Abstract
Purpose: To investigate the effect of miR-9 on the proliferation, differentiation and migration of human neural stem cells (NSCs).Methods: The expression of miR-9 was investigated by quantitative real-time polymerase chain reaction (RT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK8) assay, while cell migration was studied by Transwell assay. The effect of miR-9 on differentiation of NSCs was investigated by western blot analysis of key differentiation marker proteins. Protein expression was determined by western blotting.Results: Transfection and over-expression of miR-9 in NSCs significantly enhanced the proliferation of NSCs (p < 0.05) in a time-dependent manner, as was evident from CCK8 assay data. MiR-9 overexpression caused down-regulation of Nestin and SOX-2, and up-regulation of Tuj-1 and MAP-2. The migration of NSCs was 37 % in the cells transfected with empty vector, compared to 68 % in the cells transfected with miR-9. This effect of miR-9 on cell migration was accompanied by up-regulation of matrix metallopeptidase 9 (MMP-9) and matrix metallopeptidase 2 (MMP-2).Conclusion: These results show that miR-9 promotes the proliferation, differentiation and migration of NSCs, and thus may be an important drug target for the generation of NSCs.Keywords: Neural stem cells, MicroRNA, Mir-9, Migration, Differentiation, Proliferation
Highlights
Neural stem cells (NSCs) are basically undifferentiated cells with the capacity to divide and self-renew
It was observed that the expression of miR-9 was about 6.3 times higher in the miR-9 transfected cells, when compared to neural stem cells (NSCs) transfected with empty vector (Figure 1)
The results showed that transfection of NSCs with miR-9 led to significant and time-dependent enhancement of the proliferation of NSCs (Figure 3)
Summary
Neural stem cells (NSCs) are basically undifferentiated cells with the capacity to divide and self-renew. Thereafter, the vector containing miR-9 and the empty vector (negative control) were transfected into NSCs cells with help of FuGENE HD (Promega) as per the manufacturer’s protocol. Assessment of expression by quantitative RT-PCR and 96 h after transfection with miR-9 mimics or empty vector, and subjected to CCK-8 assay. The proteins were quantified by BSA protein assay kit, and equal amounts of protein extract from each group (miR9 and empty vector transfected NSCs) were run on SDS-PAGE and transferred to a polyvinylidene fluoride membrane. This was followed by blocking with 5% non-fat milk and incubation at room temperature for 1h.
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