Detection of lower levels of SNAP25 using multiple microarray systems and its functional significance in medulloblastoma

Abstract

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and patients with high-risk or recurrent MB respond poorly to current therapies, and have a higher related mortality. For this reason, potential molecules related to MB need be identified in order to develop targets for the development of novel therapeutics. In the present study, we compared MB microarray data obtained using different microarray systems and significant targets were selected by gene annotation and enrichment analysis. Genes for soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) annotated with the function 'vesicle' were identified and one of these proteins, synaptosomal-associated protein 25 (SNAP25), was found to have significantly lower expression levels in MB. In addition, SNAP25 was detected in a very low number of MB cells as shown by western blot analysis and immunohistochemical analyses of archived and formalin-fixed/paraffin-embedded human MB specimens. We found that SNAP25 altered the morphology and the chemotherapeutic effects of arabinofuranosyl cytidine (Ara-C) on SNAP25-expressing MB cells. On the whole, our data indicate that the expression of SNAP25 is crucial for dendrite formation and is associated with the effects of targeted chemotherapy. The detection of SNAP25 expression in MB cells may thus be essential for the chemotherapeutic application of Ara-C.

Figure 1

Venn diagram indicating the number of differentially expressed genes in medulloblastoma from the comparisons of different microarray systems. A total of 4,596 (1,656 up- and 2,940 downregulated) genes from Agilent microarray system, 12,262 (8,306 up- and 3,956 downregulated) genes from GEO GSE7578, and 14,257 (11,274 up- and 2,983 downregulated) genes from GEO GSE22139 were respectively selected according to their differential expression. Gray regions indicated the genes repeated in different experiments. Only 2,544 genes with consistent expression level differences were determined by comparing the data obtained from Agilent and Affymetrix (GEO GSE7578 and GSE22139) microarrays. GEO, Gene Expression Omnibus; GSE, GEO series.

Figure 2

Expression differences of synaptosomal-associated protein 25 (SNAP25) between medulloblastoma and normal brain cells. (A) Comparative mRNA levels of total SNAP25 and variants by RT-qPCR. SNAP25 mRNA levels were quantified in three medulloblastoma (MB) cell lines (Daoy cells: sonic hedgehog subgroup; VGH-Med; D341 Med: group 3 subgroup) and normal brain cells. Each SNAP25 mRNA level in MB cell lines was normalized with their endogenous glyceraldehyde-3-phosphate dehydrogenenase (GAPDH) mRNA level and relative to normal brain cells. All data were then log₂ transformed [log₂(total SNAP25 of normal brains), 0]. (B) Map of primers for quantifications of total SNAP25 and variants. -, indicates the conserved sequences. (C) Endogenous SNAP25 protein in MB cell lines. Protein levels of SNAP25 (25 kDa) and GAPDH (36 kDa) were detected by western blot analysis. Lanes 1 and 2, normal medulla oblongata from BioChain and Abcam, respectively; lanes 3 and 4, left and right cerebellum from BioChain; lanes 5 and 6, normal brains from Abcam and ProSci; lanes 7 to 9, Daoy, VGH-Med and D341 Med MB cell lines. T, V1, and V2 indicated the amplified regions. T, total SNAP25; V1 or var 1, variant 1; V2 or var 2, variant 2. CDS, coding sequence; 5′-UTR, 5′-untranslated region; 3′-UTR, 3′-untranslated region; ATG, start codon; TAA, stop codon.

Figure 3

Distinct protein levels of synaptosomal-associated protein 25 (SNAP25) in normal and medulloblastoma tissues of patients. Archived medulloblastoma (MB) tissues were acquired and MB lesions were identified by hematoxylin and eosin (H&E) staining (left panel). The parts of normal (N) and tumor (T) tissues were indicated and identified by the pathologists at the Department of Pathology, Sijhih Cathay General Hospital. Immunostainings of SNAP25 protein (middle panel) and the magnified views (right panel) showed the low expression in MB lesions and the high expression in normal regions. Scale bars were indicated at the lower left corner of the images.

Figure 4

Validation of overexpressed synaptosomal-associated protein 25 (SNAP25) in Daoy cells. (A) High mRNA level of SNAP25 in cells transfected with pEGFP C3-SNAP25. The increasing SNAP25 mRNA levels in the stable clones were quantified by RT-qPCR. The difference in mRNA levels was compared using the Student's t-test. Data are representative of at least 3 experiments with similar results, and a value of p<0.05 was considered to indicate significance. (B) Expression of SNAP25 protein in cells transfecdted with pEGFP C3-SNAP25. The increasing SNAP25 protein expression was immunoblotted by western blot analysis. GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Morphological changes of cells with synaptosomal-associated protein 25 (SNAP25) expression. (A) The dendrites of Daoy cells with SNAP25 expression. (B) The dendrites of VGH-Med cells with SNAP25 expression. The cells transfected with pEGFP C3 vector emitted a green fluorescence throughout the cell and the SNAP25-overexpressing cells (pEGFP C3-SNAP25) formed dendrites that emitted green fluorescence. DAPI (blue) indicated the nucleus. (C) SNAP25 expression in normal neural cells. White arrowheads indicate the dendrites. GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; TAU, Tau protein; PNM, pan neuronal marker; DAPI, 4′,6-diamidino-2-phenylindole; IBMX, 3-isobutyl-1-methylxanthine.

Figure 6

Change of susceptibility to arabinofuranosyl cytidine (Ara-C) for SNAP25-expressing Daoy cells. The half-maximal inhibitory concentration of Ara-C was determined under a dose-dependent manner (from 0.1 to 10 mM) of Ara-C following incubation for 48 h. The surviving cells were counted by CCK-8 assay. Data are the means ± SD of triplicate independent experiments. Ara-C, arabinofuranosyl cytidine; CCK-8, cell counting kit-8. SD, standard derivation.