Functional Enrichment Analysis by David in Transgenic MYCN Zebrafish Model

Abstract

AbstractAbstract 5114 IntroductionThe MYCN oncogene encodes a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that is frequently overexpressed in hematologic malignancies neoplasms (including acute leukemia, T-cell lymphoma, and so on). MYCN acts as a poor prognostic marker to promote an aggressive phenotype. However, the mechanisms of action and pathways affected by MYCN are still largely unclear. MethodsWe induced murine MYCN gene overexpression in embryonic zebrafish through heat-shock promoter and established stable germline Tg(MYCN:HSE:EGFP) zebrafish. RNA was extracted at 3 days post fertilization from wild type (WT) and transgenic zebrafish F1 generation (TG) embryo hematopoietic cells, collected by the flow cytometer, for microarray analysis. The samples were processed and subsequently analyzed in triplicate on Zebrafish Oligo Microarrays (Agilent Technologies), containing 43, 554 sets of probe, at the Advanced Throughput Inc. The microarrays were scanned in an Agilent DNA Microarray Scanner and the images were processed using Feature Extraction software. A False Discovery Rate≤0. 05 for overall interactions effect and P<0. 001 between comparisons were used to determine differentially expressed genes (DEG). Ingenuity Pathway Analysis and DAVID performed the functional analysis of DEG. ResultsMicroarray analysis revealed 626 (342 genes up-regulated and 284 genes down-regulated) DEG that showed >2-fold change in TG comparing with that of WT. Using functional enrichment analysis by DAVID, several signaling pathways were regulated in TG samples (Table 1). MAPK signaling pathway was high activated through FGF, PDGF, BDNF and CACN high expression, promoting up-regulated of Ras and MKP, enhancing phosphorylation and leading to increase of cells proliferation. TGFβ signaling was inhibited by up-regulation of IFN Ã and Smad 6/7, which negative control of TGFβR and Smad 2/3. Further, we found that MYCN enhances the expression of skp2, via decreased p21 and increased CDK2, promoting cell cycle progression (Fig. 1). In addition, overexpression of MYCN weakened the function of mismatch repair, base excision repair, while increased apoptosis pathway mediated by p53 (up-regulated Bid gene). Meanwhile, Glycolysis/gluconeogenesis pathway was significantly up-regulated in TG fish. ConclusionsOverexpression of MYCN induced up-regulation of cell proliferation and Glycolysis/gluconeogenesis pathway (as the Warburg effect in rapidly proliferating tumors), attenuation of repair function, all of which are phenomena associated with proliferation and malignancies transformation of blood cell feature. [Display omitted] We found that MYCN down-regulates p27kip1, p57kip2 and p21cip1 through up-regulate Skp2, thus up-regulates CDK2, CycA, CycB, CycD and CycE. All above changes shortened the time taken to progress through the cell cycle. Increased MARK signaling and decreased TGFβ signaling pathways also contributed to promote cell cycle. (Red star marks the up-regulated genes).Table 1Changes in Tg(MYCN:HSE:EGFP) zebrafish hematopoietic cellssignaling pathwayregulateCount*%#P-ValueGlycolysis/Gluconeogenesis+201.20.00000037Cell cycle+423.30.00000045Tryptophan metabolism+140.80.0000086Fatty acid metabolism+130.80.0000094Cardiac muscle contraction+201.20.000038Biosynthesis of unsaturated fatty acids+90.50.000057Phenylalanine metabolism+90.50.00014Tyrosine metabolism+110.70.0004Pyruvate metabolism+110.70.001p53 signaling pathway+1310.0021Fatty acid elongation in mitochondria+60.40.0022Arginine and proline metabolism+130.80.0038Valine, leucine and isoleucine degradation+100.60.0069Alanine, aspartate and glutamate metabolism+90.50.0069Butanoate metabolism+80.50.008Mismatch repair−141.15.9E-10Homologous recombination−120.90.00000097Base excision repair−1310.0000019Spliceosome−231.80.000033Nucleotide excision repair−1310.000038Pyrimidine metabolism−191.50.000056TGFβ−81.60.00063One carbon pool by folate−60.50.0021+: up-regulated; -: down-regulated.*: genes involved in the term.#: involved genes/total genes. P-Value: the threshold of EASE Score, a modified Fisher Exact P-Value, for gene-enrichment analysis (≤0. 01). Disclosures:No relevant conflicts of interest to declare.