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Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro

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Tumor Biology

Abstract

Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.

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Funding

This work was supported by the grant from Medical University of Silesia. The University had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.

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  1. Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Jednosci 8 Street, 41-200, Katowice, Sosnowiec, Poland

    Monika Paul-Samojedny, Renata Suchanek-Raif, Małgorzata Kowalczyk, Anna Fila-Daniłow, Paulina Borkowska & Jan Kowalski

  2. Department of Clinical Chemistry and Laboratory Diagnostics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Katowice, Poland

    Adam Pudełko

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  1. Monika Paul-Samojedny
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  2. Adam Pudełko
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  3. Renata Suchanek-Raif
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  4. Małgorzata Kowalczyk
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  5. Anna Fila-Daniłow
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  6. Paulina Borkowska
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  7. Jan Kowalski
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Correspondence to Monika Paul-Samojedny.

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Paul-Samojedny, M., Pudełko, A., Suchanek-Raif, R. et al. Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro. Tumor Biol. 36, 3263–3277 (2015). https://doi.org/10.1007/s13277-014-2955-0

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