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

ABSTRACT

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.

KEYWORDS:

• Mesenchymal stem cells
• Id1
• lentivirus
• RNAi
• neural differentiation

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was funded by Major Projection of Hebei North University [grant number ZD201413]; Advanced School Science Technology Research Projection of Hebei Province [grant number Z2015047].