Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Regulation of LbetaT2 gonadotrope gene expression by GnRH pulses

ABSTRACT: LbetaT2 cells exposed to different number and concentration of GnRH pulses over 4 hours during in vitro perfusion culture We used MU74Av2 arrays to profile gene expression in response to different pulse frequency and pulse amplitude RNA extracted at end of 4 h of perfusion

ORGANISM(S): Mus musculus

SUBMITTER: Nick Webster

PROVIDER: E-GEOD-63251 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Glioblastomas show heterogeneous histological features, in which tumor cells show distinct phenotypic states that confer different functional attributes. This functional and morphological heterogeneity characterizes aggressive glioblastoma; however, molecular mechanisms underlying the heterogeneity are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute the establishment of tumor heterogeneity. Using the GSC model, we investigated the differentially expressed microRNAs(miRNAs) and associated epigenetic mechanism that regulate the differentiation of GSCs. MiRNA-microarray showed that 13 and 34 miRNAs were commonly upregulated and downregulated in two independent GSC lines during differentiation, respectively. Among those miRNAs, quantitative-PCR analysis showed that miR-1275 was consistently downregulated during the GSC differentiation along with the upregulation of its target, CLDN11, a marker of oligodendroglial-lineage differentiation. Compellingly, inhibition of miR-1275 with specific antisense oligonucleotide (anti-miR-1275) in GSC increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) and loss of H3K9Ac in the pri-miR-1275 promoter were closely associated with the miR-1275 expression. Treatment of 3-Dezaneplanocin-A, an inhibitor of H3K27 methyltransferase, impaired the GSC differentiation in parallel with sustained miR-1275 expression. Our results illuminated the epigenetic regulatory pathways of miR-1275 closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity formation of glioblastomas. Inhibition of miR-1275 induces the GSC differentiation and suppresses tumor cell proliferation, miR-1275 may be a potential therapeutic target for glioblastomas. Human miRNA Microarray V3 kits (G4470C; Agilent Technologies, Santa Clara, California) were used according to the manufacturerM-bM-^@M-^Ys protocols. This microarray system contains probes for all 866 human and 89 human viral miRNAs reported from the Sanger database v12.0 (http://microrna.sanger.ac.uk/sequences/). Each miRNA species is printed 20 times with replicate probes on the array. Total RNA was isolated with TRIzol reagent (Invitrogen).M-cM-^@M-^@One hundred ng of total RNA was labeled with pCp-Cy3 (Agilent Technologies) and 15 units of T4 RNA ligase (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 16M-BM-0C for 2 hours. Labeled samples were purified with MicroBio-Spin 6 columns (Bio-Rad, Richmond, CA) and hybridized to microarray at 55 M-BM-0C with rotation at 20 rpm for 20 hours. Microarrays were scanned by an Agilent Scanner (Agilent Scan Control 7.0 software) and analyzed using Agilent Feature Extraction 10.7 software for miRNA microarray.

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Regulation of LbetaT2 gonadotrope gene expression by GnRH pulses

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