A Systematic Analysis Revealed the Potential Gene Regulatory Processes of ATRA-Triggered Neuroblastoma Differentiation and Identified a Novel RA Response Sequence in theNTRK2Gene

Abstract

Retinoic acid- (RA-) triggered neuroblastoma cell lines are widely used cell modules of neuronal differentiation in neurodegenerative disease studies, but the gene regulatory mechanism underlying differentiation is unclear now. In this study, system biological analysis was performed on public microarray data from three neuroblastoma cell lines (SK-N-SH, SH-SY5Y-A, and SH-SY5Y-E) to explore the potential molecular processes of all-trans retinoic acid- (ATRA-) triggered differentiation. RT-qPCR, functional genomics analysis, western blotting, chromatin immunoprecipitation (ChIP), and homologous sequence analysis were further performed to validate the gene regulation processes and identify the RA response element in a specific gene. The potential disturbed biological pathways (111 functional GO terms in 14 interactive functional groups) and gene regulatory network (10 regulators and 71 regulated genes) in neuroblastoma differentiation were obtained. 15 of the 71 regulated genes are neuronal projection-related. Among them, NTRK2 is the only one that was dramatically upregulated in the RT-qPCR test that we performed on ATRA-treated SH-SY5Y-A cells. We further found that the overexpression of the NTRK2 gene can trigger differentiation-like changes in SH-SY5Y-A cells. Functional genomic analysis and western blotting assay suggested that, in neuroblastoma cells, ATRA may directly regulate the NTRK2 gene by activating the RA receptor (RAR) that binds in its promoter region. A novel RA response DNA element in the NTRK2 gene was then identified by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. The novel element is sequence conservation and position variation among different species. Our study systematically provided the potential regulatory information of ATRA-triggered neuroblastoma differentiation, and in the NTRK2 gene, we identified a novel RA response DNA element, which may contribute to the differentiation in a human-specific manner.

Human platelet lysate supports SH‐SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic‐like neuronal phenotype under xenogeneic‐free culture conditions

“…Similar results were presented in a recent study, where no significant changes were found in MAP2 and SYP transcript levels in SH-SY5Y cells in response to RA treatment, despite the changes in cell morphology and MAP2 staining pattern observed by immunocytochemistry. [36] Additionally, MAP2 and SYP were not identified among the significantly regulated genes in previous microarray and RNA-seq transcriptional profiling studies of RA-treated SH-SY5Y cells. [33,37] These are presented (three biological replicates per experiment).…”

“…Similar results were presented in a recent study, where no significant changes were found in MAP2 and SYP transcript levels in SH‐SY5Y cells in response to RA treatment, despite the changes in cell morphology and MAP2 staining pattern observed by immunocytochemistry. [ 36 ] Additionally, MAP2 and SYP were not identified among the significantly regulated genes in previous microarray and RNA‐seq transcriptional profiling studies of RA‐treated SH‐SY5Y cells. [ 33,37 ] These studies highlighted the upregulation of several transcription required for neuronal differentiation, but no significant changes were detected in structural or synaptic markers at the transcriptional level, [ 33,37 ] although increases in MAP2 and SYP expression in differentiated SH‐SY5Y cells were observed by immunostaining and immunoblotting.…”

Identification of a novel gene signature for neuroblastoma differentiation using a Boolean implication network

“…Aberrant expression and activity of several GFRs, including RET, EGFR, TrkA, and TrkB, have been associated with neuroblastoma differentiation. [95][96][97][98][99][100][101][102][103][104][105][106][107][108] While further studies are clearly needed to determine the relative roles of individual intracellular and extracellular pathways and of individual genes within each cluster, our data demonstrating the association of UBE4B with neuroblastoma differentiation suggests that UBE4B-mediated signaling may play a key role in the differentiation process.…”

Modification of a Selective NTRK2 Agonist and Confirmation of Activity in a Glaucoma-on-a-Chip Model

“…SH-SY5Y cells are SKN-SH human neuroblastoma cell line [ 16 ] sub-clones and were purchased from Pasteur Institute (Tehran, Iran). Cells were cultured at an initial seeding density of 10 4 cells/cm 2 in the wells of the chip, which had been coated with laminin (0.01 mg ml ) over the Poly-D-lysine layer (0.05 mg ml ).…”