miR-381 Regulates Neural Stem Cell Proliferation and Differentiation via Regulating Hes1 Expression

Abstract

Neural stem cells are self-renewing, multipotent and undifferentiated precursors that retain the capacity for differentiation into both glial (astrocytes and oligodendrocytes) and neuronal lineages. Neural stem cells offer cell-based therapies for neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and spinal cord injuries. However, their cellular behavior is poorly understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in cell development, proliferation and differentiation through regulating gene expression at post-transcriptional level. The role of miR-381 in the development of neural stem cells remains unknown. In this study, we showed that overexpression of miR-381 promoted neural stem cells proliferation. It induced the neural stem cells differentiation to neurons and inhibited their differentiation to astrocytes. Furthermore, we identified HES1 as a direct target of miR-381 in neural stem cells. Moreover, re-expression of HES1 impaired miR-381-induced promotion of neural stem cells proliferation and induce neural stem cells differentiation to neurons. In conclusion, miR-381 played important role in neural stem cells proliferation and differentiation.

Fig 1. Neural stem cells could proliferate and differentiate into neurons and astrocytes.

(A) Representative photomicrograph of neurospheres in culture. (B) Immunocytochemical staining of purified NSCs with Nestin. (C) Immunocytochemical staining of purified protoplasmic astrocytes with GFAP. (D) Immunocytochemical staining of purified neurons with β-tubulin-III. (E) Nucleus staining of differentiated cells from NSCs with DAPI.

Fig 2. miR–381 promoted neural stem cells proliferation.

(A) The expression of miR–381 was measured by qRT-PCR. (B) CCK–8 was performed to detect the neural stem cells proliferation. (C) The mRNA expression of nestin was detected by qRT-PCR. (D) The protein expression of nestin was measured by Western blot. **p<0.01 and ***p<0.001.

Fig 3. miR–381 promoted neural stem cells differentiation to neurons.

(A) Immunocytochemical staining of purified neurons with β-tubulin-III. (B) The mRNA expression of β-tubulin-III was detected by qRT-PCR. (C) The protein expression of β-tubulin-III was measured by Western blot.***p<0.001.

Fig 4. miR–381 inhibited neural stem cells differentiation to astrocytes.

(A) Immunocytochemical staining of purified protoplasmic astrocytes with GFAP. (B) The mRNA expression of GFAP was detected by qRT-PCR. (C) The protein expression of GFAP was measured by Western blot.***p<0.001.

Fig 5. Hes1 was the direct target of miR–381 in neural stem cells.

(A) Hes1 was predicted to be target gene ofmiR–381 by TargetScan. (B) Luciferase reporter assay was done to confirm the predictions in neural stem cells. (C) The protein expression of Hes1 was measured by Western blot in neural stem cells.***p<0.001.

Fig 6. miR–381 promoted neural stem cells proliferation and differentiation to neurons by targeting Hes1.

(A) The protein expression of Hes1 was measured by Western blot in neural stem cells. (B) CCK–8 was performed to detect the neural stem cells proliferation. (C) The mRNA expression of Hes1 was measured by qRT-PCR in neural stem cells. (D) The protein expression of Hes1 was measured by Western blot in neural stem cells. (E) The mRNA expression of β-tubulin-III was measured by qRT-PCR in neural stem cells. (F)The protein expression of β-tubulin-III was measured by Western blot in neural stem cells. (G) The mRNA expression of GFAP was measured by qRT-PCR in neural stem cells. (F)The protein expression of GFAP was measured by Western blot in neural stem cells.**p<0.01 and ***p<0.001.