Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization

Abstract

BackgroundOkadaic acid (OA), a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature.ResultsIn this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h). A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY) were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure), excepting for NEFM, but their expression was similar to the controls at 48 h.ConclusionsFrom all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.

Diarrhetic effect of okadaic acid could be related with its neuronal action: Changes in neuropeptide Y

“…Also, a reduction of the levels of NPY in the intestine is directly related to the diarrheic episodes (Chandrasekharan et al, 2013). Our data supports a recent study that demonstrated the down-regulation of the NPY gene expression on the SH-SY5Y neuronal cell line (Valdiglesias et al, 2012). In vivo all those effects could produce diarrhea.…”

“…In the present study OA and DTX1 reduced the viability of the neuronal SH-SY5Y cells being DTX1 4 to 5 times more potent than OA. Together with neuronal morphological alterations, OA was also found to modify the expression of genes related to cytoskeleton and neurotransmission in neuronal cells (Valdiglesias et al, 2012). We investigated the effects of OA on the levels of NPY in SH-SY5Y cells after 1, 6, 12 and 24 h treatments.…”

In Vitro Analysis of Early Genotoxic and Cytotoxic Effects of Okadaic Acid in Different Cell Types of the MusselMytilus galloprovincialis

“…Since then, a large number of studies contributed to better understand the influence of OA (Valdiglesias et al, 2012a(Valdiglesias et al, , 2012b, showing the role played in promoting apoptosis, cytoskeleton disruption, and cell cycle alterations in mammalian cell lines . Unfortunately, the assessment of OA-induced toxicity in marine organisms, which constitute an important economic and ecological relevance, is still limited .…”

Gene expression profiling of human liver carcinoma (HepG2) cells exposed to the marine toxin okadaic acid

“…Chin et al (2000) used differential display in human glioma model T89G cells to show OA-induced upregulation of transcription factors, oxidative metabolism proteins, phosphorylation substrates, and stress response genes after 2.5 hr exposures. Valdiglesias et al (2012) used subtractive hybridization in the human neuroblastoma cell line SHSY5Y to show OA induced up and down gene regulation depending on the time of exposure from 2–48 hr with the affected genes playing roles in translation, transcription, signal transduction, metabolism, cell cycle control and apoptosis, transport, and cytoskeletal processes. The study of OA-mediated concentration effects adds to these insights.…”