Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

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7.3 Tumour Expression Correlation Results cytoma, but up-regulated in primary GBM (Fig 7.4). Finally, we found the TAGLN and TES genes to be absent or low in most GNS lines, but displaying the opposite trend in GBM tissue compared to normal brain (Fig 7.4c) or grade III astrocytoma (Fig 7.4d). Interestingly, the TES gene is located on chromosome 7, which is known to be gained in all our GNS cell lines (see 6.3 section). A strong regulatory effect must be in action in GNS cell lines to effect the down-regulation of TES. Similarly to the SYNM gene, TES has been shown to interact with the LIM domain protein Zyxin and therefore is believed to have a role in cell motility and is often found in focal adhesions [109]. The TAGLN gene encodes an actin protein found in fibrob- lasts and smooth muscle, the expression of which is down-regulated in many cell lines and may be an early marker for the onset of transformation [260]. Both TAGLN and TES have been characterised as tumour suppressors in ma- lignancies outside the brain and TES is known to often be silenced by promoter hypermethylation in GBM [30,349]. Altogether, MAF, MYL9, HMGA2, SDC2, SYNM, IRX2, TES and TAGLN may be part of a GNS expression profile that helps define the stem cell identity of these cells and the tumour that derives from them according to the cancer stem cell hypothesis. Interestingly, the expression pattern of most of these genes is mirrored in grade III astrocytomas. Of the seven genes that are down-regulated in both GNS cell lines and primary GBMs, TUSC3 is a candidate tumour suppressor known to be silenced by promoter methylation in GBM, particularly in patients over 40 years of age. Loss or down-regulation of TUSC3 has been found in other cancers, such as colon cancer, where its promoter becomes increasingly methylated with age in the healthy mucosa [12]. These data suggest that transcriptional changes in healthy aging tissue, such as TUSC3 silencing, may contribute to the more severe form of glioma in older patients. In line with its role as tumour suppressor, we found TUSC3 to be down-regulated in GNS cell lines, primary GBM and grade III astrocytoma (Fig 7.4). The PLCH1 gene is a member of the phospholipase C family of enzymes that cleave phosphatidylinositol 4,5-bisphosphate to generate second messengers in- ositol 1,4,5-trisphosphate and diacylglycerol. PLCH1 is thus involved in phos- phoinositol signaling [228], just like the frequently mutated phosphoinositide 3-kinase complex [326]. We found PLCH1 to be down-regulated in GNS cell lines and primary GBM (Fig 7.4c) and slightly up-regulated in grade III astrocytoma (Fig 7.4d). 180
7.3 Tumour Expression Correlation Results The PTEN lipid phosphatase gene is a well known tumour suppressor that is frequently deleted or mutated in GBM and its down-regulation affects the correct regulation of the RTK/PI3K/PTEN pathway in which it acts as an instrumental player [326]. PTEN is mutated in a variety of other cancers be- sides gliomas, including prostate, breast, endometrial cancer and melanoma [377,378,439,514]. We observe the expression of PTEN to be down-regulated in GNS cell lines and primary GBM (Fig 7.4c) but up-regulated in grade III astrocytoma (Fig 7.4d). The ST6GALNAC5 gene belongs to the sialyltransferase family of proteins that modify proteins and ceramides on the cell surface to alter cell-cell or cell- extracellular matrix interactions [498]. ST6GALNAC5 facilitates the transmi- gration of cancer cells through the blood-brain barrier and is known to mediate breast cancer metastasis to the brain [67]. In GBM ST6GALNAC5 has already been observed to be down-regulated with respect to normal brain tissue [250], and we observed it to be down-regulated in GNS cell lines as well as primary GBM and slightly up-regulated in grade III astrocytoma (Fig 7.4). The NDN gene is an imprinted gene expressed exclusively from the pater- nal allele that has no introns and acts as a growth suppressor. Studies in mice suggest that the Necdin protein suppresses growth in postmitotic neurons [209,487]. Necdin has also been suggested to interact with and negatively regulate HIF1α, a factor that mediates cellular homeostatic responses like angiogenesis to reduce O2 availability [342]. The expression of NDN is also known to be down-regulated due to the transcriptional control implemented by STAT3 in human melanoma, prostate cancer and breast cancer [188]. We found NDN to be strongly down-regulated in GNS cell lines and primary GBM but strongly up-regulated in grade III astrocytoma (Fig 7.4). The MAP6 gene encodes a microtubule-associated protein that is calmodulin- binding and calmodulin-regulated and is therefore involved in microtubule sta- bilisation. MAP6 has yet to be associated with any neoplasia and we observed it to be down-regulated in GNS cell lines and primary GBM, and slightly up-regulated in grade III astrocytoma (Fig 7.4). Finally, the NELL2 gene encodes a glycoprotein containing several EGF-like repeats that is found in the cytoplasm and is involved in neural cell growth and differentiation as well as oncogenesis [521]. Together with NELL1, NELL2 is predominantly expressed in neuroblastoma and other embryonal neuroepithelial tumours [305], but has yet to be characterised in glioblastoma. We observed its expression to be down- regulated in GNS cell lines and primary GBM, as well as grade III astrocytoma 181
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Transcriptional Characterization of
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This dissertation is my own work an
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Acknowledgements I would like to th
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5.9 External Dataset Expression Cor
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Introduction 1
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1.1 Glial Cells in the Central Nerv
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1.2 Glioblastoma Multiforme Introdu
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1.3 Primary and Secondary Glioblast
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1.4 Pathways Involved in Glioblasto
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1.5 Pathway Crosstalk Introduction
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Chapter 2 Neurogenesis Contents 2.1
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2.1 Radial Glia Introduction VZ adj
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2.2 Neural Stem Cells Introduction
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Chapter 3 Brain Cancer Stem Cells C
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3.1 The Cancer Stem Cell Hypothesis
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3.1 The Cancer Stem Cell Hypothesis
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3.2 Brain Cancer Stem Cells Introdu
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3.3 Glioma Culture Systems Introduc
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Chapter 4 The Non-Coding RNA World
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4.1 MicroRNA regulation Introductio
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4.1 MicroRNA regulation Introductio
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4.2 Target Prediction and Validatio
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Methods 93
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5.1 Tag-sequencing Data Processing
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5.1 Tag-sequencing Data Processing
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5.2 Array Comparative Genomic Hybri
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5.4 Quantitative Real Time-PCR Vali
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5.5 Literature Mining Methods Figur
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5.5 Literature Mining Methods How m
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5.6 Differential Isoform Expression
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5.9 External Dataset Expression Cor
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5.10 Glioblastoma Pathway Construct
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5.11 MicroRNA Target Prediction Ana
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Results 119
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6.2 Tag mapping Results Table 6.1:
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6.2 Tag mapping Results 123 Figure
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6.3 Copy Number Aberrations Results
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6.3 Copy Number Aberrations Results
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9.2. MicroRNA Target Prediction Ana
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9.3. Concluding Remarks Appendix pr
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A.1 Differential Expression Appendi
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A.2 Classified Differential Express
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.3 Quantitative RT-PCR Appendix Ta
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A.3 Quantitative RT-PCR Appendix We
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A.4 Tag-seq vs qRT-PCR Correlation
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Appendix "BEX5", "DIAPH2", "GBP3",
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End Select End If End Sub Appendix
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If NameStr.ToLower().Equals("query"
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Appendix C Long ncRNAs We detected
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C. Long ncRNAs Appendix Tag Accessi
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D.1 Pathway Interactions Appendix F
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D.2 Pathway Images Appendix Figure
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D.2 Pathway Images Appendix Figure
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E. Exon Array Data Appendix Average
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F. MicroRNA Array Data Appendix Tab
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F. MicroRNA Array Data Appendix GNS
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F. MicroRNA Array Data Appendix GNS
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Ln Natural logarithm LOH Loss of He
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3.3 Schematisation of cell cycle ph
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8.3 Workflows at the core of the pr
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7.1 Selected Gene Ontology terms an
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