The effect of Id1 gene silencing on the neural differentiation of MSCs

We aimed to construct a highly efficient, lentivirus-mediated vector for RNA interference (RNAi) silencing of the Id1 gene and to examine whether silencing of this gene promotes the neural differentiation of mesenchymal stem cells (MSCs). Three short hairpin RNA sequences targeting the coding region...

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Bibliographic Details
Published in:Biotechnology, biotechnological equipment biotechnological equipment, 2017-05, Vol.31 (3), p.554-562
Main Authors: Song, Xiao-qing, Su, Li-ning, Wei, Hui-ping, Liu, Ying-hui, Yin, Hai-feng, Li, Ji-hong, Zhu, Deng-xiang, Zhang, Ai-lan
Format: Article
Language:English
Subjects:
Cell differentiation
Coding
Differentiation
Fibroblast growth factor 2
Fluorescence
Gene expression
Gene sequencing
Gene silencing
Genetics
Green fluorescent protein
Growth factors
Id1
Id1 protein
Immunocytochemistry
lentivirus
Mesenchymal stem cells
Mesenchyme
mRNA
Multiplicity of infection
Nestin
neural differentiation
Packaging
Polymerase chain reaction
Proteins
Real time
Ribonucleic acid
RNA
RNA-mediated interference
RNAi
Stem cells
Western blotting
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Summary:We aimed to construct a highly efficient, lentivirus-mediated vector for RNA interference (RNAi) silencing of the Id1 gene and to examine whether silencing of this gene promotes the neural differentiation of mesenchymal stem cells (MSCs). Three short hairpin RNA sequences targeting the coding region of the Id1 gene and one negative control (NC) sequence were designed. After monophosphorylation, these sequences were introduced into lentiviral vectors containing enhanced green fluorescent protein (EGFP) using the restriction enzyme BsmBI to construct the recombinant vectors pWSLV-EGF-shId1 (1-3) and pWSLV-EGF-NC. These recombinant lentivirus vectors were transfected into H9C2 cells to assess the silencing effect of the shId1 (1-3) sequences on the Id1 gene. Subsequently, pWSLV-EGF-NC and pWSLV-EGF-shId1 which exhibited the strongest silencing effect were transfected into the packaging cell line 293 FT. After determining the viral titers, the cultured MSCs were infected with the lentivirus (multiplicity of infection, MOI = 10). Real-time polymerase chain reaction (PCR) and Western blotting were used to examine the amounts of Id1 mRNA and protein, respectively, in the transfected MSCs, and the results revealed that Id1 expression was markedly inhibited. Finally, MSCs were treated with the growth factors EGF and bFGF in conjunction with ligustrazine to induce their differentiation into neuron-like cells. The expression of NSE/MAP-2/Nestin was detected using immunocytochemistry and real-time PCR, which revealed that the rate of neural differentiation in the pWSLV-EGF-shId1 group was higher than that of the other groups, as was NSE/MAP-2 expression. Thus, Id1 gene silencing promotes the neural differentiation of MSCs.
ISSN:1310-2818
1314-3530
DOI:10.1080/13102818.2017.1286234