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Astrocyte elevated gene-1 (AEG-1) is an important oncogene that overexpresses in gliomas and plays a vital role in their occurrence and progression. However, few reports have shown which biomarkers could reflect the level of AEG-1 expression in vivo so far. In recent years, intracellular metabolites monitored by proton magnetic resonance spectroscopy (1H MRS) as non-invasive imaging biomarkers have been applied to the precise diagnosis and therapy feedback of gliomas. Therefore, understanding the correlation between 1H MRS metabolites and AEG-1 gene expression in U251 cells may help to identify relevant biomarkers. This study constructed three monoclonal AEG-1-knockout U251 cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR) /Cas9 technique and evaluated the biological behaviors and metabolite ratios of these cell lines. With the decline in AEG-1 expression, the apoptosis rate of the AEG-1-knockout cell lines increased. At the same time, the metastatic capacities decreased, and the relative contents of total choline (tCho) and lactate (Lac) were also reduced. In conclusion, deviations in AEG-1 expression influence the apoptosis rate and metastasis capacity of U251 cells, which the 1H MRS metabolite ratio could monitor. The tCho/creatinine(Cr) and Lac/Cr ratios positively correlated with the AEG-1 expression and malignant cell behavior. This study may provide potential biomarkers for accurate preoperative diagnosis and future AEG-1-targeting treatment evaluation of gliomas in vivo.</div

The correlation analysis among the biological behavior, AEG-1 protein relative expression level, and metabolite alterations in the U251 cell line.

The correlation analysis among the biological behavior, AEG-1 protein relative expression level, and metabolite alterations in the U251 cell line.</p

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Astrocyte elevated gene-1 (AEG-1) is an important oncogene that overexpresses in gliomas and plays a vital role in their occurrence and progression. However, few reports have shown which biomarkers could reflect the level of AEG-1 expression in vivo so far. In recent years, intracellular metabolites monitored by proton magnetic resonance spectroscopy (1H MRS) as non-invasive imaging biomarkers have been applied to the precise diagnosis and therapy feedback of gliomas. Therefore, understanding the correlation between 1H MRS metabolites and AEG-1 gene expression in U251 cells may help to identify relevant biomarkers. This study constructed three monoclonal AEG-1-knockout U251 cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR) /Cas9 technique and evaluated the biological behaviors and metabolite ratios of these cell lines. With the decline in AEG-1 expression, the apoptosis rate of the AEG-1-knockout cell lines increased. At the same time, the metastatic capacities decreased, and the relative contents of total choline (tCho) and lactate (Lac) were also reduced. In conclusion, deviations in AEG-1 expression influence the apoptosis rate and metastasis capacity of U251 cells, which the 1H MRS metabolite ratio could monitor. The tCho/creatinine(Cr) and Lac/Cr ratios positively correlated with the AEG-1 expression and malignant cell behavior. This study may provide potential biomarkers for accurate preoperative diagnosis and future AEG-1-targeting treatment evaluation of gliomas in vivo.</div

PCR short-chain primers for TA cloning analysis.

Astrocyte elevated gene-1 (AEG-1) is an important oncogene that overexpresses in gliomas and plays a vital role in their occurrence and progression. However, few reports have shown which biomarkers could reflect the level of AEG-1 expression in vivo so far. In recent years, intracellular metabolites monitored by proton magnetic resonance spectroscopy (1H MRS) as non-invasive imaging biomarkers have been applied to the precise diagnosis and therapy feedback of gliomas. Therefore, understanding the correlation between 1H MRS metabolites and AEG-1 gene expression in U251 cells may help to identify relevant biomarkers. This study constructed three monoclonal AEG-1-knockout U251 cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR) /Cas9 technique and evaluated the biological behaviors and metabolite ratios of these cell lines. With the decline in AEG-1 expression, the apoptosis rate of the AEG-1-knockout cell lines increased. At the same time, the metastatic capacities decreased, and the relative contents of total choline (tCho) and lactate (Lac) were also reduced. In conclusion, deviations in AEG-1 expression influence the apoptosis rate and metastasis capacity of U251 cells, which the 1H MRS metabolite ratio could monitor. The tCho/creatinine(Cr) and Lac/Cr ratios positively correlated with the AEG-1 expression and malignant cell behavior. This study may provide potential biomarkers for accurate preoperative diagnosis and future AEG-1-targeting treatment evaluation of gliomas in vivo.</div

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Astrocyte elevated gene-1 (AEG-1) is an important oncogene that overexpresses in gliomas and plays a vital role in their occurrence and progression. However, few reports have shown which biomarkers could reflect the level of AEG-1 expression in vivo so far. In recent years, intracellular metabolites monitored by proton magnetic resonance spectroscopy (1H MRS) as non-invasive imaging biomarkers have been applied to the precise diagnosis and therapy feedback of gliomas. Therefore, understanding the correlation between 1H MRS metabolites and AEG-1 gene expression in U251 cells may help to identify relevant biomarkers. This study constructed three monoclonal AEG-1-knockout U251 cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR) /Cas9 technique and evaluated the biological behaviors and metabolite ratios of these cell lines. With the decline in AEG-1 expression, the apoptosis rate of the AEG-1-knockout cell lines increased. At the same time, the metastatic capacities decreased, and the relative contents of total choline (tCho) and lactate (Lac) were also reduced. In conclusion, deviations in AEG-1 expression influence the apoptosis rate and metastasis capacity of U251 cells, which the 1H MRS metabolite ratio could monitor. The tCho/creatinine(Cr) and Lac/Cr ratios positively correlated with the AEG-1 expression and malignant cell behavior. This study may provide potential biomarkers for accurate preoperative diagnosis and future AEG-1-targeting treatment evaluation of gliomas in vivo.</div

The construction process and results of the three monoclonal AEG-1-knockout cell lines.

(A) Schematic diagram of reconstructed pX459 plasmid. (B) Enzyme digestion showed they cannot be cut by Bbs I because the original Bbs I site had a DNA fragment insert. (C) All the plasmids had the donor DNA inserted with the correct open reading frames. (D) TA cloning analysis to identify Indel mutations, three forms of gene mutations were caused.</p

Fig 2 -

(A) Evaluation of AEG-1 gene expression in the stable AEG-1 gene knockout cell lines. (B)Flow cytometry showed a representative series figure of cell apoptosis. (C)A representative series of cell cycle FACS histograms is shown. (D)The apoptotic rate among the five cell lines. * P<0.05, ** P<0.01, vs control (Cas9) group. (E) The percentage of cells at the G1 and G2 stages of the cell cycle is shown for the five cell lines. * P<0.05, ** P<0.01, vs control (Cas9) group. (F)The invasion and migration abilities among the five cell lines. * P<0.05, ** P<0.01, *** P<0.001, vs control (Cas9) group.</p

Related data of the Transwell invasion chamber experiments and scratch test.

Related data of the Transwell invasion chamber experiments and scratch test.</p

¹H MRS spectra of the five cell lines.

Metabolites labelled are as follows: Lac: lactate(1.33–1.34ppm), Succ: succinate(1.92ppm), Ace: acetate (2.40ppm),Cr: creatine(3.04ppm), tCho: total choline(3.21–3.23ppm).</p

Sequences of the AEG-1 sgRNA oligos.

Astrocyte elevated gene-1 (AEG-1) is an important oncogene that overexpresses in gliomas and plays a vital role in their occurrence and progression. However, few reports have shown which biomarkers could reflect the level of AEG-1 expression in vivo so far. In recent years, intracellular metabolites monitored by proton magnetic resonance spectroscopy (1H MRS) as non-invasive imaging biomarkers have been applied to the precise diagnosis and therapy feedback of gliomas. Therefore, understanding the correlation between 1H MRS metabolites and AEG-1 gene expression in U251 cells may help to identify relevant biomarkers. This study constructed three monoclonal AEG-1-knockout U251 cell lines using the clustered regularly interspaced short palindromic repeat (CRISPR) /Cas9 technique and evaluated the biological behaviors and metabolite ratios of these cell lines. With the decline in AEG-1 expression, the apoptosis rate of the AEG-1-knockout cell lines increased. At the same time, the metastatic capacities decreased, and the relative contents of total choline (tCho) and lactate (Lac) were also reduced. In conclusion, deviations in AEG-1 expression influence the apoptosis rate and metastasis capacity of U251 cells, which the 1H MRS metabolite ratio could monitor. The tCho/creatinine(Cr) and Lac/Cr ratios positively correlated with the AEG-1 expression and malignant cell behavior. This study may provide potential biomarkers for accurate preoperative diagnosis and future AEG-1-targeting treatment evaluation of gliomas in vivo.</div