Article
Analysis of meningioma specimens regarding the gene expression levels of PI3K-Akt-mTOR and Receptor-Tyrosine-Kinase signalling pathway proteins and their WHO grade dependent variations using qPCR
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Published: | May 13, 2014 |
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Objective: It is known from different human tumors, that PI3K-Akt-mTOR and Receptor-Tyrosine-Kinase signalling pathways, which lead to an increase of tumor proliferation and a reduction of apoptosis, in most cases are constantly activated or even over-activated, resulting in further tumor progression. To analyze the gene expression of these receptor proteins in meningeomas, the cDNA of surgical specimens of all three WHO grades and transcripts of normal dural tissue were quantitatively examined using real-time polymerase chain reaction (qPCR). Furthermore we were interested in possible WHO grade dependent expression variations of the target genes.
Method: After mRNA transcription of 75 meningioma samples (WHO I°-III°) into cDNA, the expression of the candidate genes PDGFRbeta, VEGFR-2, pan-Ras, B-Raf, ERK1, ERK2, Akt1 and mTOR was measured by qPCR and analyzed applying the delta-delta ct method for approximation, referring to reference genes in comparison to reference specimens of the dura mater.
Results: During selection of appropriate reference genes, which should present a high stability in their expression for standardisation of the target genes, we found that the commonly used housekeeping genes HPRT, SDHA, GAPDH and YWHAZ are not suitable in meningiomas, because of their instable and regulated gene expression measured in the transcripts. More valuable reference could be provided by Vimentin as a marker identifying mesenchymal tissue and EMA (epithelial membrane protein), a key marker of meningiomas. Additionally we found differences in expression of all target genes (PDGFRbeta, VEGFR-2, pan-Ras, B-Raf, ERK1, ERK2, Akt1 and mTOR) compared to dural specimens. Surprisingly there were no significant WHO grade dependent expression variations of the target genes in the tested meningiomas.
Conclusions: Analyzing mRNA of a larger group of surgical specimens of meningiomas (WHO I°-III°) by quantitative RT-PCR with regard to the gene expression of proteins of the PI3K-Akt-mTor- and the Receptor-Tyrosine-Kinase signaling cascades, we found that there are expression differences compared to dural tissue. But contrasting the groups of transcripts classified by their WHO grade, no significant variations could be found.