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RNA-seq transcriptome analysis OF formalin-fixed paraffin-embedded canine meningioma

ABSTRACT: Meningiomas are the most commonly reported primary intracranial tumor in dogs and humans. Canine meningiomas closely resemble human meningiomas in histological appearance as well as biological behavior. Because of the similarities between canine and human meningiomas, dogs have been proposed as models and surrogates for studying and treating human cancers including meningioma. However, little is known about specific pathways and individual genes that are involved in the development and progression of canine meningioma. In addition, there is a paucity of suitable fresh material available for studies of differential expression (DE) of genes in cases of veterinary neoplasia. We report here the use of formalin-fixed paraffin embedded (FFPE) specimens as much as a decade old, to provide RNA suitable for transcriptome analysis using next-generation sequencing (NGS). RNA was extracted from 13 canine meningiomas – 12 from (FFPE) and one flash-frozen. These represented 6 grade I and 7 grade II/III. RNA was also collected from flash-frozen meningeal tissue (arachnoid) from 3 control dogs. RNA from FFPE was of sufficient quality to successfully identify 125 significantly DE genes, the majority of which were related to oncogenic processes. 12 genes (AQP1, BMPER, FBLN2, FRZB, MEDAG, MYC, PAMR1, PDGFRL, PDPN, PECAM1, PERP, ZC2HC1Cc) were validated using qPCR. Among the DE genes were oncogenes, tumor suppressors, transcription factors, VEGF-related genes, and members of the WNT pathway. Our work demonstrates that RNA of sufficient quality can be extracted from FFPE samples to provide biologically relevant transcriptome analyses using a NGS technique, such as RNA-seq.

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE95048 | GEO | 2017/10/30

SECONDARY ACCESSION(S): PRJNA375808

REPOSITORIES: GEO

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Project description:Purpose: In this study, we try to investigate the possible signaling pathways involved in the tumorigenesis of fibroblastic and anaplastic meningiomas. We also attempt to investigate EGFL6 gene expression in brain arachnoidal tissues and various tumors and to measure EGFL6 levels in serum samples from healthy people and patients with various tumors by using ELISA. Experimental Design: Differential gene expression profiles between meningiomas and brain arachnoidal tissues were established by using Affymetrix GeneChip Human U133 Plus 2.0 Array. KEGG pathway analysis was performed to identify potential gene pathways that may be involved in the pathogenesis of meningiomas. Quantitative real-time PCR (qRT-PCR) was performed to validate the differentially expressed genes in the KEGG pathways. EGFL6 mRNA levels were also determined in brain arachnoidal tissues, meningiomas, and other tumors by qRT-PCR. EGFL6 levels were measured in serum samples from healthy people and patients with various tumors by using ELISA. Results: Fibroblastic meningioma exhibited upregulated PI3K/Akt and TGFÎ² signaling pathways, and accelerated G1/S progression cell cycle. KEGG analysis also demonstrated that focal adhesion and ECM-receptor interaction pathways were activated in anaplastic meningioma. Benign meningiomas had significantly higher levels of EGFL6 mRNA than brain arachnoidal tissues and atypical and anaplastic meningiomas (P<0.001). EGFL6 gene was also highly expressed in ovarian cancer, but expressed lowly in all other investigated tumors. EGFL6 was hardly detectable in serum samples of healthy people. The mean serum EGFL6 concentration was 675, 118, and 126 pg/ml in patients with benign, atypical, and anaplastic meningiomas respectively. Patients with ovarian cancers also had high serum EGFL6 levels (mean concentration: 617 pg/ml). Patients with all other investigated tumors, however, had low levels of serum EGFL6 with mean concentration less than 240 pg/ml. Conclusion: We proposed that deregulation of cell cycle and PI3K/Akt pathways might play important roles in the tumorigenesis of fibroblastic meningioma. It was also suggested that the activated integrin-mediated signaling pathways were involved in the pathogenesis of anaplastic meningioma. We presented evidence that EGFL6 might serve as a novel serum biomarker for benign meningioma and ovarian cancer. It was also suggested that EGFL6 could help discriminate benignancy or malignancy of meningiomas before surgery or at early time points. Differential gene expression profiles between meningiomas and brain arachnoidal tissues were established by using Affymetrix GeneChip Human U133 Plus 2.0 Array. KEGG pathway analysis was performed to identify potential gene pathways that may be involved in the pathogenesis of meningiomas. Quantitative real-time PCR (qRT-PCR) was performed to validate the differentially expressed genes in the KEGG pathways. EGFL6 mRNA levels were also determined in brain arachnoidal tissues, meningiomas, and other tumors by qRT-PCR. EGFL6 levels were measured in serum samples from healthy people and patients with various tumors by using ELISA.

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RNA-seq transcriptome analysis OF formalin-fixed paraffin-embedded canine meningioma

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