Unknown,Transcriptomics,Genomics,Proteomics

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Expression data after BCLAF1-L knockdown in human RKO cells

ABSTRACT: Bcl-2-accociated transcription factor 1(BCLAF1) has been reported to be involved in diverse biological processes. Alternative splicing leads to mutiple transcript virants in human cells and we are interested in functions of its full length isoform(BCLAF1-L) in human colon cancer cells, thus to understanding its role in colon cancer progression. We used microarray to detail the global programme of gene expression after BCLAF1-L knockdown(sh-BCLAF1-L#1) in RKO cells and identified distinct classes of up-regulated or down-regulated genes impaired by its inhibition, when comparing with the control group(sh-Luci). RKO cells were infected with retroviruses either expressing control (sh-Luci) or BCLAF1-L knockdown (sh-BCLAF1-L#1). Medium was replaced 24h after infection, and infected cells were selected by the addition of puromycin (2ug/ml) for 72-96h. Then cells were havested for RNA extraction and hybridization on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Feng Ying

PROVIDER: E-GEOD-52990 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

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Expression data after BCLAF1-L knockdown in human RKO cells

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