Small RNA seq of patient-derived glioma cells (GBM1,GBM2, GG3)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE290047 | GEO | 2025/10/29
REPOSITORIES: GEO
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Project description:Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the miRNAs inolved in regulating glioma cell invasion thus appear of major relevance for the development of novel and advanced therapies. miRNA expression profiling on microdissected human tumor biopsy specimens was performed to identify differentially expressed miRNAs in invading glioblastoma cells in vivo. miRNA was extracted from the infiltrative rim (INF) as well as from the central tumor areas (CEN) of 7 patients with malignant glioma. The Micro Fluidic Card System (Applied Biosystems) was used according to the manufacturerM-bM-^@M-^Ys protocol for miRNA expression profiling.
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Project description:<p>We used massively parallel, paired-end sequencing of expressed transcripts (RNA-seq) to detect novel gene fusions in short-term cultures of glioma stem-like cells freshly isolated from nine patients carrying primary glioblastoma multiforme (GBM). The culture of primary GBM tumors under serum-free conditions selects cells that retain phenotypes and genotypes closely mirroring primary tumor profiles as compared to serum-cultured glioma cell lines that have largely lost their developmental identities.</p>
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Project description:cell culture:The human glioma cell line U87MG was obtained from the Cell Resource Center, Peking Union Medical College (Beijing, China), and U251MG was acquired from the American Type Culture Collection (Manassas, VA). Temozolomide (TMZ) resistant U87MG cells (U87TR) and TMZ resistant U251MG cells (U251TR) of glioblastoma (GBM) sub-cell lines, were established through repetitive exposure to increasing TMZ concentrations in vitro in our laboratory. Cells were cultured in DMEM culture medium supplemented with 10% FBS with a standard humidified incubator under 5% CO2 at 37°C.
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Project description:In this study, we explored the transcriptomic consequences of strong activation of the Notch pathway in embryonic human neural stem cells and in gliomas. For this we used a forced expression of the Notch intracellular domain (NICD). Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signalling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration which was accompanied by the sharp reduction of neural stem cell transcription factor expression (ASCL1, OLIG2, SOX2) while HEY1/2, KLF9, SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRb and a-smooth muscle actin (aSMA)) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (HB-EGF, IL8, PLGF), metalloprotease (MMP9) and adhesion proteins (VCAM-1, ICAM-1, ITGA9). In xenotransplantation experiments, contrasting with the infiltrative and poorly-vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly-disseminating but highly-vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. Embryonic human neural progenitors obtained from Lonza, the U87 glioma cell line, and glioma cancer stem cells (Gb4 and Gb7) characterized in Guichet et al. (Glia, 2013, 61(2), 225-39) were infected with lentiviruses expressing YFP or YFP-IRES-NICD (activated form of Notch receptor). After 48h, RNA were extracted for Affymetrix microarray analysis.
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