Abstract
Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.
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Abbreviations
- BDNF:
-
Brain-derived neurotrophic factor
- db-cAMP:
-
Dibutyryl-cyclic AMP
- DEG:
-
Differentially expressed gene
- FDZs:
-
Frizzled receptors
- FPKM:
-
Fragments per kilobase per million mapped reads
- IPA:
-
QIAGEN’s ingenuity® pathway analysis
- log2FC:
-
Log2 fold change (ratio between averaged FPKM of RA-NBM differentiated cells and the averaged FPKM of untreated cells)
- NGF:
-
Nerve growth factor
- NGFR:
-
Nerve growth factor receptor
- NRP1:
-
Neuropilin-1
- NTF4:
-
Neurotrophin-4
- NTRK2:
-
Neurotrophic tyrosine kinase receptor type 2
- PLXNA4:
-
Plexin A4
- pNF-H:
-
Phosphorylated heavy-chain neurofilament proteins
- pNF-M:
-
Phosphorylated medium-chain neurofilament proteins
- RA:
-
All trans retinoic acid
- RA-NBM:
-
9-day-long differentiation paradigm in which SH-SY5Y cells were pre-differentiated in RA medium (6 days) and subsequently treated with neurobasal medium enriched with neurotrophic factors (3 days)
- RA-BDNF:
-
9-day-long differentiation paradigm in which SH-SY5Y cells were pre-differentiated in RA medium (6 days) and subsequently treated with serum-free medium containing rhBDNF (3 days)
- SEMA:
-
Semaphorin
- SLITRK6:
-
SLIT and NTRK-like family
- SYN1:
-
Synapsin I
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Acknowledgments
Dr. A. Ferrarini and Dr. E. Dal Molin from the Department of Biotechnology of Verona University are fully acknowledged for their experienced support with RNA-seq analysis. We are grateful to the scientists and technicians of the Laboratory of Neuropathology, University of Verona School of Medicine. We also thank Dr. Enzo Scifo (University of Toronto, Canada) for critical comments on the manuscript.
Author contributions
F.P: designed and performed experiments, interpreted data, prepared figures and tables, and wrote the manuscript; L.B.: performed experiments and analysed data; F.DiL.: analysed and interpreted data; M.B.: analysed data; E.Z.: performed experiments and analysed data; R.C.: analysed and interpreted data; M.D.: designed experiments, interpreted data, revised the manuscript; F.M.S.: conceived the study, and revised the manuscript; M.L. designed the study, analysed and interpreted data, prepared figures, revised the manuscript; A.S.: conceived and designed the study, analysed and interpreted data, drafted and revised the manuscript.
Funding
This study was funded by European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No 281234.
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Pezzini, F., Bettinetti, L., Di Leva, F. et al. Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells. Cell Mol Neurobiol 37, 665–682 (2017). https://doi.org/10.1007/s10571-016-0403-y
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DOI: https://doi.org/10.1007/s10571-016-0403-y