{'fraction_non_alphanumeric': 0.03054989816700611, 'fraction_numerical': 0.015196616011279963, 'mean_word_length': 5.234375, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3

Abstract Background Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. Methods Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). Results Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan–Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). Conclusion In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

For the viability assay, cells were plated at density of 2x10 4 /ml in triplicate in 96 well plates. Cell viability was monitored by counting the cells and confirmed by using the CellTiter-Blue Viability Assay (Promega Inc). Cell proliferation was evaluated by  incorporation using BrdU Cell proliferation ELISA kit (Abcam) according to manufacturer's instruction.
The motility was evaluated by plating in Corning FluoroBlok TM Multiwell Inserts System (Corning Life Sciences), according to manufacturer's instruction. Briefly, 3x10 3 GSCs were added to the upper chambers. The plates were incubated for 48h at 37°C, then the fluorescent dye calcein acetoxymethyl ester was added to the lower chamber for 30 min before microscope observation.
Colony formation ability was evaluated by plating a single cell/well in 96 well plates. After 3-4 weeks, each well was examined and the number of spheres/cell aggregates were counted.

Intracranial and subcutaneous implantation of glioblastoma stem-like cells (GSCs) in immunocompromised mice and analysis of brain xenografts
For intracranial grafting, the animal skulls were immobilized in a stereotactic head frame and a burr hole was made 2 mm right of the midline and 1 mm behind the coronal suture. The tip of a 10 l-Hamilton microsyringe was placed at a depth of 3.5 mm from the dura and the cells were injected over 10 minutes. After grafting, the mice were kept under pathogen-free conditions in positive-pressure cabinets (Tecniplast). The body weight and neurological status were assessed weekly. Kaplan-Meier analysis of mice outcome was done assuming as event occurrence either the drop of body weight to less than 80% of initial weight or appearance of neurological signs. Twelve weeks after implantation, the mice were deeply anesthetized and transcardially perfused with 0.1 M PBS (pH 7.4), then treated with 4% paraformaldehyde in 0.1 M PBS. The brain was removed, stored in 30% sucrose buffer overnight at 4°C, and serially cryotomed at 20 m on the coronal plane. Sections were collected in distilled water, mounted on slides, and cover-slipped with Eukitt. Images were obtained with a Laser Scanning Confocal Microscope (IX81, Olympus Inc). The density of tumor cells was assessed by counting the number of GFP-expressing GSCs in 10 non-superimposing high-power fields across the striatum, amygdaloid area, anterior commissure, and optic chyasm of the right brain hemisphere.
For subcutaneous implantation, cells were resuspended in 0.1 ml of cold PBS and the suspension was mixed with an equal volume of cold Matrigel (BD Bioscience). Mice were kept under pathogen-free conditions in positive-pressure cabinets (Tecniplast) and observed daily for the visual appearance of tumors at injection sites. Tumor diameter was measured using calipers and calculated as the mean value between the shortest and the longest diameters. At 8 weeks after grafting, the mice were sacrificed with an overdose of barbiturate.
For the immunohistochemistry of subcutaneous grafts, paraffinized sections (5-m thick) were treated with heat-induced antigen retrieval technique by microwave oven processing (2 cycles of 5 min, 750 W) in citrate buffer. The sections were incubated with antibody for 1 h, and immunodetection was performed using the avidin-biotin complex peroxidase method (Dako) with fresh diaminobenzidine as a chromagen. Alternate sections were stained with hematoxylin and eosin (H&E) for morphological analysis. Immunoreaction was performed utilizing antiserum directed against GFAP and Ki67 (both from Dako).

Statistical methods
Statistical analysis was performed using GraphPad-Prism 5 software (GraphPad Software) or STATVIEW v.5.0 (SAS Institute). Kaplan-Meier survival curves were plotted and differences in survival between groups of patients were compared using the log-rank test. Statistical comparison of continuous variables was performed by the Mann-Whitney test, as appropriate. Multivariate analysis was performed using the Cox proportional hazards model, which was adjusted for the major prognostic factors that included MEG3 and MEG8 expression, age (≤ 70 years versus > 70 years), Karnofsky performance status (KPS; < 70 versus ≥ 70) and MGMT methylation status.
Comparison of categorical variables was performed by chi-square statistic, using the Fisher's exact test.
Differences in density of GSCs between MEG3 and control brain xenografts were evaluated using Student's t-test. Statistical significance was assigned to p-values<0.05.
Heatmaps of MLPA results were performed in 2 or 3 technical replicates using the number of methylated alleles, as provided by the Coffalyser software.
Euclidean Distance Metric was used to cluster data obtained from Illumina array 450k.