Antisense lncRNA CHROMR is linked to glioma patient survival.

Background: Natural non-coding antisense transcripts (ncNATs) are long non-coding RNAs (lncRNA) transcribed from the opposite strand of a separate protein coding or non-coding gene. As such, ncNATs can increase overlapping mRNA (and the coded protein) levels by stabilizing mRNA, absorbing inhibitory miRNAs and protecting the mRNA from degradation, or conversely decrease mRNA (or protein) levels by directing the mRNA towards degradation or inhibiting protein translation. Recently, growing numbers of ncNATs were shown to be dysregulated in cancerous cells, however, actual impact of ncNATs on cancer progression remains largely unknown. We therefore investigated gene expression levels of natural antisense lncRNA CHROMR (Cholesterol Induced Regulator of Metabolism RNA) and its sense protein coding gene PRKRA (Protein Activator of Interferon Induced Protein Kinase EIF2AK2) in gliomas. Next, we checked CHROMR effect on the survival of glioma patients. Methods: We performed RNA-seq on post-surgical tumor samples from 26 glioma patients, and normal brain tissue. Gene expression in TPM values were extracted for CHROMR and PRKRA genes. These data were validated using the TCGA and GTEx gene expression databases. Results: The gene expression level of ncNAT lncRNA CHROMR in glioma tissue was significantly higher compared to healthy brain tissue, while the expression of its sense counterpart protein coding PRKRA mRNA did not differ between glioma and healthy samples. Survival analysis showed lower survival rates in patients with low mRNA PRKRA/lncRNA CHROMR gene expression ratio compared to high ratio showing a link between lncRNA CHROMR and glioma patient survival prognosis. Conclusion: Here we show that elevated levels of lncRNA CHROMR (i.e., low ratio of mRNA PRKRA/lncRNA CHROMR) is associated with poor prognosis for glioma patients.

Identification and comparison of m6A modifications in glioblastoma non-coding RNAs with MeRIP-seq and Nanopore dRNA-seq.

The most prominent RNA modification - N6-methyladenosine (m6A) - affects gene regulation and cancer progression. The extent and effect of m6A on long non-coding RNAs (lncRNAs) is, however, still not clear. The most established method for m6A detection is methylated RNA immunoprecipitation and sequencing (MeRIP-seq). However, Oxford Nanopore Technologies recently developed direct RNA-seq (dRNA-seq) method, allowing m6A identification at higher resolution and in its native form. We performed whole transcriptome sequencing of the glioblastoma cell line U87-MG with both MeRIP-seq and dRNA-seq. For MeRIP-seq, m6A peaks were identified using nf-core/chipseq, and for dRNA-seq - EpiNano pipeline. MeRIP-seq analysis revealed 5086 lncRNAs transcripts, while dRNA-seq identified 336 lncRNAs transcripts from which 556 and 198 were found to be m6A modified, respectively. While 24 lncRNAs with m6A overlapped between two methods. Gliovis database analysis revealed that the expression of the major part of identified overlapping lncRNAs was associated with glioma grade or patient survival prognosis. We found that the frequency of m6A occurrence in lncRNAs varied more than 9-fold throughout the provided list of 24 modified lncRNAs. The highest m6A frequency was detected in MIR1915HG, THAP9-AS1, MALAT1, NORAD1, and NEAT1 (49-88nt), while MIR99AHG, SNHG3, LOXL1-AS1, ILF3-DT showed the lowest m6A frequency (445-261nt). Taken together, (1) we provide a high accuracy list of 24 m6A modified lncRNAs of U87-MG cells; (2) we conclude that MeRIP-seq is more suitable for an initial m6A screening study, due to its higher lncRNA coverage, whereas dRNA-seq is most useful when more in-depth analysis of m6A quantity and precise location is of interest.Abbreviations: (dRNA-seq) direct RNA-seq, (GBM) glioblastoma, (LGG) low-grade glioma, (lncRNAs) long non-coding RNAs, (m6A) N6-methyladenosine, (MeRIP-seq) methylated RNA immunoprecipitation and sequencing, (ncRNA) non-coding RNA, (ONT) Oxford Nanopore Technologi; Lietuvos Mokslo Taryba.

Glioblastoma Molecular Classification Tool Based on mRNA Analysis: From Wet-Lab to Subtype.

Most glioblastoma studies incorporate the layer of tumor molecular subtype based on the four-subtype classification system proposed in 2010. Nevertheless, there is no universally recognized and convenient tool for glioblastoma molecular subtyping, and each study applies a different set of markers and/or approaches that cause inconsistencies in data comparability and reproducibility between studies. Thus, this study aimed to create an applicable user-friendly tool for glioblastoma classification, with high accuracy, while using a significantly smaller number of variables. The study incorporated a TCGA microarray, sequencing datasets, and an independent cohort of 56 glioblastomas (LUHS cohort). The models were constructed by applying the Agilent G4502 dataset, and they were tested using the Affymetrix HG-U133a and Illumina Hiseq cohorts, as well as the LUHS cases. Two classification models were constructed by applying a logistic regression classification algorithm, based on the mRNA levels of twenty selected genes. The classifiers were translated to a RT-qPCR assay and validated in an independent cohort of 56 glioblastomas. The classification accuracy of the 20-gene and 5-gene classifiers varied between 90.7-91% and 85.9-87.7%, respectively. With this work, we propose a cost-efficient three-class (classical, mesenchymal, and proneural) tool for glioblastoma molecular classification based on the mRNA analysis of only 5-20 genes, and we provide the basic information for classification performance starting from the wet-lab stage. We hope that the proposed classification tool will enable data comparability between different research groups.

Transcriptome-wide analysis of glioma stem cell specific m6A modifications in long-non-coding RNAs.

The interest in chemical RNA modifications is rapidly growing in the field of molecular biology. Dynamic and reversible alterations of N6-methyladenosine (m6A) RNA modification are responsible for a platter of structural and functional changes in healthy and cancerous cell states. Although many studies reported the link between tumor initiation/progression and m6A modulators, there are few studies exploring transcriptome-wide m6A profile of non-coding RNAs. The aim of current study was to identify glioma stem cell (GSC) specific m6A landscape of long non-coding RNAs (lncRNAs) applying MeRIP-seq approach. MeRIP-seq analysis assigned 77.9% of m6A peaks to mRNAs and 8.16% to lncRNAs. GSCs and differentiated cells showed 76.4% conservation of m6A peaks, while 19.4% were unique to GSCs. Seven novel GSC-specific m6A modified lncRNAs were identified: HRAT92, SLCO4A1-AS1, CEROX1, PVT1, AGAP2-AS1, MIAT, and novel lncRNA gene ENSG00000262223. Analysis disclosed a strong negative correlation between lncRNAs m6A modification rate and expression. MeRIP-seq analysis revealed m6A modifications on previously reported glioma-associated lncRNAs: LINC000461, HOTTIP, CRNDE, TUG1, and XIST. Moreover, current study disclosed that most highly m6A modified lncRNAs primarily contain m6A modifications close to 3' and 5' ends. Our results provide basis and insight for further studies of m6A modifications in non-coding transcriptome of GSCs.

Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia.

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures-from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide-by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation.

Mesenchymal and Proneural Subtypes of Glioblastoma Disclose Branching Based on GSC Associated Signature.

Glioblastomas (GBM)-the most common, therapy-resistant, and lethal tumors driven by populations of glioma stem cells (GSCs) are still on the list of the most complicated pathologies. Thus, deeper understanding and characterization of GSCs is indispensable to find suitable targets and develop more effective therapies. In the present study, we applied native glioblastoma cells and GSCs sequencing, screened for GSC-specific targets and checked if the signature is related to GBM patient pathological, clinical data as well as molecular subtypes applying TCGA cohort. Data analysis revealed that tumors of proneural and mesenchymal subtypes are branching in separate clusters based on screened gene expression. Samples of the same subtype revealed significantly different patient survival prognosis as well as recurrence chance between the clusters. Recently, different subpopulations of mesenchymal GSC demonstrating different properties were shown, which indicates possible internal heterogeneity of GBM subtypes as well. Current findings also revealed branching of molecular GBM subtypes that were significantly linked to patient outcome and that might be decided by distinct GSC subpopulations.

The Role of CASC2 and miR-21 Interplay in Glioma Malignancy and Patient Outcome.

Recently long non-coding RNAs (lncRNAs) were highlighted for their regulatory role in tumor biology. The novel human lncRNA cancer susceptibility candidate 2 (CASC2) has been characterized as a potential tumor suppressor in several tumor types. However, the roles of CASC2 and its interplay with miR-21 in different malignancy grade patient gliomas remain unexplored. Here we screened 99 different malignancy grade astrocytomas for CASC2, and miR-21 gene expression by real-time quantitative polymerase chain reaction (RT-qPCR) in isocitrate dehydrogenase 1 (IDH1) and O-6-methylguanine methyltransferase (MGMT) assessed gliomas. CASC2 expression was significantly downregulated in glioblastomas (p = 0.0003). Gliomas with low CASC2 expression exhibited a high level of miR-21, which was highly associated with the higher glioma grade (p = 0.0001), IDH1 wild type gliomas (p < 0.0001), and poor patient survival (p < 0.001). Taken together, these observations suggest that CASC2 acts as a tumor suppressor and potentially as a competing endogenous RNA (ceRNA) for miR-21, plays important role in IDH1 wild type glioma pathogenesis and patients' outcomes.

Unique Interplay Between Molecular miR-181b/d Biomarkers and Health Related Quality of Life Score in the Predictive Glioma Models.

In the last decade, an increasing amount of research has been conducted analyzing microRNA expression changes in glioma tissue and its expressed exosomes, but there is still sparse information on microRNAs or other biomarkers and their association with patients' functional/psychological outcomes. In this study, we performed a combinational analysis measuring miR-181b and miR-181d expression levels by quantitative polymerase chain reaction (qPCR), evaluating isocitrate dehydrogenase 1 (IDH1) single nucleotide polymorphism (SNP), and O-6-methylguanine methyltransferase (MGMT) promoter methylation status in 92 post-surgical glioma samples and 64 serum exosomes, including patients' quality of life evaluation applying European Organization for Research and Treatment of Cancer (EORTC) questionnaire for cancer patients (QLQ-30), EORTC the Brain Cancer-Specific Quality of Life Questionnaire (QLQ-BN20), and the Karnofsky performance status (KPS). The tumoral expression of miR-181b was lower in grade III and glioblastoma, compared to grade II glioma patients (p < 0.05). Additionally, for the first time, we demonstrated the association between miR-181 expression levels and patients' quality of life. A positive correlation was observed between tumoral miR-181d levels and glioma patients' functional parameters (p < 0.05), whereas increased exosomal miR-181b levels indicated a worse functional outcome (p < 0.05). Moreover, elevated miR-181b exosomal expression can indicate a significantly shorter post-surgical survival time for glioblastoma multiforme (GBM) patients. In addition, both tumoral and exosomal miR-181 expression levels were related to patients' functioning and tumor-related symptoms. Our study adds to previous findings by demonstrating the unique interplay between molecular miR-181b/d biomarkers and health related quality of life (HRQOL) score as both variables remained significant in the predictive glioma models.

A SEMA3 Signaling Pathway-Based Multi-Biomarker for Prediction of Glioma Patient Survival.

Glioma is a lethal central nervous system tumor with poor patient survival prognosis. Because of the molecular heterogeneity, it is a challenge to precisely determine the type of the tumor and to choose the most effective treatment. Therefore, novel biomarkers are essential to improve the diagnosis and prognosis of glioma tumors. Class 3 semaphorin proteins (SEMA3) play an important role in tumor biology. SEMA3 transduce their signals by using neuropilin and plexin receptors, which functionally interact with the vascular endothelial growth factor-mediated signaling pathways. Therefore, the aim of this study was to explore the potential of SEMA3 signaling molecules for prognosis of glioma patient survival. The quantitative real-time PCR method was used to evaluate mRNA expression of SEMA3(A-G), neuropilins (NRP1 and NRP2), plexins (PLXNA2 and PLXND1), cadherins (CDH1 and CDH2), integrins (ITGB1, ITGB3, ITGA5, and ITGAV), VEGFA and KDR genes in 59 II-IV grade glioma tissues. Seven genes significantly associated with patient overall survival were used for multi-biomarker construction, which showed 64%, 75%, and 68% of accuracy of predicting the survival of 1-, 2-, and 3-year glioma patients, respectively. The results suggest that the seven-gene signature could serve as a novel multi-biomarker for more accurate prognosis of a glioma patient's outcome.

Identification of Astrocytoma Blood Serum Protein Profile.

High-grade astrocytomas are some of the most common and aggressive brain cancers, whose signs and symptoms are initially non-specific. Up to the present date, there are no diagnostic tools to observe the early onset of the disease. Here, we analyzed the combination of blood serum proteins, which may play key roles in the tumorigenesis and the progression of glial tumors. Fifty-nine astrocytoma patients and 43 control serums were analyzed using Custom Human Protein Antibody Arrays, including ten targets: ANGPT1, AREG, IGF1, IP10, MMP2, NCAM1, OPN, PAI1, TGFß1, and TIMP1. The decision tree analysis indicates that serums ANGPT1, TIMP1, IP10, and TGFß1 are promising combinations of targets for glioma diagnostic applications. The accuracy of the decision tree algorithm was 73.5% (75/102), which correctly classified 79.7% (47/59) astrocytomas and 65.1% (28/43) healthy controls. The analysis revealed that the relative value of osteopontin (OPN) protein level alone predicted the 12-month survival of glioblastoma (GBM) patients with the specificity of 84%, while the inclusion of the IP10 protein increased model predictability to 92.3%. In conclusion, the serum protein profiles of ANGPT1, TIMP1, IP10, and TGFß1 were associated with the presence of astrocytoma independent of its malignancy grade, while OPN and IP10 were associated with GBM patient survival.

Oncosuppressive Role of RUNX3 in Human Astrocytomas.

BACKGROUND: Gliomas are the most common and aggressive among primary malignant brain tumours with significant inter- and intratumour heterogeneity in histology, molecular profile, and patient outcome. However, molecular targets that could provide reliable diagnostic and prognostic information on this type of cancer are currently unknown. Recent studies show that certain phenotypes of gliomas such as malignancy, resistance to therapy, and relapses are associated with the epigenetic alterations of tumour-specific genes. Runt-related transcription factor 3 (RUNX3) is feasible tumour suppressor gene since its inactivation was shown to be related to carcinogenesis. AIM: The aim of the study was to elucidate RUNX3 changes in different regulation levels of molecular biology starting from epigenetics to function in particular cases of astrocytic origin tumours of different grade evaluating significance of molecular changes of RUNX3 for patient clinical characteristics as well as evaluate RUNX3 reexpression effect to GBM cells. METHODS: The methylation status and protein expression levels of RUNX3 were measured by methylation-specific PCR and Western blot in 136 and 72 different malignancy grade glioma tissues, respectively. Lipotransfection and MTT were applied for proliferation assessment in U87-MG cells. RESULTS: We found that RUNX3 was highly methylated and downregulated in GBM. RUNX3 promoter methylation was detected in 69.4% of GBM (n=49) as compared to 0 to 17.2% in I-III grade astrocytomas (n=87). Weighty lower RUNX3 protein level was observed in GMB specimens compared to grade II-III astrocytomas. Correlation test revealed a weak but significant link among Runx3 methylation and protein level. Kaplan-Meier analysis showed that increased RUNX3 methylation and low protein level were both associated with shorter patient survival (p<0.05). Reexpression of RUNX3 in U87-MG cells significantly reduced glioma cell viability compared to control transfection. CONCLUSIONS: The results demonstrate that RUNX3 gene methylation and protein expression downregulation are glioma malignancy dependent and contribute to tumour progression.

Significance of Amphiregulin (AREG) for the Outcome of Low and High Grade Astrocytoma Patients.

Background: Amphiregulin (AREG) is one of the ligands of the epidermal growth factor receptor which levels was shown to have a tight coherence with various types of cancer. AREG was also designated to be a promising marker for several types of cancer however precious little data about AREG role in the most frequent and generally lethal human brain tumours - astrocytomas reported up to date. The aim of the study was to investigate how AREG changes at epigenetic and expression levels reflect on astrocytoma malignancy and patient outcome. Methods: In total 205 low and high grade astrocytoma samples (15 pilocytic astrocytomas, 56 diffuse astrocytomas, 32 anaplastic astrocytomas and 102 glioblastomas) were used for target mRNA, protein expression and DNA methylation analysis applying qRT-PCR, Western-Blot and MS-PCR assays, respectively. Results: Present research revealed that AREG expression level and methylation in cancer tissue is dependent on the grade of astrocytoma. GBM tissue disclosed elevated AREG mRNA expression but reduced AREG protein level as compared to grade II and grade III astrocytomas (p<0.001). Increased methylation frequency was also more abundant in GBM (74%) than grade I, II and III astrocytomas (25%, 34%, and 36%, respectively). The survival analysis revealed relevant differences in patient overall survival between AREG methylation, mRNA and protein expression groups. Kaplan-Meier analysis encompassing only malignant tumours showed similar results indicating that AREG is associated with astrocytoma patient survival independently from astrocytoma grade. Conclusions: Current findings demonstrate that AREG appearance is associated with patient survival as well as astrocytomas malignancy indicating its influence on tumour progression and suggest its applicability as a promising marker.

Association of miR-34a Expression with Quality of Life of Glioblastoma Patients: A Prospective Study.

MiR-34a acts as tumor-suppressor by targeting many oncogenes related to proliferation, apoptosis, and invasion of gliomas. We studied the relationships between health-related quality of life (HRQOL), depression, and miR-34a expression status in patients with newly diagnosed glioblastoma (GBM). A comprehensive HRQOL assessment was completed by 38 patients with glioblastoma prior to surgical resection and included the European Organization for Research and Treatment of Cancer (EORTC) questionnaire for cancer patients (QLQ-C30) and the Brain Cancer-Specific Quality of Life Questionnaire (QLQ-BN20), the Patient Health Questionnaire-9 (PHQ-9), the Karnofsky performance index (KPS), and The Glasgow Outcome Scale (GOS). The miR-34a expression in glioblastoma tissue was measured using quantitative reverse transcription PCR. Our findings show that lower miR-34a expression is significantly associated with higher tumor volume, worse physical functioning, lower KPS, and greater depressive symptom severity of GBM patients. Moreover, analysis reveals that miR-34a effects might be gender specific, as stronger relationships between miR-34a and patient functioning measures were observed in males when compared to females. Despite the fact that, due to small sample size, our results should be considered as preliminary, our study suggests that miR-34a is associated with tumor burden and can be important for health-related quality of life, functional status, and mood symptoms of glioblastoma patients.

MMP2 is associated with glioma malignancy and patient outcome.

Gliomas are fast growing and usually manifest in an aggressive infiltrative model. MMP2 overexpression is associated with brain tumor malignancy and metastasis formation. The aim of this study was to investigate the influence of MMP2 on glioma formation and clinical outcomes by performing analysis at the DNA, RNA, and protein levels. Methylation status and mRNA level were evaluated in 162 samples; the MMP2 protein level was analyzed in 28 patient preoperative and postoperative blood samples using protein microarray analysis and conventional ELISA. The MMP2 MSP analysis revealed a gradually increasing gene promoter demethylation frequency, and the Kaplan-Meier analysis showed that the methylated gene promoter is related to longer overall survival (Log-rank test X 2 = 12.508, df = 1, P < 0.0001). Relative mRNA expression was significantly downregulated when the promoter was methylated. Pairwise comparison analysis showed statistically significant (Mann-Whitney test, P < 0.05) differences in the MMP2 expression median when comparing different glioma grades. The Kaplan-Meier analysis revealed that low MMP2 expression was associated with better survival (Log-rank test X 2 = 7.732, df = 1, P = 0.005). At the protein level, MMP2 expression in patient sera showed no differences between malignancy grades and patient preoperative and postoperative states, while the ELISA assay showed the tendency of accumulating MMP2 protein in higher malignancy patient sera samples. The Kaplan-Meier analysis showed the tendency of having a shorter survival time with a higher MMP2 protein level in patient sera. MMP2 has a significant role in glioma pathogenesis and could be used as a potential molecular marker for tumor progression.

Signal transducer and activator of transcription 3 (STAT3) promoter methylation and expression in pituitary adenoma.

BACKGROUND: Pituitary adenoma (PA) is a benign brain tumor that can cause neurological, endocrinological and ophthalmological aberrations. Till now there is a need to identify factors that can influence the tumor invasiveness and recurrence. The aim of this study was to evaluate the associations between the signal transducer and activator of transcription 3 (STAT3) promoter methylation, mRNA expression and the invasiveness or recurrence of PAs and patient clinical characteristics. METHODS: Study participants comprised of 102 subjects with a diagnosis of PA: 54 functioning and 48 non-functioning, 58 invasive and 30 non-invasive PAs and 14 relapses. The bisulfite treatment of tumor DNA and methylation-specific polymerase chain reaction (MS-PCR) method was used to determine the STAT3 gene promoter methylation. For the STAT3 mRNA expression, the first-strand cDNA was produced from total RNA by using reverse transcriptase and quantitative real-time PCR (qRT-PCR) was performed. RESULTS: In 10.78% (11/102) of PA tissues STAT3 gene promoter was methylated. A gender of male and patient group older than 60 years were significantly associated with reduced STAT3 mRNA expression (Mann-Whitney test, p = 0.025, p = 0.047, respectively). However, no more statistical differences were found between STAT3 promoter methylation, mRNA expression and patient clinical characteristics or PA invasiveness or recurrence. CONCLUSIONS: Further investigations are needed to clarify the influence of STAT3 gene promoter methylation and mRNA expression changes in PAs.

Testin (TES) as a candidate tumour suppressor and prognostic marker in human astrocytoma.

Astrocytomas are one of the most common brain tumours; however, the current methods used to characterize these tumours are inadequate. The establishment of molecular markers may identify variables required to improve tumour characterization and subtyping, and may aid to specify targets for improved treatment with essential prognostic value for patient survival. One such candidate is testin (TES), which was reported to have prognostic value for glioblastoma patients. However, the role of TES protein in gliomagenesis is currently unknown. In the present study, the methylation status of the TES promoter was investigated in post-operative astrocytoma tumours of different malignancy grade, and its association with the survival of astrocytoma patients was evaluated. In addition, the expression of TES protein was investigated in the same set of astrocytoma tumours tissue, and the association of protein expression with glioma patients survival was evaluated. The methylation status of TES was assessed by methylation-specific polymerase chain reaction in 138 different grade astrocytoma samples. Western blot analysis was used to characterize the expression pattern of TES in 86 different grade astrocytoma specimens: 13 of pathological grade I, 31 of pathological grade II, 17 of pathological grade III and 25 of pathological grade IV (glioblastoma). Statistical analyses were conducted to investigate the association between tumour molecular pattern, patient clinical variables and overall survival. The methylation analysis of the TES promoter exhibited a distinct profile between astrocytomas of different malignancy grade (P<0.001). Furthermore, gene promoter methylation was significantly associated with patients' age, survival and pathological grade (P<0.001). The protein expression level of TES was significantly lower in glioblastoma (grade IV astrocytoma) than in lower grade (II-III) astrocytoma tissue (P=0.028 and P=0.04, respectively). Additionally, short overall survival of patients was markedly associated with low TES protein expression (P=0.007). However, no association between TES methylation and TES protein expression was noticed. The present study demonstrated that decreased expression of TES may be important in tumour progression and prognosis in human astrocytomas. TES may be a useful marker for predicting the clinical outcome of astrocytoma patients.

High CHI3L1 expression is associated with glioma patient survival.

BACKGROUND: Survival of glioma patients with the same tumor histology and grade can vary significantly, and some low-grade gliomas transform to a more malignant phenotype. There is a need of molecular signatures, which are better predictors of the patient diagnosis, outcome of treatment, and prognosis than the diagnosis provided by histopathology. We propose CHI3L1 mRNA expression as a prognostic biomarker for patients with glioma. METHODS: We measured CHI3L1 expression with quantitative real time-polymerase chain reaction (qRT-PCR) in the cohort of 98 patients with different grade glioma: 10 grade I pylocytic astrocytomas, 30 grade II diffuse astrocytomas, 20 grade III anaplastic astrocytomas, and 38 grade IV astrocytomas (glioblastomas). Statistical analyses were conducted to investigate the association between CHI3L1 mRNA expression levels and patient clinical variables. RESULTS: We demonstrated that mRNA expression of CHI3L1 was evidently higher in glioblastoma than in lower grade glioma tissues. We evaluated correlations between CHI3L1 expression, clinicopathological characteristics, and the outcomes of the patients. Patients with high CHI3L1 expression had a shorter overall survival (p < 0.001). CONCLUSIONS: Findings presented in our study showed that increased mRNA level of CHI3L1 could be associated with the progression of astrocytoma and poor patient survival not only for glioblastoma, but for lower grade astrocytoma tumors as well. Further investigation will be required to evaluate CHI3L1 value as a molecular marker for astrocytoma prognoses and for novel treatment strategies against all grade astrocytomas.

High level of Sema3C is associated with glioma malignancy.

BACKGROUND: Malignant gliomas are characterized by the tendency of cancerous glial cells to infiltrate into normal brain tissue, thereby complicating targeted treatment of this type of cancer. Recent studies suggested involvement of Sema3C (semaphorin 3C) protein in tumorigenesis and metastasis in a number of cancers. The role of Sema3C in gliomagenesis is currently unclear. In this study, we investigated how expression levels of Sema3C in post-operative glioma tumors are associated with the malignancy grade and the survival of the patient. FINDINGS: Western blot analysis was used for detection of Sema3C protein levels in 84 different grade glioma samples: 12 grade I astrocytomas, 30 grade II astrocytomas, 17 grade III astrocytomas, and 25 grade IV astrocytomas (glioblastomas). Sema3C mRNA levels in gliomas were analysed by real-time PCR. Several statistical methods have been used to investigate associations between Sema3C protein and mRNA levels and clinical variables and survival outcome. The results demonstrated that protein levels of Sema3C were markedly increased in glioblastomas compared to grade I-III astrocytoma tissues and were significantly associated with the shorter overall survival of patients. High accumulation of Sema3C positively associated with the age of patients and pathological grade, but did not correlate with patient's gender. Sema3C mRNA levels showed no association with either grade of glioma or patient survival. CONCLUSIONS: The data presented in this work suggest that the increased levels of Sema3C protein may be associated with the progression of glioma tumor and has a potential as a prognostic marker for outcome of glioma patients. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1564066714158642.

Glioma Malignancy-Dependent NDRG2 Gene Methylation and Downregulation Correlates with Poor Patient Outcome.

AIMS: NDRG2 (N-myc downstream regulated gene 2) gene is involved in important biological processes: cell differentiation, growth and apoptosis. Several molecular studies have shown NDRG2 as a promising diagnostic marker involved in brain tumor pathology. The aim of the study was to investigate how changes in epigenetic modification and activity of NDRG2 reflect on glioma malignancy and patient outcome. METHODS: 137 different malignancy grade gliomas were used as the study material: 14 pilocytic astrocytomas grade I, 45 diffuse astrocytomas grade II, 29 anaplastic astrocytomas grade III, and 49 grade IV astrocytomas (glioblastomas). Promoter methylation analysis has been carried out by using methylation-specific PCR, whereas RT-PCR and Western-blot analyses were used to measure NDRG2 expression levels. RESULTS: We demonstrated that NDRG2 gene methylation frequency increased whereas expression at both mRNA and protein levels markedly decreased in glioblastoma specimens compared to the lower grade astrocytomas. NDRG2 transcript and protein levels did not correlate with the promoter methylation state, suggesting the presence of alternative regulatory gene expression mechanisms that may operate in a tissue-specific manner in gliomas. Kaplan-Meier analyses revealed significant differences in survival time in gliomas stratified by NDRG2 methylation status and mRNA and protein expression levels. CONCLUSIONS: Our findings highlight the usefulness of combining epigenetic data to gene expression patterns at mRNA and protein level in tumor biomarker studies, and suggest that NDRG2 downregulation might bear influence on glioma tumor progression while being associated with higher malignancy grade.

Promoter methylation of AREG, HOXA11, hMLH1, NDRG2, NPTX2 and Tes genes in glioblastoma.

Epigenetic alterations alone or in combination with genetic mechanisms play a key role in brain tumorigenesis. Glioblastoma is one of the most common, lethal and poor clinical outcome primary brain tumors with extraordinarily miscellaneous epigenetic alterations profile. The aim of this study was to investigate new potential prognostic epigenetic markers such as AREG, HOXA11, hMLH1, NDRG2, NTPX2 and Tes genes promoter methylation, frequency and value for patients outcome. We examined the promoter methylation status using methylation-specific polymerase chain reaction in 100 glioblastoma tissue samples. The value for clinical outcome was calculated using Kaplan-Meier estimation with log-rank test. DNA promoter methylation was frequent event appearing more than 45 % for gene. AREG and HOXA11 methylation status was significantly associated with patient age. HOXA11 showed the tendency to be associated with patient outcome in glioblastomas. AREG gene promoter methylation showed significant correlation with poor patient outcome. AREG methylation remained significantly associated with patient survival in a Cox multivariate model including MGMT promoter methylation status. This study of new epigenetic targets has shown considerably high level of analyzed genes promoter methylation variability in glioblastoma tissue. AREG gene might be valuable marker for glioblastoma patient survival prognosis, however further analysis is needed to clarify the independence and appropriateness of the marker.