Yonsei Med J.  2016 Nov;57(6):1461-1467. 10.3349/ymj.2016.57.6.1461.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

• Song J
• Oh Y
• Kim JY
• Cho KJ
• Lee JE

• KMID: 2427165
• DOI: http://doi.org/10.3349/ymj.2016.57.6.1461

Abstract

PURPOSE
Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis.
MATERIALS AND METHODS
This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression.
RESULTS
Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a.
CONCLUSION
Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Keyword

Neural stem cell (NSCs); microRNA let-7a (let-7a); agmatine; DCX; TLX; ERK

MeSH Terms

Agmatine/*pharmacology
Cell Differentiation/*drug effects
Cell Proliferation
Humans
MicroRNAs/*metabolism
Neural Stem Cells/cytology/*drug effects
Neurogenesis/*drug effects
Neurons/cytology/drug effects/metabolism
MicroRNAs
Agmatine

Figure

• 

  Fig. 1 Alterations in NeuN and DCX protein levels in NSCs by agmatine treatment. (A) The protein levels of NeuN increased in the agmatine treatment group. In particular, the 100 µM agmatine treatment significantly increased NeuN protein levels in NSCs. (B) DCX protein levels increased in the agmatine treatment group. Similar to NeuN, 100 µM agmatine significantly increased DCX protein levels in NSCs. β-actin was used as an internal control. Data are expressed as mean±SEM. *p<0.05, †p<0.001 compared to agmatine 0 uM group. Agm, agmatine treatment; NSCs, neural stem cells.

• 

  Fig. 2 Effects of alterations of let-7a levels in neural stem cells. A TaqMan assay was performed to check let-7a level. The level of let-7a decreased in the agmatine group compared with the let-7a mimic group. U6 was used as an internal control. Data are expressed as mean±SEM. *p<0.05, †p<0.001 compared to normal control group. let-7a mimic, let-7a mimic treatment/let-7a overexpression group; let-7a inhibitor, let-7a inhibitor treatment/let-7a suppression group; Agm, 100 µM agmatine treatment group; Veh, Lipofectamine treatment group.

• 

  Fig. 3 The expression of DCX and TLX mRNA levels in neural stem cells. (A) Immunochemical images indicated that DCX-positive cells decreased in the let-7a mimic group. Scale bar: 200 µm. NC: the normal control group, 4', 6-diamidino-2-phenylindole (DAPI): blue, DCX: red. (B) Under agmatine treatment, the let-7a suppression group (let-7a inhibitor group) expressed lower DCX mRNA levels. GAPDH was used as an internal control. (C) The let-7a mimic group with agmatine had decreased TLX mRNA expression compared to the let-7a mimic group. GAPDH was used as an internal control. (D) Inhibition of TLX expression by agmatine decreased let-7a levels. U6 was used as an internal control. Data are expressed as mean±SEM. *p<0.05, †p<0.001 compared to normal control group, ‡p<0.001 compared to let-7a mimic group. let-7a mimic, let-7a mimic treatment/let-7a overexpression group; let-7a inhibitor, let-7a inhibitor treatment/let-7a suppression group; Normal, normal control group/no treatment; Agm, 100 µM agmatine treatment group; siTLX, TLX silencing group; siTLX+Agm, TLX silencing with agmatine treatment group.

• 

  Fig. 4 Assessment of c-Myc mRNA and ERK phosphorylation in neural stem cells. (A) c-Myc mRNA expression was reduced in the let-7a mimic treatment with agmatine group in comparison with let-7a mimic group. β-actin was used as an internal control. (B) ERK phosphorylation significantly increased in the let-7a mimic treatment with agmatine group compared to let-7a mimic group. GAPDH was used as an internal control. Data are expressed as mean±SEM. *p<0.05, †p<0.001 compared to normal control group, ‡p<0.05 compared to let-7a mimic group. let-7a mimic, let-7a mimic treatment/let-7a overexpression group; let-7a inhibitor, let-7a inhibitor treatment/let-7a suppression group; Agm, 100 µM agmatine treatment group; Veh, Lipofectamine treatment group; p-ERK, ERK phosphorylation.

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