Abstract
BackgroundGlutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells.Methods Microarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G.ResultsMiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression.ConclusionsGlutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.
Highlights
Glutaminase isoenzymes GLS and Glutaminaseisoenzyme 2 (GLS2) play apparently opposing roles in cancer: GLS acts as an onco‐ protein, while GLS2 (GAB isoform) has context specific tumour suppressive activity
To investigate the role of GLS and LongerGLS2 transcript (GAB) isoforms in miRNAs regulation, we first selected the 23 miRNAs that strongly changed their expression levels when GLS was silenced or GAB was overexpressed (Table 2)
Among miRNAs having notable expression differences between T98G-GAB(+) vs. T98G-pcDNA cells, and LN229-GAB(+) vs. LN229-pcDNA cells, miRNA140-3p were increased 8.8- and 3.5-fold, respectively, as assessed by Quantitativereverse transcription-polymerase chain reaction (qRT-PCR). Studies dealing with this particular miRNA in GBM or other brain tumours have not been yet published, our results showing a marked increase in the less-malignant and more differentiated GAB(+) cell line are in agreement with the downregulation of miRNA-140-3p found in lung cancer [21]
Summary
Glutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an onco‐ protein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. The GLS gene encodes two isoforms, known as kidney (K-type) glutaminase or KGA, and a shorter spliced form named glutaminase C or GAC [4] These two isoenzymes are usually referred to as GLS [3]. The GLS2 gene codes for the liver (L-type) isozymes, named LGA, as well as for a longer isoform named GAB originally described in breast cancer cells [2]. Both isoenzymes are collectively designated as GLS2 [3]
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