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Series GSE267374 Query DataSets for GSE267374
Status Public on May 18, 2024
Title Transcriptomic analysis of human vascular endothelial cell line (EA.hy926) exposed to okadaic acid
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary As the climate changes due to global warming, the frequency of appearance of marine-derived toxins increases. In particular, the need to investigate marine-derived toxins for which toxicity has not yet been identified is emerging. Blood vessels are passageways through which compounds entering the body move to various organs and are the sites first exposed to compounds. Therefore, it is important to know the effects of compounds on blood vessels when they enter the body. In this study, the human vascular endothelial cell line EA.hy926 was exposed to the marine toxins OA (IC20; 42.9 nM) and the mechanism of toxicity was investigated at the transcriptome level. Quantseq 3' mRNA sequencing was performed by extracting RNA from EA.hy926 cells exposed to the toxins. Differentially expressed genes were determined based on fold change ≥ 1.5 and P value < 0.05.
Overall design Cell culture The EA.hy926 cell line (human vascular endothelial cell line) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco®, USA) supplemented with 10% fetal bovine serum (FBS; Gibco®, USA), 1% Penicillin/Streptomycin (P/S; Gibco®, USA), 3.7 g/L sodium bicarbonate (Sigma-Aldrich, USA), 1 mM sodium pyruvate (Gibco®, USA) and maintained at 37℃ under 5% CO2. The cells were grown in a 100 mm culture dish and subcultured every 3 d by detaching with 0.25% (w/v) trypsin-EDTA (Gibco®, USA) at a split ratio of approximately 1:3. Cell viability assay The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT; Sigma-Aldrich, USA) assay is based on the principle that the yellow MTT solution is reduced to purple by the dehydrogenase of living cells that exhibit mitochondrial activity. EA.hy926 cells were seeded at a density of 20×104 cells per well in 96-well plates and incubated for 24 h. Cells were exposed to OA (National Research Council, Canada) at concentrations of 4.0625–1040 nM, respectively. DMEM was used as a solvent for OA. After 24 h of exposure, the medium was removed, 200 μL of MTT solution was dispensed into each well and incubated in the dark at 37°C for 3 h. Afterward, the medium was removed and 200 μL of DMSO was dispensed to dissolve the formazan crystals. Absorbance value were measured at a wavelength of 570 nm using a spectrophotometer (Varioskan LUX Multimode Microplate Reader, Thermo Fisher, USA). Cell viability of the control group was set to 100%, and the survival of the exposure group was calculated accordingly. IC20 was defined as the concentration that reduced cell viability by 20% compared to that in the control group. IC20 values were determined using linear-dose curves. Total RNA isolation EA.hy926 cells were treated with OA at IC20 concentration for 24 h, and total RNA was extracted using TRIzol™ reagent (Invitrogen, USA). Total RNA was purified using the RNeasy Mini Kit (Qiagen, USA). The RNase-free Kit (Qiagen, USA) was used to remove genomic DNA during RNA purification. The RNA integrity number was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Fisher, USA). Library preparation and sequencing To obtain gene information, Quant sequencing was performed using EA.hy926 cells exposed to OA (IC20). Sequencing was conducted using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Austria). Two micrograms of total RNA were prepared and oligo dT primers containing Illumina-compatible sequences at the 5' end were hybridized to RNA and reverse transcription carried out. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. The double-stranded library was purified by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library was purified from the PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, USA). Data analysis of RNA-seq QuantSeq 3’ mRNA sequencing reads were aligned using Bowtie2 (Langmead and Salzberg, 2012). Bowtie2 indices were either generated from the genome assembly sequence or the representative transcript sequences for aligning to the genome and transcriptome. The alignment file was used for assembling transcripts, estimating their abundances, and detecting differential expression of genes. DEGs were determined based on counts from unique and multiple alignments based on coverage, in Bedtools (Quinlan and Hall, 2010). The Read count (RC) were processed based on the quantile normalization method using the EdgeR tool in R (Robinson and Mccarthy and Smyth, 2010) using the Bioconductor package (Gentleman et al., 2004). Gene classification was based on searches performed by DAVID Bioinformatics Resources ( and Medline databases ( Data mining and graphic visualization were performed using ExDEGA v5.1.1.4 (Ebiogen Inc., Korea).
Contributor(s) Park J, Kim JH, Kim Y
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Submission date May 14, 2024
Last update date May 18, 2024
Contact name Jeong-In Park
Phone +82 10-6598-2052
Organization name Incheon National University
Street address 119, Academy-ro
City Incheon
ZIP/Postal code 22012
Country South Korea
Platforms (1)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (6)
GSM8264900 Control of EA.hy926 cell treated to OA 1
GSM8264901 Control of EA.hy926 cell treated to OA 2
GSM8264902 Control of EA.hy926 cell treated to OA 3
BioProject PRJNA1111366

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