miR-29a modulates neuronal differentiation through targeting REST in mesenchymal stem cells.
OBJECTIVE
To investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.


METHODS
Rat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.


RESULTS
MSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.


CONCLUSIONS
miR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Ping
P
Duan
Hematology Department in the First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou, Henan, China.
Shiling
S
Sun
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Bo
B
Li
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Chuntian
C
Huang
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Yan
Y
Xu
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Xuefei
X
Han
Department of Physiology, Xinxiang Medical University, Xinxiang, Henan, China.
Ying
Y
Xing
Institute of Basic Medicine, Zhengzhou University, Zhengzhou, Henan, China.
Wenhai
W
Yan
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000502:physiology*; D035683:MicroRNAs / Q000378:metabolism*; D039842:Neural Cell Adhesion Molecule L1 / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000628:therapy; D009474:Neurons / Q000502:physiology*; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D057905:Stem Cell Research; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D000099229:RE1-Silencing Transcription Factor
D035683:MicroRNAs; D039842:Neural Cell Adhesion Molecule L1; D012097:Repressor Proteins; D050825:Synaptosomal-Associated Protein 25; D000099229:RE1-Silencing Transcription Factor; C526366:MIRN29 microRNA, rat; C494210:Snap25 protein, rat