Nan fang yi ke da xue xue bao = Journal of Southern Medical University · 2017
[Effect of nucleolin silencing on differentiation of rat neural stem cells in vitro and the molecular mechanism].
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Chen Chong-Feng, Yang Huai-Cai, Liu Guo-Sheng
Аннотация
OBJECTIVE: To investigate the effect of nucleolin silencing on the differentiation of rat neural stem cells (NSCs) and the role of Wnt signaling pathway in mediating such effect.
METHODS: Adenovirus vectors expressing small interfering RNA (siRNA) against nucleolin were constructed, verified, and packaged in HEK293A cells. The adenovirus was then transfected into NSCs isolated from neonatal SD rats and the differentiation of the NSCs was examined by detecting the expressions of neuron specific encloase (NSE) and glial fibrillary acidic protein (GFAP) using immunocytochemistry. The expressions of nucleolin, nestin, Wnt3, and β-catenin in the cells were determined with Western blotting.
RESULTS: Restriction endonuclease and sequencing analysis verified successful construction of the adenoviral vector expressing nucleolin siRNA (nucleolin-siRNA2). Infection of rat NSCs with nucleolin-siRNA2 significantly lowered nucleolin protein expression as compared with that in negative and blank control groups (P<0.05). The percentages of NSE-positive cells and GFAP-positive cells were significantly higher in NSCs infected with nucleolin-siRNA (P<0.01); the infection also resulted in obviously lowered expression of nestin protein and increased expressions of Wnt3 protein and β-catenin nucleoprotein in the cells.
CONCLUSIONS: Nucleolin silencing by adenovirus-mediated RNA interference induces the differentiation of NSCs into neurons and astrocytes, which is related with the activation of Wnt signaling pathway.
METHODS: Adenovirus vectors expressing small interfering RNA (siRNA) against nucleolin were constructed, verified, and packaged in HEK293A cells. The adenovirus was then transfected into NSCs isolated from neonatal SD rats and the differentiation of the NSCs was examined by detecting the expressions of neuron specific encloase (NSE) and glial fibrillary acidic protein (GFAP) using immunocytochemistry. The expressions of nucleolin, nestin, Wnt3, and β-catenin in the cells were determined with Western blotting.
RESULTS: Restriction endonuclease and sequencing analysis verified successful construction of the adenoviral vector expressing nucleolin siRNA (nucleolin-siRNA2). Infection of rat NSCs with nucleolin-siRNA2 significantly lowered nucleolin protein expression as compared with that in negative and blank control groups (P<0.05). The percentages of NSE-positive cells and GFAP-positive cells were significantly higher in NSCs infected with nucleolin-siRNA (P<0.01); the infection also resulted in obviously lowered expression of nestin protein and increased expressions of Wnt3 protein and β-catenin nucleoprotein in the cells.
CONCLUSIONS: Nucleolin silencing by adenovirus-mediated RNA interference induces the differentiation of NSCs into neurons and astrocytes, which is related with the activation of Wnt signaling pathway.