Valdiglesias et al. BMC Genomics 2012, 13:46 http://www.biomedcentral.com/1471-2164/13/46 RESEARCH ARTICLE Open Access Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization Vanessa Valdiglesias1,2, Juan Fernández-Tajes2, Eduardo Pásaro1, Josefina Méndez2 and Blanca Laffon1* Abstract Background: Okadaic 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. Results: In 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. Conclusions: From 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. Background has become a serious problem for public health and for Okadaic acid (OA) is a marine toxin produced by sev- the economy of aquaculture industries in several parts eral dinoflagellate species. It was firstly isolated from the of the world [3]. black sponge Halichondria okadai [1] and is frequently OA was found to be a very potent tumour promoter found in several types of molluscs usual in the human in two-stage carcinogenesis experiments in vivo invol- diet as those from Mytilus or Ostrea genus. The inges- ving mouse skin [4] or mucosa of the rat glandular sto- tion of OA-contaminated shellfish results in a syndrome mach [5]. OA was also reported to induce different known as diarrhoeic shellfish poisoning (DSP) which is genotoxic, cytotoxic, and embryotoxic effects such as characterized by severe gastrointestinal symptoms micronuclei [6-8], oxidative DNA damage [9,10], DNA including nauseas, vomit, diarrhoea and abdominal ache strand breaks and alterations in DNA repair [11], mito- [2]. Although fatalities associated with DSP-contami- tic spindle alterations [12,13], apoptosis [14,15], cell nated shellfish have not been reported, this intoxication cycle disruptions [15,16], anomalies of the embryonic development [17] and teratogenicity [18]. Besides, despite the fact that DSP toxins are not classified as * Correspondence: [email protected] 1Toxicology Unit, Psychobiology Department, University of A Coruña, Edificio neurotoxins [19], some previous studies have already de Servicios Centrales de Investigación, Campus Elviña s/n, 15071 A Coruña, reported neurotoxic effects induced by OA including Spain neuronal apoptosis and cytoskeleton alterations [20,21], Full list of author information is available at the end of the article © 2012 Valdiglesias et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Valdiglesias et al. BMC Genomics 2012, 13:46 Page 2 of 14 http://www.biomedcentral.com/1471-2164/13/46 deficits in spatial memory [22] and also cognitive deficits was quantified and quality checked using the NANO- in rodents [23]. DROP™ 1000 spectrophotometer (Thermo Scientific, On the basis on these and other previous studies, OA Madrid, Spain). One microgram of total RNA from each represents other potential threats to human health sample was used as template to synthesize the first besides DSP, even at concentrations within the nanomo- strand cDNA with the SMART™ PCR cDNA Synthesis lar range. It is well-known that OA can inhibit specifi- Kit (Clontech, Madrid, Spain), a method for producing cally the serine/threonine protein phosphatases 1 (PP1) high quality cDNA from a low amount of starting mate- and 2A (PP2A) [24]; the number of physiological pro- rial [29]. The double stranded cDNA was amplified with cesses in which those phosphatases are involved is the same Kit according to the manufacturer’s protocol. immense, including regulation of glycogen metabolism and coordination of the cell cycle and gene expression Construction of subtracted cDNA libraries [25]. So this role of phosphatase inhibition by OA could SSH was carried out with the PCR-Select™ cDNA sub- explain most of the cell effects induced by this toxin traction kit (Clontech, Madrid, Spain), as described by [26]. However the number of controversial data in the the manufacturer. Briefly, the double stranded cDNAs literature continues increasing and further investigations obtained from the step described above were digested on biochemical and molecular OA action mechanisms with the restriction enzyme RsaI to obtain blunt-ends are required since the fact that non-phosphatase targets that are necessary for adaptor ligation. cDNA subtrac- for OA are not known does not mean that they do not tion was carried out in two directions for the different exist [3]. In fact, the existence of OA binding proteins exposure times. The forward subtracted libraries were other than phosphatases was demonstrated in several made with the control cells cultured for 3, 24 or 48 h as marine organisms [27,28]. the driver and OA-exposed cells (also cultured for 3, 24 In this study, a suppression subtractive hybridization or 48 h) as the tester. These forward reaction libraries (SSH) approach was used to identify genes differentially were designed to produce clones that are overexpressed expressed in SHSY5Y cells in response to OA exposure or up-regulated in OA-treated cells. The reverse at different times (3, 24 and 48 h). Sequences obtained libraries were made in the same way, but in this case by SSH were used to search for homology/identity to the adapter ligated cDNA from OA-exposed cells were nucleotide and protein databases. Furthermore, differen- the driver and control cells were the tester. The reverse tial expression patterns of 5 selected genes were also reaction library was designed to produce clones under- studied in OA-treated SHSY5Y cells at 3, 24 and 48 h expressed or down-regulated in OA-treated cells. In by real-time PCR. either case the driver cDNA was added in excess during each hybridization to remove common cDNAs by hybrid Methods selection and leaving overexpressed and novel tester Cell culture and OA treatment cDNA to be recovered and cloned. The subtracted ® SHSY5Y cells (human neuroblastoma cell line) were cDNA fragments were then inserted into yT&A clon- obtained from the European Collection of Cell Cultures ing vector, transformed into Escherichia coli ECOS707 and cultured in nutrient mixture EMEM/F12 (1:1) med- (strain JM109) competent cells, and plated on LB agar ium with 1% non-essential aminoacids, 1% antibiotic plates containing 100 μg/ml ampicillin, 100 μlIPTG ® and antimycotic solution and supplemented with 10% (100 mM) and 20 μl X-gal (50 mg/ml). The yT&A heat-inactivated foetal bovine serum, all from Invitrogen cloning vector and the E. coli ECOS707 competent cells (Barcelona, Spain). The cells were incubated in a humi- were purchased from Yeastern Biotech. Co., Ltd., (Tai- dified atmosphere with 5% CO2 at 37°C. OA (CAS No. pei, Taiwan). From the six libraries, a total of 384 white 78111-17-8) was purchased from Sigma-Aldrich Co. recombinant colonies (corresponding to four 96-well (Madrid, Spain) and dissolved in DMSO prior use. plates) were picked. Flasks with approximately 90% of confluence and 4 × 106 cells were chosen for the experiments. For the treat- Sequencing of the subtracted cDNA clones and ments, cells were incubated at 37°C for 3, 24 or 48 h in bioinformatics analysis the presence of OA (100 nM) or the control dimethyl- Sequencing of all the cDNA clones from the six SSH ® sulfoxide (DMSO) at 1% of final volume. libraries was carried out using the BigDye Terminator v3.1 (Applied Biosystems) and an AB3730 sequencer Total RNA isolation and cDNA synthesis for SSH (Applied Biosystems) at Secugen (Madrid, Spain). After After OA treatments, total RNA was isolated from excluding redundant and false-positive sequences, ® SHSY5Y cells with TRIZOL reagent (Invitrogen, nucleic acid homology searches were performed against Madrid, Spain) following the manufacturer’sinstruc- nucleotide databases at the National Center for Biotech- tions, and then dissolved in nuclease-free water. RNA nology Information