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Research ArticleExperimental Studies

Expression of ENO1 Is Up-regulated in Low-grade Glioma and Positively Correlated With Meningioma Grade

DANG THUY DIEM DINH, SASKIA KUHL, LUKAS GÖRTZ, ROLAND GOLDBRUNNER and MARCO TIMMER
Anticancer Research May 2022, 42 (5) 2319-2326; DOI: https://doi.org/10.21873/anticanres.15711
DANG THUY DIEM DINH
Laboratory for Neurooncology and Experimental Neurosurgery, Department of General Neurosurgery, Center for Neurosurgery, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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SASKIA KUHL
Laboratory for Neurooncology and Experimental Neurosurgery, Department of General Neurosurgery, Center for Neurosurgery, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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LUKAS GÖRTZ
Laboratory for Neurooncology and Experimental Neurosurgery, Department of General Neurosurgery, Center for Neurosurgery, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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ROLAND GOLDBRUNNER
Laboratory for Neurooncology and Experimental Neurosurgery, Department of General Neurosurgery, Center for Neurosurgery, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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MARCO TIMMER
Laboratory for Neurooncology and Experimental Neurosurgery, Department of General Neurosurgery, Center for Neurosurgery, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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  • For correspondence: marco.timmer@uk-koeln.de
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Abstract

Background: α-Enolase (ENO1) is a glycolytic enzyme involved in the Warburg effect which cancer cells utilize to satisfy their higher need for nutrients. Up-regulation of ENO1 has been detected in several tumor types, including melanoma and endometrial, gastric and colorectal cancer. In these tumors, ENO1 may function as prognostic marker. Therefore, it was our interest to determine the expression of ENO1 in glioma and meningioma and whether chemotherapy of glioma alters ENO1 expression. Material and Methods: Tumor samples and control tissues were obtained during neurosurgery. All tumor samples were grouped according to WHO classification. Quantitative polymerase chain reaction and western blot were used to detect the expression of ENO1 in glioma and meningioma. All assays were carried out in triplicates; β-actin was used as a housekeeping gene. For western blots, all samples were incubated with mouse monoclonal anti-ENO1 followed by secondary horseradish peroxidase-linked anti-mouse antibody, with β-actin as a loading control. Immunofluorescence (n=33) was performed to determine the presence of ENO1 in tumor and control tissues using primary antibody to ENO1 and anti-Cy3 as secondary antibody. Results: The expression of ENO1 mRNA was significantly higher in the control group compared to glioma (p<0.0001) and its protein was also significantly up-regulated in low-grade glioma in comparison to high-grade (p<0.0001). ENO1 expression in grade II and III meningiomas was increased compared to grade I (p=0.016 and p=0.0010, respectively) and in grade III compared to grade II (p=0.0363). Conclusion: Our findings suggest that ENO1 might be a marker for meningioma progression and that ENO1 is up-regulated in low-grade glioma.

Key Words:
  • Glioma
  • meningioma
  • ENO1
  • α-enolase
  • glioblastoma
  • MBP1

Gliomas are the most common malignant primary brain tumors; 14.7% of central nervous system tumors registered from 2011-2015 in the US were glioblastoma (GBM) and 10.2% were other malignant gliomas (1). Gliomas are graded based on histological characteristics and, since 2016, also on molecular ones (2). In contrast, meningiomas are mostly benign tumors which originate from the meninges and represent 37.1% of all primary central nervous system tumors (1). The World Health Organization (WHO) divides gliomas into grades I-IV and meningiomas into grades I-III (3). The current therapy for glioma consists of surgery, radiotherapy, and chemotherapy (4). However, the 5-year-survival rate, especially for GBM, is still poor (5.6%) (1). For meningioma, surgery, radiosurgery, and fractionated radiotherapy are possible treatments; pharmacotherapy is still experimental (5). Hence different therapeutic approaches and prognostic markers are still researched for these tumor types.

Like other tumors, glioma and meningioma show high glycolytic activity because of their high needs for nutrients. Total glycolysis is increased in cancer cells compared to normal tissue because they reorganize their metabolic pathways so that glycolysis can take place under both hypoxic and normoxic conditions. The peculiarity of cancer cells to gain energy through fermentation, despite normoxia, is known as the Warburg effect and was first reported by Otto Warburg in 1926 as aerobic glycolysis. This effect has already been described for glioma and meningioma in previous studies (6-9).

α-Enolase (ENO1) is a glycolytic enzyme converting 2-phospho-D-glycerate to phosphoenolpyruvate and contributes to the Warburg effect. Besides promoting glycolysis, ENO1 is also expressed on the cell surface and acts as plasminogen receptor. In numerous tumors, such as endometrial carcinoma (10), melanoma (11), gastric cancer (12) and colorectal cancer (13), increased ENO1 expression has been described. ENO1 up-regulation is generally associated with increased cell proliferation, migration and invasion, and poorer clinical outcome (10, 11, 14-18). However, some studies showed controversial results. For instance, down-regulation of ENO1 in non-small lung cancer (NSCLC) correlated with poorer overall survival (19). In clear-cell renal carcinoma, ENO1 was found to be down-regulated with increasing tumor stage and grade. Furthermore, increased overall survival was directly positively correlated with ENO1 expression (20). The current available data underline the fact that the function of ENO1 in tumors is not fully understood.

ENO1 has also been researched in glioma. ENO1 promoted cell growth, migration and invasion in glioma, and the expression of ENO1 was negatively correlated with the survival rate of patients (21, 22). In meningioma it has been reported that ENO1 acts as tumor-associated autoantigen (23, 24).

The aim of our study was to examine the expression of ENO1 in glioma and meningioma dependent on their malignancy, i.e., WHO histological tumor grade. It was also of interest whether the impact of chemotherapy on glioma can be seen from the expression level of ENO1.

Materials and Methods

Patients. All tissue samples were obtained during surgery from the Department of Neurosurgery at the University of Cologne. After surgery, tissues were frozen at –80°C with liquid nitrogen and stored at the same temperature until further procedures. The samples were independently classified, according to the WHO 2007 classification, by two neuropathologists.

The resected gliomas were divided into grade II glioma, grade III glioma, secondary GBM with and without chemotherapy, and primary GBM with and without chemotherapy. The resected meningiomas were divided into grade I, II and III meningioma. Peritumoral brain tissues, confirmed by neuropathologists to be tumor-free, were used as controls.

Quantitative real-time polymerase chain reaction (qPCR). For each glioma group undergoing qPCR, 10 samples were analyzed. The control group also contained 10 samples. Each meningioma group analyzed contained 20 samples.

The RNA was extracted from frozen tumor samples via RNeasy Kit (Qiagen, Hilden, Germany). The cDNA was synthesized using QuantiTec Reverse Transcription Kit (Qiagen). qPCR was then performed in Cycler Rotor-Gene-Q (Qiagen) with SYBR Green PCR Kit (Qiagen) for quantification. The samples were heated to 95°C for 5 min, followed by a two-step protocol (95°C for 5 s, 60°C for 10 s). PCR was carried out in 35 cycles. β-Actin served as the housekeeping gene. As primers, QuantiTec Primer Assay for ENO1 and β-actin (Qiagen) were used. All qPCRs were performed in triplicates.

Western blot. The number of samples used for each group were as follows:

control, n=7; each meningioma group, n=10; grade II glioma, n=7; grade III glioma, n=12; secondary GBM, n=9; secondary GBM treated with chemotherapy, n=10; primary GBM=10; primary GBM after chemotherapy, n=8.

Proteins were isolated from the samples before performing western blot. The samples were resuspended in RIPA with protease inhibitor (Roche Diagnostic, Basel, Switzerland) and then incubated on ice for 30 min. After sonicating and centrifugation, the supernatant was stored at –80°C. For determining the protein concentration, Bradford Assay (Bio-Rad, Hercules, CA, USA) was carried out.

After adding sodium dodecyl sulfate sample buffer and sample reducing agent (NuPAGE; Thermo Fisher Scientific, Waltham, MA, USA) to aliquots containing 50 μg protein, they were heated at 70°C for 10 min. Precast protein gels (NuPAGE 4-12% Bis-Tris 1 mm gel) from Thermo Fisher Scientific were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins on the gel were then blotted to a nitrocellulose membrane in a chamber system of Thermo Fisher Scientific (XCell SureLock Mini-Cell). The membrane was incubated in a solution of 5% dry non-fat milk, 3% bovine serum albumin (BSA) and Tris-buffered saline with 0.05% Tween 20 as detergent (TBST) on a shaker. The primary antibody, anti-ENO1 (Antibodies Online, Aachen, Germany) was dissolved in the previously mentioned solution at a dilution of 1:1,000 and membranes were incubated overnight at 4°C on a shaker. After washing the membrane with TBST, it was incubated with a secondary anti-mouse horseradish peroxidase-conjugated antibody dissolved in TBST (1:10,000).

To detect bands, Western Bright ECL spray (Advansta, San Jose, CA, USA) was applied to the membrane and ChemiDoc XRS+ System (Bio-Rad) was used as an imager. The density of the bands was measured with Image Lab software (Bio-Rad). The antibody was stripped from the membrane using a detergent (0.05 M Tris, 2% sodium dodecyl sulfate, pH6.7, β-mercaptoethanol) and heat. The membrane was incubated in β-actin antibody, diluted in TBST (1: 10,000). Anti-mouse horseradish peroxidase-conjugated antibody was again used as secondary antibody. Each run was performed in triplicates. To normalize the expression level of ENO1, the signal of ENO1 was divided by that of β-actin.

Immunofluorescence. The samples used for immunofluorescence were 10 μm-thick cryo slices. TBST was used to wash the slices. The samples were incubated for 2 h in Dulbecco’s phosphate-buffered saline with 5% goat-serum. The primary antibody, anti-ENO1 (ab85086; Abcam, Cambridge, UK) was diluted in 1% BSA and 0,1% Triton (1:50). Each slice was incubated overnight with 50 μl of this solution at 4°C. Cy3-Conjugated anti-rabbit (106831; Jackson ImmunoResearch, Cambridge, UK) was used as secondary antibody. The secondary antibody was dissolved in 1% BSA (1:500). Each slice was then incubated for 90 min protected from light. 4’6-Diamidino-2’-phenylindole, dihydrochloride, was used to stain the nuclei. The slides were covered in ProLong (Thermo Fisher Scientific) and coverslips. The staining was analyzed qualitatively with fluorescence microscopy (Axiovert 200M with Apotome, Carl Zeiss, Jena, Germany).

Statistical analysis. For statistical analysis, Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used. Results beyond the boxplot’s whiskers (1.5 times the interquartile range) were considered outliers and excluded from statistical evaluation. The Gaussian distribution was determined by Shapiro–Wilk normality test. When normally distributed, an unpaired t-test was used to compare two groups with each other. For comparison of more than two groups, a one-way analysis of variance test and Tukey test were performed. When the distribution was not Gaussian, the groups were analyzed with Kruskal–Wallis test and Mann–Whitney test. p-Values of 0.05 or less were considered significant. Data are reported as means±standard error of the mean.

Results

ENO1 expression was higher in control tissues than in glioma tissue. To determine the expression level of ENO1 in glioma, qPCR and western blot were performed. The qPCR data showed ENO1 mRNA expression to be significantly higher in the control group compared to untreated glioma (40.37±6.64 vs. 14.61±7.12; p<0.0001; Figure 1A). Our western blot data showed similar results, however, without significant difference (Figure 1D).

Figure 1.
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Figure 1.

Expression of α-enolase (ENO1) mRNA in gliomas as measured by quantitative polymerase chain reaction and protein by western blotting. A: The ENO1 mRNA level was elevated in control tissues compared to gliomas. B: ENO1 mRNA expression in different glioma grades. After chemotherapy (CTx), primary (Pri.) and secondary (Sec.) glioblastoma (GBM) had a higher ENO1 level than untreated GBM. C: Low-grade gliomas had a higher ENO1 mRNA level than high-grade glioma. D: Control tissues had higher ENO1 protein levels than glioma tissues. E: ENO1 protein level in different glioma grades. Grade II glioma showed significant up-regulation of ENO1 compared to grade III and non-treated primary and secondary GBM. F: Low-grade gliomas presented elevated ENO1 protein level compared to high-grade gliomas. G: Blotting membrane showing from left to right: control, grade II, grade III, primary GBM, primary GBM with chemotherapy, secondary GBM, secondary GBM treated with chemotherapy. ENO1 bands were detected at 48 kDA. β-Actin was uses as reference gene and was detected at 42 kDA. H: Representative immunofluorescence shows presence of ENO1 in gliomas. Control tissue showed the highest signal of ENO1. Secondary and primary GBM showed weakest staining. Significantly different at: *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.

ENO1 was up-regulated in low-grade glioma compared to high-grade gliomas. We observed the ENO1 protein level to be significantly elevated in grade II glioma (1.90±0.50) compared to grade III (1.35±0.39; p=0.0071), secondary GBM (1.02±0.16; p=0.0007) and primary GBM (1.00±0.26; p=0.0002). Grade III glioma had higher ENO1 protein level than secondary GBM (1.35±0.39 vs. 1.02±0.16; p=0.0409) and primary GBM (1.35±0.39 vs. 1.00±0.26; p=0.0242; Figure 1E).

We further divided the groups into low- and high-grade glioma and compared their ENO1 protein level. Low-grade glioma showed significant up-regulation of ENO1 protein compared to high-grade glioma (1.90±0.050 vs. 1.140±0.33; p<0.0001; Figure 1F).

Our PCR data showed a similar tendency. There was no significant difference between grade II and III (16.64±7.25 vs. 15.96±8.524; p>0.9999) but grade II and grade III showed higher ENO1 mRNA expression than secondary GBM (16.64±7.25 vs. 9.05±3.94; p=0.6170; 15.96±8.52 vs. 9.05±3.94; p=0.7194; Figure 1B). When samples were divided into low- and high-grade glioma, the ENO1 mRNA level was slightly higher in low-grade (16.64±7.25 vs. 13.89±7.17; p=0.3054; Figure 1C).

ENO1 expression differed in primary and secondary GBM after treatment with chemotherapy. Our western blot data revealed that secondary GBM which underwent chemotherapy had significantly higher ENO1 mRNA and protein expression than non-treated secondary GBM (26.61±16.10 vs. 9.50±3.94; p= 0.0034; Figure 1B) (1.50±0.34 vs. 1.02±0.16; p=0.0079; Figure 1E). On the mRNA and protein levels, the data for treated and non-treated primary GBM were very much alike (17.08±7.59 vs. 15.31±6.73; p=0.9994; Figure 1B) (1.60±1.13 vs. 1.00±0.26; p=0.016; Figure 1E).

ENO1 is found in the cytoplasm of glioma cells. To show the localization and presence of ENO1 in glioma tissues, immunofluorescence was performed. The tissues stained showed ENO1 presence in the cytoplasm of cells. The staining signal seemed stronger in control tissue, and grade II and III glioma compared to secondary and primary GBM (Figure 1H). There were barely different staining signals between treated and untreated primary and secondary GBM (data not shown).

ENO1 mRNA and protein expression differed in meningioma. Via qPCR, we measured the mRNA level of ENO1 in meningioma. Meningioma had slightly higher expression than the controls (Figure 2A). When dividing the meningioma samples accordingly to their WHO classification, we observed up-regulation of ENO1, correlating with increasing meningioma grade. Grade II and III meningioma had significantly higher mRNA levels compared to grade I (13.58±5.82 vs. 9.37±8.84; p=0.0161; 20.17±10.70 vs. 9.37±8.84; p=0.0010; Figure 2B). ENO1 expression in grade III meningioma was significantly elevated compared with grade II (p=0.0363).

Figure 2.
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Figure 2.

Expression of α-enolase (ENO1) mRNA in meningiomas as measured by quantitative polymerase chain reaction and protein by western blotting. A: Meningioma had higher ENO1 mRNA levels than control tissues. B: ENO1 mRNA level increased with increasing tumor grade. C: Meningiomas had less ENO1 protein than control tissue. D: On the protein level, there were no differences in ENO1 level between the different grades. E: Blotting membrane showing from left to right: control, grade I, grade II, and grade III. β-Actin (42 kDA) was used as protein loading control. ENO1 bands were detected at 48 kDa. F: Immunohistochemistry showing representative examples. GBM: Glioblastoma; CTx: chemotherapy; Prim.: primary; sec.: secondary. Significantly different at: *p≤0.05, **p≤0.01 and ***p≤0.001.

On the protein level, our results were controversial. In comparison to control samples, the ENO1 level in meningioma was significantly lower than that of the control samples (1.26±0.26 vs. 1.00±0.15; p=0.0019; Figure 2C). Divided by grade, ENO1 protein in meningioma samples did not differ significantly (grade I: 1.02±0.16; grade II: 0.96±0.12; grade III: 1.00± 0.17; Figure 2D).

Through immunofluorescence, we localized this enzyme to the cytoplasm. ENO1 was present in all grades of meningioma. We were not able to see a clear difference between meningioma grades. However, in the control tissue, the signal was weaker than in meningioma tissue (Figure 2F).

ENO1 was up-regulated in glioma compared to meningioma. We compared meningioma samples with glioma samples concerning ENO1 expression. The qPCR data showed ENO1 expression to be similar in meningioma and glioma (14.28±9.64 vs. 14.61±7.12; p=0.4538; Figure 3A), whereas the western blot data showed the protein level in glioma to be significantly elevated compared to meningioma (1.27±0.46 vs. 1.00±0.15; p=0.0099; Figure 3B).

Figure 3.
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Figure 3.

Comparison of α-enolase (ENO1) expression in meningiomas and gliomas. A: At the mRNA level, no differences were seen. B: Gliomas had a higher ENO1 protein level than meningiomas. **Significantly different at p≤0.01.

Discussion

Since cancer cells have higher anabolic needs, they reroute their metabolic pathway, so that glycolysis can take place under hypoxic and normoxic conditions. Thus, cancer cells gain more energy. This is called the Warburg effect (9, 25). ENO1 is a glycolytic enzyme and contributes to the Warburg effect. Hence, it has been of interest whether ENO1 might be a prognostic marker for tumors (14).

Up-regulation of ENO1 has been reported in numerous tumors. The elevated expression of ENO1 is associated with higher proliferation, migration and invasion, and poorer clinical outcome. The exact way by which ENO1 leads to advantageous conditions for cancer cells is not yet clear. Different mechanisms have been reported beside glycolytic activity, such as by regulating the 5’ AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in colorectal cancer or modulating the focal adhesion kinase (FAK)-mediated phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT) pathway in NSCLC (11-13, 15, 26). In addition, ENO1 can also function as plasminogen receptor (16). As a plasminogen receptor, the protein has been described in tumors such as prostate cancer and pancreatic ductal adenocarcinoma, leading to cell adhesion, migration proliferation and tumor metastasis (17, 18, 26, 27). Furthermore, it can act as a tumor-associated antigen and trigger an immune response, which has been observed in various tumor types (28-31).

However, there are some controversial studies. In clear-cell renal carcinoma, ENO1 was found to be down-regulated with increasing tumor stage and grade, and the overall survival correlated positively with its expression (20). Different ENO1 expression was not only observed in different tumor types, there were also studies investigating the same type of tumor and having contradictory results. Chang et al. discovered reduced expression of ENO1 in NSCLC and a poorer overall survival of patients with reduced ENO1 level (19). On the other hand, Fu et al. reported up-regulation of ENO1 expression in NSCLC (15). Data concerning endometrial carcinoma also vary. Lomnytska et al. detected reduced ENO1 expression in endometrial cancer compared to normal endometrium and atypical hyperplasia of endometrium (32), whereas Zhao et al. found ENO1 mRNA and protein levels to be increased (10). The current available data show that the exact function and behavior of ENO1 in tumors are not fully understood yet.

In our research, we concentrated on the expression level of ENO1 in glioma and meningioma and whether a correlation between the expression and the tumor grade could be seen. Our data suggest a negative correlation between ENO1 expression and glioma grade. The lower the tumor grade, the higher the ENO1 expression, which is in contrast to the findings of Song et al. They observed up-regulation of ENO1 mRNA and protein levels in glioma tissues compared to normal brain tissues and, furthermore, ENO1 expression increased with increasing tumor grade (they compared WHO grade I+II with grade III+I) (22). There were other studies which confirmed elevated ENO1 expression in glioma in general and also confirmed this especially in GBM. However, the studies revealing up-regulation of ENO1 in GBM used cell lines such as U87 and U251 and not patient samples of glioma like we did (21, 33, 34). We further have to consider that our control tissue was not normal brain tissue but peritumoral brain tissue. These might be reasons for our distinct results compared to previous studies.

On the contrary, different studies have found that there is another ENO1 gene product, MYC promoter-binding protein 1 (MBP1). MBP1 is a product resulting from alternative translation and acts as transcriptional repressor via binding to the MYC P2 promoter. MYC is a proto-oncogene which is overexpressed in many different tumor types and is associated with the control of cell proliferation, differentiation, and apoptosis. It is not only this alternate ENO1 gene product but also ENO1 protein itself can bind that can bind the MYC P2 promoter, however, less effectively than MBP1. Long-term inactivation of MYC led to cell-growth arrest, enhanced apoptosis, and tumor regression (35-37). The expression level of MYC is also related to the histological grade of glioma. MYC expression increases with increasing tumor grade (38). Hence it might be possible that lower expression of ENO1 in high-grade glioma leads to reduced repression of MYC and therefore higher cell proliferation, which is typical for high-grade glioma, and vice versa for low-grade glioma. However, this is only a hypothesis. Our data further revealed an alteration of ENO1 expression in primary and secondary GBM after chemotherapy, which indicates that chemotherapy affects GBM and changes ENO1 expression.

For meningioma, our data imply an increasing ENO1 mRNA level with increasing tumor grade, as has been documented for many different tumor types (11-18). Our control tissues showed only a slightly lower ENO1 mRNA level, but here as well, we must consider that peritumoral brain tissues were used. In contrast, ENO1 protein level was higher in control than in meningioma tissues. Between the meningioma groups, no differences in protein leveIs were seen. This suggests a post-transcriptional change which results in a different protein level compared to the mRNA level. MYC overexpression has also been reported for grade II and III meningioma; however, in this case, our data do not indicate a connection between ENO1 expression and its function as MYC repressor (39).

Footnotes

  • Authors’ Contributions

    DTDD: Performed experiments, wrote the article, and performed statistical analysis; SK: helped with experiments and article writing; Lukas Görtz: corrected the article, and performed statistical analysis; Roland Goldbrunner: correction of the article; MT: concept, article, performed statistical analysis, and supervised.

  • Conflicts of Interest

    None.

  • Received June 5, 2021.
  • Revision received March 15, 2022.
  • Accepted March 17, 2022.
  • Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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Anticancer Research: 42 (5)
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Expression of ENO1 Is Up-regulated in Low-grade Glioma and Positively Correlated With Meningioma Grade
DANG THUY DIEM DINH, SASKIA KUHL, LUKAS GÖRTZ, ROLAND GOLDBRUNNER, MARCO TIMMER
Anticancer Research May 2022, 42 (5) 2319-2326; DOI: 10.21873/anticanres.15711

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Expression of ENO1 Is Up-regulated in Low-grade Glioma and Positively Correlated With Meningioma Grade
DANG THUY DIEM DINH, SASKIA KUHL, LUKAS GÖRTZ, ROLAND GOLDBRUNNER, MARCO TIMMER
Anticancer Research May 2022, 42 (5) 2319-2326; DOI: 10.21873/anticanres.15711
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Keywords

  • Glioma
  • meningioma
  • ENO1
  • α-enolase
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  • MBP1
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