulated luciferase activities controlled by wild-type CDH13 3′UTR, but did not affect luciferase activity controlled by mutant CDH13 3′UTR. The luciferase activity was measured by dual-luciferase reporter assay (Promega) and was normalized to Renilla luciferase activity. *P<0.05, **P<0.01.</p

An Axis Involving SNAI1, microRNA-128 and SP1 Modulates Glioma Progression

<div><p>Background</p><p>Glioblastoma is an extraordinarily aggressive disease that requires more effective therapeutic options. Snail family zinc finger 1, dysregulated in many neoplasms, has been reported to be involved in gliomas. However, the biological mechanisms underlying SNAI1 function in gliomas need further investigation.</p><p>Methods</p><p>Quantitative real-time PCR was used to measure microRNA-128 (miR-128) expression level and western blot was performed to detect protein expression in U87 and U251 cells and human brain tissues. Cell cycle, CCK-8, transwell and wound-healing assays were performed. Dual-luciferase reporter assay was used for identifying the mechanism of SNAI1 and miR-128b regulation. The mechanism of miR-128 targeting SP1 was also tested by luciferase reporter assay. Immunohistochemistry and in situ hybridisation staining were used for quantifying SNAI1, SP1 and miR-128 expression levels in human glioma samples.</p><p>Results</p><p>The Chinese Glioma Genome Atlas (CGGA) data revealed that SNAI1 was up-regulated in glioma and we confirmed the findings in normal and glioma tissues. SNAI1 depletion by shRNA retarded the cell cycle and suppressed proliferation and invasion in glioma cell lines. The CGGA data showed that the Pearson correlation index between SNAI1 and miR-128 was negatively correlated. SNAI1 suppressed miR-128b expression by binding to the miR-128b specific promoter motif, and miR-128 targeted SP1 via binding to the 3′-untranslated region of SP1. Moreover, introduction of miR-128 anti-sense oligonucleotide alleviated the cell cycle retardation, proliferation and invasion inhibition induced by SNAI1 shRNA. Immunohistochemistry and in situ hybridisation analysis of SNAI1, SP1 and miR-128 unraveled their expression levels and correlations in glioma samples.</p><p>Conclusions</p><p>We propose that the SNAI1/miR-128/SP1 axis, which plays a vital role in glioma progression, may come to be a clinically relevant therapeutic target.</p></div