Normal Brain Tissue Samples Research Articles

Reduced expression of microRNA-134 correlates with malignancy and poor prognosis in human glioma

MicroRNA-134 (miR-134) has been demonstrated to be dysregulated in glioma tissues. However, its clinical significance in this tumor type has not been fully elucidated. This study was designed to explore the association of miR-134 expression with clinicopathological features and prognosis in human glioma. Using real-time quantitative polymerase chain reaction, miR-134 expression was detected in 162 glioma specimens with various World Health Organization (WHO) grades and compared to the expression in 36 normal brain tissue samples. Glioma tissues exhibited significantly reduced expression of miR-134 (mean 2.15±standard deviation 0.82 versus 4.37±1.16, p<0.001) compared with normal brain tissues. In addition, miR-134 expression was notably associated with WHO grade (p<0.001) and Karnofsky Performance Scale score (KPS; p=0.02) in glioma tissues. Low miR-134 expression occurred more frequently in glioma tissues with high WHO grades and low KPS scores. In univariate analysis, both progression-free survival (PFS) and overall survival (OS) of glioma patients with low miR-134 expression were significantly shorter than those with high miR-134 expression (both p<0.001). Additionally, glioma patients with high WHO grades, low KPS scores and subtotal resection attained significantly poorer PFS (p<0.001, 0.02 and 0.01, respectively) and OS (p<0.001, 0.01 and 0.01, respectively). In multivariate analysis, miR-134 expression, WHO grade, KPS score and extent of resection were identified as the independent prognostic factors for both PFS and OS. Collectively, our data prove that the reduced expression of miR-134 may predict aggressive progression and poor prognosis in human gliomas. miR-134 may represent both a prognostic marker and a novel therapeutic target for this malignancy.

Tenascin-C: a novel candidate marker for cancer stem cells in glioblastoma identified by tissue microarrays.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133+/TNC+ and CD133–/TNC+ cell populations is much higher than that of the CD133+/TNC– and CD133–/TNC– populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC+ population is identified as a CSC population overlapping with part of the CD133– cell population.

Clinical and prognostic significance of high-mobility group box-1 in human gliomas.

The objective of this study was to explore the expression and the clinical and prognostic significance of high-mobility group box-1 (HMGB1) in human gliomas. The expression of HMGB1 in 15 samples of normal brain tissue and 65 samples of different-grade glioma tissue was assayed using immunohistochemistry and western blot analysis. The associations between the differences in expression and pathology grades were analyzed statistically. Uni- and multivariate analyses were performed to investigate the prognostic value of HMGB1 expression and its expression levels. The positive rates of HMGB1 expression in normal brain and glioma tissue were 20.0% (3/15) and 76.9% (50/65), respectively. The expression of HMGB1 in glioma tissue was higher than that in normal tissue (P<0.05). The positive rates of HMGB1 expression in low-grade gliomas (LGGs, grades I and II) and high-grade gliomas (HGGs, grades III and IV) were 63.0% (17/27) and 86.8% (33/38), respectively, and the positive rates in HGG were higher than those in LGG (P=0.024). Western blot analysis showed that HMGB1 was also expressed in normal brain tissue. The expression levels in HGG were significantly higher than those in LGG (P<0.001). HMGB1-positive patients had significantly shorter overall survival times compared with HMGB1-negative patients (P=0.026). Increasing levels of HMGB1 expression significantly correlated with reduced survival times when all patients with glioma were considered (P=0.045). In conclusion, HMGB1 positivity and protein expression levels are of significant clinical and prognostic value in human gliomas. Detecting HMGB1 in human gliomas may be useful for assessing the degree of malignancy, and HMGB1 would appear to be a promising target in the clinical management of patients with glioma.

MiR-21 up-regulation mediates glioblastoma cancer stem cells apoptosis and proliferation by targeting FASLG.

To investigate whether miR-21 can affect the apoptosis and proliferation of glioblastoma cancer stem cells (GSCs) from down-regulating FASLG. The expression of miRNA-21 was detected by quantitative real-time PCR in normal brain tissue and glioblastoma samples, and the changes of miRNA-21 expression between GSCs and non-GSCs were also detected. The apoptosis and proliferation ability of miR-21 in GSCs were analyzed by MTT and flow cytometry assay after anti-miR-21 transfection. For the regulation mechanism analysis of miR-21, TargetScan, PicTar and microRNA were selected to predict some potential target genes of miR-21. The predicted gene was identified to be the direct and specific target gene of miR-21 by luciferase activities assay and western blot. RNA interference technology was used to confirm the apoptosis and proliferation effects of miR-21 were directly induced by FASLG. The expression of miR-21 increased significantly in glioblastoma contrast to normal brain tissue, and miR-21 up-regulated in GSCs remarkably. The proliferation of GSCs cell could be inhibited with high-expression of miR-21 and this effect could be restored by miR-21 knocked down. Mechanism analysis revealed that FASLG was a specific and direct target gene of miR-21. The advanced effects of anti-miR-21 on GSCs apoptosis and proliferation were mediated by expression of silenced FASLG. In summary, aberrantly expressed miR-21 regulates GSCs apoptosis and proliferation partly through directly down-regulating FASLG protein expression in GSCs and this might offer a new potential therapeutic stratagem for glioblastoma.

Abstract 5268: The expression of lemur tyrosine kinase-3 in high grade pediatric tumors

Abstract High grade gliomas are the most common primary brain tumors in adults, but also occur in childhood, with a poor prognosis in all age groups . Lemur tyrosine kinase 3 (LMTK3) is a transmembrane tyrosine kinase that seems to play a pivotal role in oncogenesis in breast cancer. It regulates the estrogen receptor alpha and is evolutionarily selected for in humans compared to non-human primates. LMTK3 expression levels were strongly associated with survival and therapy response.This study assesses the role of LMTK3 in pediatric high grade brain tumors at both the protein and mRNA levels. We initially assessed by immunohistochemistry (IHC) the expression levels of LMTK3 in well described cohorts of patient specimens with brain cancer. First, we performed an IHC analysis using a brain tumor tissue microarray (TMA) purchased from US Biomax (#T175), in order to examine the LMTK3 levels in a variety of brain tumors. Preliminary analysis revealed variable LMTK3 expression levels in brain tissue, scored as negative, low, moderate and high. We demonstrate that LMTK3 appears to be expressed at low levels at normal brain tissue samples and epithelial meningioma tissues (benign tumors). On the contrary, LMTK3 was expressed at different levels in cases with grade 2 or grade 4 gliomas. Further immunohistochemical analysis was therefore undertaken on a selection of TMAs containing pediatric high grade gliomas. Genome wide gene expression analysis (using the Affymetrix U133plus2 chip) previously undertaken on a cohort of pediatric high grade gliomas demonstrated measurable LMTK3 mRNA expression in many samples. Our preliminary analysis was correlated with various tumor characteristics that are available in Nottingham datasets (including demographic and overall survival data). In conclusion, LMTK3 is expressed in pediatric high grade gliomas and may have differential expression depending on the histological grade of the tumor. This work will lead to larger scale studies to elucidate the exact role of LMTK3 in high grade brain neoplasms. Citation Format: Nektarios K. Mazarakis, Stuart Smith, Aikaterini Vraka, Donald Macarthur, Richard Grundy, Justin Stebbing, George Giamas. The expression of lemur tyrosine kinase-3 in high grade pediatric tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2014-5268

Abstract 4354: miR-603 targets WIF1 to promote glioma tumorigenesis via Wnt/β-catenin pathway

Abstract Glioma is the most common and deadly brain tumor. In spite of progressive treatments, which include surgery, chemotherapy and radiotherapy, patient prognosis has not improved significantly. Thus emerges the urgent need to discover novel targets that effectively control glioma. The aim of our work is to investigate the possible roles of miR-603 in glioma tumorigenesis. We first examined miR-603 expression in glioma patient samples and cell lines using RT-PCR. Then, the effect of miR-603 on glioma cells was determined by MTS assay, neurosphere formation assay, EdU assay and cell cycle analysis. Western blot, immunofluorescence staining and luciferase assay were conducted to analyze the regulation of Wnt/β-catenin signaling pathway by miR-603. Lastly, nude mice were subject to intracranial xenograft of U87 cells to determine miR-603's function on glioma oncogenesis in vivo. Herein, we report, for the first time, that miR-603 is highly expressed in glioma tissue and cell lines (U87vlll, U87, T98G, LN229, U373 and U251), suggesting its prominence in glioma tumorigenesis (p &amp;lt; 0.01). Transfection of miR-603 inhibitor into U87 and U373 glioma cells revealed a significant inhibition of cell proliferation, cell cycle progression and neurosphere formation. Conversely, overexpression of miR-603 remarkably promoted these effects. From publicly available algorithms, we identified WIF1 as a potential target of miR-603. WIF1 is known to be a Wnt/β-catenin pathway inhibitor that impedes cancer cell proliferation. As expected, western blot analysis showed that WIF1 expression was markedly decreased in U87 and U373 cells transfected with miR-603, and increased in cells transfected with miR-603-inhibitor. Moreover, we confirmed WIF1 is a bona fide target of miR-603 by 3′UTR luciferase reporter assay. The negative correlation between WIF1 and miR-603 expression was shown by Pearson correlation in four normal brain tissue samples and 43 primary glioma tissue samples. Next, we found that overexpression of miR-603 could attenuate nuclear β-catenin levels and TOPflash luciferase activity, indicating that miR-603 inhibits Wnt/β-catenin signaling pathway. In accordance with our results, WIF1 and TCF4 depletion using specific siRNA dramatically suppressed the cell proliferation, cell cycle progression and neurosphere formation induced by miR-603, thus confirming that WIF1 and Wnt/β-catenin signaling pathway are functionally involved in miR-603 regulation of glioma cancer cell growth. Furthermore, nude mice stereotactically implanted with Lentivirus-miR-603-U87 cells in the right cerebral hemisphere displayed significant inhibition of WIF1 expression, shortened survival time and increased tumor size when compared with mice implanted with Lentivirus-miR-control-U87 cells. Collectively, miR-603 regulates glioma development via target WIF1, and could potentially be used in therapeutic applications for glioma treatment. Note: This abstract was not presented at the meeting. Citation Format: Mian Guo, Guangzhi Wang, Kevork Khadarian, Yongri Zheng, Albert D. Ha, Jia Shen. miR-603 targets WIF1 to promote glioma tumorigenesis via Wnt/β-catenin pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4354. doi:10.1158/1538-7445.AM2014-4354

WE‐E‐17A‐10: Singular Value Decomposition Identifies Transcript Length Distribution Functions from DNA Microarray Data and Reveals Evolutionary Forces Globally Affecting Glioblastoma Multiforme Metabolism

Purpose:To search for evolutionary forces that might act upon eukaryotic mRNA transcript length, e.g., as a normal cell is transformed to a tumor cell.Methods:We use the singular value decomposition (SVD) to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as “asymmetric generalized coherent states” from the DNA microarray data and with no a‐priori assumptions [Alter and Golub, Proc. Natl. Acad. Sci. USA 103 (32), pp. 11828‐11833 (2006); doi: 10.1073/pnas.0604756103].Results:Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM) or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity [Bertagnolli, Drake, Tennessen and Alter, PLoS One 8 (11), article e78913 (2013); doi: 10.1371/journal.pone.0078913].Conclusion:These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length‐dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.This research was supported by the Utah Science, Technology and Research (USTAR) Initiative, National Human Genome Research Institute (NHGRI) R01 Grant HG‐004302 and National Science Foundation (NSF) CAREER Award DMS‐0847173

Reductions in the expression of miR-124-3p, miR-128-1, and miR-221-3p in pediatric astrocytomas are related to high-grade supratentorial, and recurrent tumors in Mexican children

Astrocytomas are the most frequent type of tumor of the central nervous system in children. Hence, it is important to describe markers that may improve our understanding of their behavior. Mature microRNAs (miRNAs) may be such biological markers. They are small molecules of RNA that regulate gene expression post-transcriptionally. Due to their importance in cancer, the objective of the present study was to determine the profile of expression of precursor and mature forms of miR-124-3p, miR-128-1, and miR-221-3p using RT-qPCR in pediatric samples. A total of 57 astrocytomas embedded in paraffin were selected. As controls, the study included 13 samples of normal brain tissue. Three of eight miRNAs were selected after a preliminary screening. All the miRNAs showed higher levels of expression in normal brain tissue. The expression of miR-124-3p and miR-128-1 decreased in astrocytomas than in normal brain tissue in all grades (p < 0.05 in both cases), and this reduction was most evident in GIV (407- and 1,469-fold, respectively); however, the expression of the precursor forms pre-miR-128-1 and pre-miR-221 was higher in GIV (3.5-fold) than in GI. The levels of miR-128-1 were higher in infratentorial tumors than in supratentorial cases (p = 0.006). Finally, the expression of miR-221-3p was higher in non-recurrent tumors and live patients (p = 0.0185 and p = 0.0004, respectively). The low expression of these miRNAs may constitute a potential marker of astrocytomas that correlates with localization, possibly due to alterations in the maturation processes of these miRNAs that produced low mature forms in patients with recurrent pediatric astrocytomas.

DNA methylation alterations in grade II- and anaplastic pleomorphic xanthoastrocytoma

BackgroundPleomorphic xanthoastrocytoma (PXA) is a rare WHO grade II tumor accounting for less than 1% of all astrocytomas. Malignant transformation into PXA with anaplastic features, is unusual and correlates with poorer outcome of the patients.MethodsUsing a DNA methylation custom array, we have quantified the DNA methylation level on the promoter sequence of 807 cancer-related genes of WHO grade II (n = 11) and III PXA (n = 2) and compared to normal brain tissue (n = 10) and glioblastoma (n = 87) samples. DNA methylation levels were further confirmed on independent samples by pyrosequencing of the promoter sequences.ResultsIncreasing DNA promoter hypermethylation events were observed in anaplastic PXA as compared with grade II samples. We further validated differential hypermethylation of CD81, HCK, HOXA5, ASCL2 and TES on anaplastic PXA and grade II tumors. Moreover, these epigenetic alterations overlap those described in glioblastoma patients, suggesting common mechanisms of tumorigenesis.ConclusionsEven taking into consideration the small size of our patient populations, our data strongly suggest that epigenome-wide profiling of PXA is a valuable tool to identify methylated genes, which may play a role in the malignant progression of PXA. These methylation alterations may provide useful biomarkers for decision-making in those patients with low-grade PXA displaying a high risk of malignant transformation.

Over-expression of ARHI decreases tumor growth, migration, and invasion in human glioma

This study was conducted to evaluate the role of tumor suppressor gene ras homologue member I (ARHI) in human glioma tumors. We examined expression of ARHI in human glioma tumors and normal brain tissue and also in 4 different glioma cell lines. Furthermore, the effects of ARHI over-expression produced by cellular transfection on the growth of human glioma U251 cells cultured in vitro were also studied. Expression of ARHI was evaluated in samples of glioma tumors obtained from 59 patients who underwent surgery at the Department of Neurosurgery, Fujian Medical University Union Hospital, Fuzhou, China. Ten samples of normal brain tissue were used as controls. Additionally, in vitro studies were conducted in which a recombinant vector carrying ARHI cDNA was constructed and transfected into U251 glioma cells with reduced expression of ARHI. Following transfection, the MTT assay, flow cytometry, TUNEL procedure, Transwell assay, and wound-healing test were employed to evaluate the biological functions of ARHI in U251 glioma cells in vitro. Analyses of mRNA and protein expression revealed that ARHI was significantly down-regulated in glioma tissues as well as in 4 malignant glioma cell lines. Over-expression of ARHI resulted in suppression of glioma cell proliferation, arrest of cell cycle progression, reduction in cell migration and invasion, and promotion of cell apoptosis. Collectively, our data highlight the importance of ARHI in glioma progression and provide the first biological basis for ARHI as a novel candidate target for gene therapy of glioma.

MiR-210 up-regulation inhibits proliferation and induces apoptosis in glioma cells by targeting SIN3A.

BackgroundThe aim of this study was to determine whether miR-210 can affect the apoptosis and proliferation of human U251 glioma cells from down-regulating SIN3A.Material/MethodsThe expression of miRNA-210 was detected by quantitative real-time PCR in normal brain tissue and glioma samples. The apoptosis and proliferation ability of U251 cells were analyzed by MTT and flow cytometry assay after anti-miR-210 transfection. For the regulation mechanism analysis of miR-210, TargetScan, PicTar, and microRNA were selected to predict some potential target genes of miR-210. The predicted gene was identified to be the direct and specific target gene of miR-210 by luciferase activities assay and Western blot. RNA interference technology was used to confirm that the apoptosis and proliferation effects of miR-210 were directly induced by SIN3A.ResultsThe expression of miR-210 increased significantly in glioma in comparison with normal brain tissue. The silence of miR-210 expression could inhibit the proliferation of U251 cells and induce the apoptosis. Mechanism analysis revealed that SIN3A was a specific and direct target gene of miR-210. The siRNA-SIN3A could down-regulate the expression of SIN3A protein, which was up-regulated in U251 cells by anti-miR-210 transfection, and our experiments found that silence of SIN3A could inhibit the apoptosis and sharply increase the proliferation of U251 cells. The regulation effects of anti-miR-210 on apoptosis and proliferation can be reversed respectively by the expression silence of SIN3A.ConclusionsAberrantly expressed miR-210 regulates human U251 glioma cells apoptosis and proliferation partly through directly down-regulating SIN3A protein expression. This might offer a new potential therapeutic stratagem for glioma.

SVD Identifies Transcript Length Distribution Functions from DNA Microarray Data and Reveals Evolutionary Forces Globally Affecting GBM Metabolism

To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD) to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as “asymmetric generalized coherent states” from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM) or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

SAMSN1 Is Highly Expressed and Associated with a Poor Survival in Glioblastoma Multiforme

ObjectivesTo study the expression pattern and prognostic significance of SAMSN1 in glioma. MethodsAffymetrix and Arrystar gene microarray data in the setting of glioma was analyzed to preliminarily study the expression pattern of SAMSN1 in glioma tissues, and Hieratical clustering of gene microarray data was performed to filter out genes that have prognostic value in malignant glioma. Survival analysis by Kaplan-Meier estimates stratified by SAMSN1 expression was then made based on the data of more than 500 GBM cases provided by The Cancer Genome Atlas (TCGA) project. At last, we detected the expression of SAMSN1 in large numbers of glioma and normal brain tissue samples using Tissue Microarray (TMA). Survival analysis by Kaplan-Meier estimates in each grade of glioma was stratified by SAMSN1 expression. Multivariate survival analysis was made by Cox proportional hazards regression models in corresponding groups of glioma. ResultsWith the expression data of SAMSN1 and 68 other genes, high-grade glioma could be classified into two groups with clearly different prognoses. Gene and large sample tissue microarrays showed high expression of SAMSN1 in glioma particularly in GBM. Survival analysis based on the TCGA GBM data matrix and TMA multi-grade glioma dataset found that SAMSN1 expression was closely related to the prognosis of GBM, either PFS or OS (P<0.05). Multivariate survival analysis with Cox proportional hazards regression models confirmed that high expression of SAMSN1 was a strong risk factor for PFS and OS of GBM patients. ConclusionSAMSN1 is over-expressed in glioma as compared with that found in normal brains, especially in GBM. High expression of SAMSN1 is a significant risk factor for the progression free and overall survival of GBM.

Optical imaging for brain tissue characterization using relative fluorescence lifetime imaging

An autofluorescence lifetime wide-field imaging system that can generate contrast in underlying tissue structures of normal and malignant brain tissue samples with video rate acquisition and processing time is presented. Images of the investigated tissues were acquired with high resolution (∼35 μm) using an algorithm to produce contrast based on differences in relative lifetimes. Sufficient contrast for delineation was produced without the computation of fluorescence decay times or Laguerre coefficients. The imaged tissues were sent for histological analysis that confirmed the detected imaged tissues morphological findings and correlations between relative lifetime maps and histology identified.

Telomere Length Modulation in Human Astroglial Brain Tumors

BackgroundTelomeres alteration during carcinogenesis and tumor progression has been described in several cancer types. Telomeres length is stabilized by telomerase (h-TERT) and controlled by several proteins that protect telomere integrity, such as the Telomere Repeat-binding Factor (TRF) 1 and 2 and the tankyrase-poli-ADP-ribose polymerase (TANKs-PARP) complex.ObjectiveTo investigate telomere dysfunction in astroglial brain tumors we analyzed telomeres length, telomerase activity and the expression of a panel of genes controlling the length and structure of telomeres in tissue samples obtained in vivo from astroglial brain tumors with different grade of malignancy.Materials and MethodsEight Low Grade Astrocytomas (LGA), 11 Anaplastic Astrocytomas (AA) and 11 Glioblastoma Multiforme (GBM) samples were analyzed. Three samples of normal brain tissue (NBT) were used as controls. Telomeres length was assessed through Southern Blotting. Telomerase activity was evaluated by a telomere repeat amplification protocol (TRAP) assay. The expression levels of TRF1, TRF2, h-TERT and TANKs-PARP complex were determined through Immunoblotting and RT-PCR.ResultsLGA were featured by an up-regulation of TRF1 and 2 and by shorter telomeres. Conversely, AA and GBM were featured by a down-regulation of TRF1 and 2 and an up-regulation of both telomerase and TANKs-PARP complex.ConclusionsIn human astroglial brain tumours, up-regulation of TRF1 and TRF2 occurs in the early stages of carcinogenesis determining telomeres shortening and genomic instability. In a later stage, up-regulation of PARP-TANKs and telomerase activation may occur together with an ADP-ribosylation of TRF1, causing a reduced ability to bind telomeric DNA, telomeres elongation and tumor malignant progression.

Clinical significance of FOXP3 expression in human gliomas

Studies have demonstrated that the transcription factor forkhead box P3 (FOXP3) is expressed not only in regulatory T cells, but also in some cancer cells. This study aims to clarify whether or not FOXP3 expression occurs in human gliomas and investigate the clinical significance of this expression in gliomas. We detected FOXP3 protein expression in 40 glioma samples, 3 normal brain tissue samples, and 4 normal tonsil tissue samples using immunohistochemical staining and western blot. The expression of FOXP3 protein was also detected in five glioma cell lines by western blot. We also evaluated the association of FOXP3 expression with clinical pathological grades, prognosis, and recurrence. Western blot analysis showed that the expression of FOXP3 protein was upregulated in high-grade glioma (HGGS) samples compared with low-grade samples. The cell line U87 showed the highest FOXP3 expression, while U373 had the lowest expression. Immunohistochemical analysis detected FOXP3 protein in 35 out of the 40 (87.5%) glioma samples and high levels of FOXP3 were observed in 26 out of the 27 (96.3%) high-grade gliomas samples. Statistical analysis suggested that the upregulation of FOXP3 is significantly correlated with the histologic grade of gliomas (P<0.05) and that patients with high expression of FOXP3 protein exhibit a poorer prognosis than those with low FOXP3 expression. Our findings suggest that FOXP3 expression in glioma cells has a crucial function in the development of HGGS and is associated with the malignant biological behavior of HGGS.

MicroRNA383 regulates expression of PRDX3 in human medulloblastomas

To investigate the effects of microRNA-383 (miR-383) on PRDX3 gene expression, cell proliferation and apoptosis of human medulloblastma. PRDX3 and miR-383 RNA expression was detected by real-time quantitative RT-PCR in human medulloblastoma tumor tissue samples, Daoy cell line and normal brain tissue samples. Western blot was used to detect protein expression of PRDX3. Synthetic miR-383 mimics were transfected into Daoy cells by lipofectamine. Using Cell Counting Kit-8 (CCK-8) method, flow cytometry was used to investigate the cell proliferation and apoptosis, cells reactive oxgen species(ROS), mitochondrial membrane potential changes in each experimental groups. Of 15 cases of human medulloblastoma tumor, 13 cases had miR-383 expression levels significantly lower than that of normal brain tissue, and 14 had PRDX3 mRNA expression levels significantly higher than that of normal brain tissue. The expression levels of miR-383 and PRDX3 in Daoy cells were 0.353 and 1.315 times than those of normal brain tissue, respectively. The protein expression levels of PRDX3 were higher in human medulloblatoma tumors and Daoy cells than that of normal brain tissue. Transfected miR-383 mimics increased the expression level of miR-383 after 24 h and 48 h was significantly higher than that of the control. In contrast, PRDX3 gene mRNA and protein expression levels were significantly decreased at 48 h compared with the control group. Using CCK-8 assay, the cell proliferation rate in the experimental group was significantly lower than that of the control group (P < 0.05). Annexin V-FITC assay demonstrated that early apoptosis rate of the experimental group (11.60 ± 0.30)% was significantly higher than those of the control group (2.3 ± 0.20)% and negative control group (10.37 ± 0.25)% (P = 0.000) after 48 h of transfection. The intracellular ROS levels after transfection at 24 and 48 h significantly increased than those of the control group. Mitochondrial membrane potential level at 24 h after transfection significantly decreased, comparing with the blank control group and the negative control group. Compared with normal brain tissue, decreased expression of miR-383 but elevated expression of PRDX3 are medulloblastoma tumour and Daoy cell lines. Up-regulation of miR-383 knockdowns the expression of PRDX3, inhibits proliferation and promotes apoptosis of Daoy cells, leading to increased intracellular ROS and decreased levels of mitochondrial membrane potential.

Differences in carotenoid content of normal elderly and Alzheimerˈs brains

Alzheimerˈs Disease (AD) is a severe neurodegenerative disease that inflicts &gt;4,500,000 US elderly. Antioxidants, such as carotenoids, have been implicated in the prevention of degenerative diseases and recent data correlates blood and macular carotenoid levels with cognition.PurposeTo measure and compare the major carotenoids in normal elderly and AD human brain.MethodsSamples of normal and AD brain tissue from frontal lobe cortex and occipital cortex were examined. Sections were dissected into gray and white matter, extracted with organic solvents, and analyzed by HPLC.ResultsAt least 16 carotenoids, 3 tocopherols, and retinol were present in human brain. Xanthophylls accounted for &gt;70% of carotenoids in the brain. Mean concentrations of carotenoids ranged from 3.5–15.3 pmol/g. Combining all brains, lutein (LUT, P&lt;0.001) and zeaxanthin (ZX, P&lt;0.02) were higher in gray vs white matter. ZX and LUT were lower in AD brain (P&lt;0.002, P&lt;0.04, respectively). Healthy brain had 2× more ZX and 30% more LUT than AD brain. Retinol and α‐tocopherol were lower in AD brain, (p&lt;0.002, P&lt;0.006).ConclusionsAD status is negatively correlated with levels of ZX, LUT, vitamins A and E in specific brain tissue.

Gene expression profiling of grade II oligodendrogliomas and ependymomas

Grade II gliomas are morphologically and clinically heterogeneous tumors for which histopathological typing remains the major tool for clinical classification. To what extent the major histological subtypes (astrocytic, oligodendroglial, oligoastrocytic and ependymal tumors) constitute is largely unresolved. Morphological classification can often be ambiguous and would be facilitated by specific subtype markers. Gene expression profiling is becoming an established method to characterize different types of cancers, and it has been used extensively for glioblastoma analysis and for low-grade glioma to glioblastoma comparison. However, few studies of low-grade oligodendrogliomas and ependymomas have been reported. Using oligonucleotide-based microarray analysis, we compared the transcriptional profiles of grade II oligodendrogliomas and grade II ependymomas (two types of grade II gliomas). An essential initial step toward more objective tumor classification is the establishment of taxonomy for tumors based on their gene expression profiles. We aimed to identify expression profiles that differentiate these two types of grade II gliomas. After comparing the expression signature of a tumor to that of normal tissue, differentially expressed genes were used to perform hierarchal clustering, principal component analysis and prediction analysis. Hierarchal clustering and principal component analysis divided nine different tumor tissue samples and four normal brain tissue samples into categories that corresponded well with clinical pathology analysis, grade II oligodendrogliomas, grade II ependymomas and normal tissues. By using expression data from the most informative gene clusters, we constructed a tumor-clustering model. Class prediction using nearest shrunken centroid analysis withcross-validation identified 53 differentially expressed genes between the two types of grade II gliomas. Key words: Grade II ependymoma, grade II oligodendrogliomas, microarray, differentially expressed genes, clustering, classification, prediction.

Kinesin family member 2C (KIF2C/MCAK) is a novel marker for prognosis in human gliomas

ObjectiveGliomas are a group of center nervous system tumors of glial origin. Most are characterized by diffuse infiltrative growth in the surrounding brain. Patients almost always ultimately die from their disease. The drastically increased knowledge about the molecular abnormalities, these are linked with tumor grade and patient prognosis. The aim of this study was to examine the role of KIF2C and AURKB as prognostic factors in gliomas. Materials and methodsA total 40 tumor samples and 6 normal brain tissue samples were collected. Biomarker KIF2C and AURKB were tested by qRT-PCR. Using Western blot validate the results. ResultsWe found that KIF2C and AURKB genes were higher expression in glioma samples when were compared with normal brain tissues. KIF2C and AURKB expression were associated with histopathological grades. We found that KIF2C as a potential independent prognostic marker for patient with glioma by Cox analysis. ConclusionThe expression level of KIF2C mRNA could be a potential independent marker to evaluate patient prognosis.