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Differentiation Induces Dramatic Changes in miRNA Profile, Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

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Abstract

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.

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Abbreviations

AD:

Alzheimer’s disease

BDNF:

brain-derived neurotrophic factor

CDKs:

cyclin-dependent kinases

DTT:

dithiothreitol

FBS:

fetal bovine serum

HPRT:

hypoxanthine-guanine phosphoribosyl transferase

IDV:

integrated density value

MAP1B:

microtubule-associated protein 1b

miRNA:

microRNA

mRNA:

messenger RNA

NFL:

neurofilament

NGF:

nerve growth factor

NTC:

non-targeted control

PCR:

polymerase chain reaction

PSD95:

postsynaptic density protein 95

RA:

retinoic acid

RQ:

relative quantification

RT:

reverse transcription

RTF:

reprogramming transcription factor

IR:

infrared

PBS:

phosphate-buffered saline

SNAP25:

synaptosomal-associated protein 25

SYP:

synaptophysin

SYN1:

synapsin 1

PVDF:

polyvinylidene fluoride

TLDA:

Taqman low-density array

TH:

tyrosine hydroxylase

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Acknowledgments

Funding for the work carried out in the present study has been provided by CSIR network projects (InDepth and miND) and Department of Biotechnology project (GAP-254). Mr. Abhishek Jauhari and Ankur Kumar Srivastava are grateful to UGC, New Delhi, and Ms. Ankita Pandey is grateful to CSIR, New Delhi, for providing research fellowships. The technical assistance of Mr. B S Pandey and Mr. Puneet Khare is also gratefully acknowledged. The CSIR-IITR communication reference number is 3397.

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    Authors and Affiliations

    1. Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research (IITR), Vishvigyan Bhawan, 31 Mahatma Gandhi Marg, Lucknow, UP, 226001, India

      Abhishek Jauhari, Tanisha Singh, Ankita Pandey, Parul Singh, Nishant Singh, Ankur Kumar Srivastava, Aditya Bhushan Pant, Devendra Parmar & Sanjay Yadav

    2. Academy of Scientific and Innovative Research (AcSIR), IITR Campus, Lucknow, India

      Abhishek Jauhari & Ankur Kumar Srivastava

    3. Department of Biochemistry, School of Dental Sciences, Babu Banarasi Das University, Lucknow, India

      Tanisha Singh, Parul Singh & Nishant Singh

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    1. Abhishek Jauhari
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    Correspondence to Sanjay Yadav.

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    Abhishek Jauhari and Tanisha Singh contributed equally to this work.

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    Jauhari, A., Singh, T., Pandey, A. et al. Differentiation Induces Dramatic Changes in miRNA Profile, Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence. Mol Neurobiol 54, 4986–4995 (2017). https://doi.org/10.1007/s12035-016-0042-9

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