Cellular and Molecular Neurobiology

, Volume 37, Issue 4, pp 665–682 | Cite as

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells

  • Francesco Pezzini
  • Laura Bettinetti
  • Francesca Di Leva
  • Marzia Bianchi
  • Elisa Zoratti
  • Rosalba Carrozzo
  • Filippo M. Santorelli
  • Massimo Delledonne
  • Maciej Lalowski
  • Alessandro Simonati
Original Research

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.

Keywords

SH-SY5Y differentiation RNA-seq analysis Axonal guidance signalling Semaphorins Neuronal markers Axonal elongation 

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

Notes

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Neuroscience, Biomedicine and MovementUniversity of VeronaVeronaItaly
  2. 2.Department of BiotechnologiesUniversity of VeronaVeronaItaly
  3. 3.Unit for Neuromuscular and Neurodegenerative Disorders, Laboratory of Molecular MedicineIRCCS Bambino Gesù Children’s HospitalRomeItaly
  4. 4.Applied Research on Cancer-Network (ARC-NET)University of VeronaVeronaItaly
  5. 5.Unit for Neuromuscular and Neurodegenerative Disorders, Laboratory of Molecular MedicineIRCCS Stella MarisCalambrone-PisaItaly
  6. 6.Medicum, Biochemistry/Developmental Biology Meilahti Clinical Proteomics Core FacilityUniversity of HelsinkiHelsinkiFinland
  7. 7.Aptuit s.r.l.VeronaItaly

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