TaqMan Low Density Arrays on Glioma Stem-like Cell lines (GSC-1 to 11)
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ABSTRACT: Determination of pluripotency and differentiation-related genes expression in GSC-1 to 11 11 GSCs lines and human ES (H1) cell line as reference
Project description:We compared whole genome expression profiles of GSCs with normal human cortex, human neural stem cells (hNSC) from fetal cortex, glioblastoma (GBM) primary, and recurrent tumors to find GSC-specific plasma membrane transcripts. All of the expression profiles were batch normalized by a robust multichip average (RMA) algorithm using Geospiza GeneSifter (PerkinElmer) online microarray database and analysis software. The data was then exported into Microsoft Office Excel 2010 and organized for GSC transcripts with raw intensity values 10 fold or higher over normal brain, hNSCs, GBM primary and recurrent tumor samples. The reverse sorting algorithm was done to obtain downregulated GSC trascripts.
Project description:We compared whole genome expression profiles of GSCs with normal human cortex, human neural stem cells (hNSC) from fetal cortex, glioblastoma (GBM) primary, and recurrent tumors to find GSC-specific plasma membrane transcripts.
Project description:Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs potentially regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). ALDH1A3 is the dominant isomer of the ALDH gene family in Mesenchymal subtype of GSC cells and is highly upregulated compared to other subtype of GSCs. ALDH1A3 is an important enzyme in the synthesis of Retinoic Acid, which regulates various downstream pathways and the transcription of numerous genes. Microarray analysis of the GSCs before or after depletion of ALDH1A3 provides important information to determine the genes regulated by ALDH1A3 in the mesenchymal subtype of GSCs. We used microarrays to analyze the transcriptome change after the depletion of ALDH1A3 in GSC-326 cells that express very high levels of ALDH1A3.
Project description:At the cellular level, the malignant characteristics of GBM are largely attributed to GBM stem cells (GSCs). These cells, endowed with stem-like properties, can self-renew, generate diverse cancerous cell populations, and initiate tumors in vivo. The GSC lines, NCH644 and NCH421k, when cultured as neurospheres (NS) — a condition that preserves their stem-like state, as evidenced by the expression of stem cell markers like SOX2, CD133, and Nestin — were found to align with the PN subclass and CL-B/C respectively.
Project description:Abstract from Knabel et al. PLoS One (2015): Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include mir29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of mir-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders. The Taqman MicroRNA Array (Taqman Array Rodent MicroRNA A Cards v2.0, ABI) was run, using manufacturer's protocols, on 3 groups of mice with varying amounts fibrotic injury. The groups include carbon tetrachloride intraperitinially injected bi-weekly for 1, 4, and 8 weeks with n=4, 3, and 2 respectively for each group. The RNA was isolated from whole liver using the MiRvana miRNA Isolation Kit (Ambion) according to manufacturer's protocols. The fold_change.txt contains the following data columns; ID_Ref: mmu-miR being quantified not-treated (FC): Fold Change compared to not-treated group using Geometric mean normalization 1 week (FC): Fold Change compared to not-treated group using Geometric mean normalization 4 weeks (FC): Fold Change compared to not-treated group using Geometric mean normalization 8 weeks (FC): Fold Change compared to not-treated group using Geometric mean normalization
Project description:Using induced pluripotent stem cell (iPSC) technology, we reprogrammed GBM derived cells (GBM-DCs) into embryonic-like cells termed as induced core-GSCs (ic-GSCs). The aim of this experiment was to characterize the transcriptomic profile of ic-GSCs using RNA-seq. For this experiment, we selected three biological replicates of GBM-DCs (GBM-DC1, GBM-DC2 and GBM-DC3) and three independent clonal lines of ic-GSCs (ic-GSC#1, ic-GSC#3 and ic-GSC#7).
Project description:Using induced pluripotent stem cell (iPSC) technology, we reprogrammed GBM derived cells (GBM-DCs) into embryonic-like cells termed as induced core-GSCs (ic-GSCs). The aim of this experiment was to characterize the DNA methylation profile of ic-GSCs using the Infinium MethylationEPIC array BeadChip (850K, Illumina). For this experiment, we selected three biological replicates of GBM-DCs (GBM-DC1, GBM-DC2 and GBM-DC3) and three independent clonal lines of ic-GSCs (ic-GSC#1, ic-GSC#3 and ic-GSC#7).
Project description:Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However, molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further, Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway, comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3, were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover, inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last, radiation treatment of PN GSCs up-regulated Mes-associated markers and downregulated PN-associated markers, whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together, our data suggest that two subtypes of GSCs, harboring distinct metabolic signaling pathways, represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3- mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature.
Project description:Plasma samples from 10 colonrectal cancer patients (CRCs) (including 5 stage II and 5 stage III patients) and 10 normal controls.An amount of 5 to 10 milliliters of whole blood were obtained from each participant.The plasma was obtained by centrifugation at 1200g for 10min at 4°C.To complete the removal of residual cellular components, plasma samples were recentrifuged at 12,000g for a further 10min at 4°C.A volume of 600?L of each plasma samples from CRC group or normal control group was picked out and uniformly mixed. qPCR miRNA expression profiling. Plasma samples form 10 colonrectal cancer patients(CRCs) (including 5 stage II and 5 stage III patients) and 10 normal controls were used and treated as indicated in the summary. Equal amount total RNA from each donor was pooled prior to gene expression analysis.